• Don't forget to check out Neurotransmitter.net's research index for a list of dozens of other resources created by Shawn Thomas.
• The articles are retrieved from PubMed Central and PubMed on a daily basis.
• Free full text articles are also available for the following subjects: biology, molecular and cellular biology, neuroscience, genetics and proteomics, psychiatry, pharmacology, endocrinology, cancer, biotechnology, informatics, and miscellaneous.
  
• You can jump directly to articles from Microbiology and Molecular Biology Reviews, Applied and Environmental Microbiology, Applied Microbiology, Bacteriological Reviews, BMC Immunology, BMC Infectious Diseases, BMC Microbiology, Clinical and Diagnostic Laboratory Immunology, Clinical and Molecular Allergy, Clinical Microbiology Reviews, Infection and Immunity, Journal of Bacteriology, Journal of Clinical Microbiology, Journal of Immune Based Therapies and Vaccines, Journal of Virology, Medical Immunology, Microbiology and Immunology, International Microbiology, Retrovirology, Virology Journal, Microbial Cell Factories, Antimicrobial Agents and Chemotherapy, and Malaria Journal.
• You can view earlier articles from these journals as well: skip 0, skip 30, skip 60, or skip 120 of the most recent articles.
• Confused by the terminology? Try searching The On-Line Medical Dictionary (OMD).

Web www.neurotransmitter.net

---

Recent Articles in Microbiology and Molecular Biology Reviews

---

Woese CR
A new biology for a new century.
Microbiol Mol Biol Rev. 2004 Jun;68(2):173-86.
Biology today is at a crossroads. The molecular paradigm, which so successfully guided the discipline throughout most of the 20th century, is no longer a reliable guide. Its vision of biology now realized, the molecular paradigm has run its course. Biology, therefore, has a choice to make, between the comfortable path of continuing to follow molecular biology's lead or the more invigorating one of seeking a new and inspiring vision of the living world, one that addresses the major problems in biology that 20th century biology, molecular biology, could not handle and, so, avoided. The former course, though highly productive, is certain to turn biology into an engineering discipline. The latter holds the promise of making biology an even more fundamental science, one that, along with physics, probes and defines the nature of reality. This is a choice between a biology that solely does society's bidding and a biology that is society's teacher. [Abstract/Link to Full Text]

Perkins-Balding D, Ratliff-Griffin M, Stojiljkovic I
Iron transport systems in Neisseria meningitidis.
Microbiol Mol Biol Rev. 2004 Mar;68(1):154-71.
Acquisition of iron and iron complexes has long been recognized as a major determinant in the pathogenesis of Neisseria meningitidis. In this review, high-affinity iron uptake systems, which allow meningococci to utilize the human host proteins transferrin, lactoferrin, hemoglobin, and haptoglobin-hemoglobin as sources of essential iron, are described. Classic features of bacterial iron transport systems, such as regulation by the iron-responsive repressor Fur and TonB-dependent transport activity, are discussed, as well as more specific features of meningococcal iron transport. Our current understanding of how N. meningitidis acquires iron from the human host and the vaccine potentials of various components of these iron transport systems are also reviewed. [Abstract/Link to Full Text]

Vimr ER, Kalivoda KA, Deszo EL, Steenbergen SM
Diversity of microbial sialic acid metabolism.
Microbiol Mol Biol Rev. 2004 Mar;68(1):132-53.
Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals. [Abstract/Link to Full Text]

Forsburg SL
Eukaryotic MCM proteins: beyond replication initiation.
Microbiol Mol Biol Rev. 2004 Mar;68(1):109-31.
The minichromosome maintenance (or MCM) protein family is composed of six related proteins that are conserved in all eukaryotes. They were first identified by genetic screens in yeast and subsequently analyzed in other experimental systems using molecular and biochemical methods. Early data led to the identification of MCMs as central players in the initiation of DNA replication. More recent studies have shown that MCM proteins also function in replication elongation, probably as a DNA helicase. This is consistent with structural analysis showing that the proteins interact together in a heterohexameric ring. However, MCMs are strikingly abundant and far exceed the stoichiometry of replication origins; they are widely distributed on unreplicated chromatin. Analysis of mcm mutant phenotypes and interactions with other factors have now implicated the MCM proteins in other chromosome transactions including damage response, transcription, and chromatin structure. These experiments indicate that the MCMs are central players in many aspects of genome stability. [Abstract/Link to Full Text]

Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE, Read ND, Seiler S, Bell-Pedersen D, Paietta J, Plesofsky N, Plamann M, Goodrich-Tanrikulu M, Schulte U, Mannhaupt G, Nargang FE, Radford A, Selitrennikoff C, Galagan JE, Dunlap JC, Loros JJ, Catcheside D, Inoue H, Aramayo R, Polymenis M, Selker EU, Sachs MS, Marzluf GA, Paulsen I, Davis R, Ebbole DJ, Zelter A, Kalkman ER, O'Rourke R, Bowring F, Yeadon J, Ishii C, Suzuki K, Sakai W, Pratt R
Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism.
Microbiol Mol Biol Rev. 2004 Mar;68(1):1-108.
We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease. [Abstract/Link to Full Text]

Neuhaus FC, Baddiley J
A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in gram-positive bacteria.
Microbiol Mol Biol Rev. 2003 Dec;67(4):686-723.
Teichoic acids (TAs) are major wall and membrane components of most gram-positive bacteria. With few exceptions, they are polymers of glycerol-phosphate or ribitol-phosphate to which are attached glycosyl and D-alanyl ester residues. Wall TA is attached to peptidoglycan via a linkage unit, whereas lipoteichoic acid is attached to glycolipid intercalated in the membrane. Together with peptidoglycan, these polymers make up a polyanionic matrix that functions in (i) cation homeostasis; (ii) trafficking of ions, nutrients, proteins, and antibiotics; (iii) regulation of autolysins; and (iv) presentation of envelope proteins. The esterification of TAs with D-alanyl esters provides a means of modulating the net anionic charge, determining the cationic binding capacity, and displaying cations in the wall. This review addresses the structures and functions of D-alanyl-TAs, the D-alanylation system encoded by the dlt operon, and the roles of TAs in cell growth. The importance of dlt in the physiology of many organisms is illustrated by the variety of mutant phenotypes. In addition, advances in our understanding of D-alanyl ester function in virulence and host-mediated responses have been made possible through targeted mutagenesis of dlt. Studies of the mechanism of D-alanylation have identified two potential targets of antibacterial action and provided possible screening reactions for designing novel agents targeted to D-alanyl-TA synthesis. [Abstract/Link to Full Text]

Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK
RNA interference: biology, mechanism, and applications.
Microbiol Mol Biol Rev. 2003 Dec;67(4):657-85.
Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes. [Abstract/Link to Full Text]

Nikaido H
Molecular basis of bacterial outer membrane permeability revisited.
Microbiol Mol Biol Rev. 2003 Dec;67(4):593-656.
Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions. [Abstract/Link to Full Text]

González JE, Marketon MM
Quorum sensing in nitrogen-fixing rhizobia.
Microbiol Mol Biol Rev. 2003 Dec;67(4):574-92.
Members of the rhizobia are distinguished for their ability to establish a nitrogen-fixing symbiosis with leguminous plants. While many details of this relationship remain a mystery, much effort has gone into elucidating the mechanisms governing bacterium-host recognition and the events leading to symbiosis. Several signal molecules, including plant-produced flavonoids and bacterially produced nodulation factors and exopolysaccharides, are known to function in the molecular conversation between the host and the symbiont. Work by several laboratories has shown that an additional mode of regulation, quorum sensing, intercedes in the signal exchange process and perhaps plays a major role in preparing and coordinating the nitrogen-fixing rhizobia during the establishment of the symbiosis. Rhizobium leguminosarum, for example, carries a multitiered quorum-sensing system that represents one of the most complex regulatory networks identified for this form of gene regulation. This review focuses on the recent stream of information regarding quorum sensing in the nitrogen-fixing rhizobia. Seminal work on the quorum-sensing systems of R. leguminosarum bv. viciae, R. etli, Rhizobium sp. strain NGR234, Sinorhizobium meliloti, and Bradyrhizobium japonicum is presented and discussed. The latest work shows that quorum sensing can be linked to various symbiotic phenomena including nodulation efficiency, symbiosome development, exopolysaccharide production, and nitrogen fixation, all of which are important for the establishment of a successful symbiosis. Many questions remain to be answered, but the knowledge obtained so far provides a firm foundation for future studies on the role of quorum-sensing mediated gene regulation in host-bacterium interactions. [Abstract/Link to Full Text]

O'Donoghue P, Luthey-Schulten Z
On the evolution of structure in aminoacyl-tRNA synthetases.
Microbiol Mol Biol Rev. 2003 Dec;67(4):550-73.
The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases. [Abstract/Link to Full Text]

Van Hamme JD, Singh A, Ward OP
Recent advances in petroleum microbiology.
Microbiol Mol Biol Rev. 2003 Dec;67(4):503-49.
Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times (<60 s) are being used effectively. Microbes are being injected into partially spent petroleum reservoirs to enhance oil recovery. However, these microbial processes have not exhibited consistent and effective performance, primarily because of our inability to control conditions in the subsurface environment. Microbes may be exploited to break stable oilfield emulsions to produce pipeline quality oil. There is interest in replacing physical oil desulfurization processes with biodesulfurization methods through promotion of selective sulfur removal without degradation of associated carbon moieties. However, since microbes require an environment containing some water, a two-phase oil-water system must be established to optimize contact between the microbes and the hydrocarbon, and such an emulsion is not easily created with viscous crude oil. This challenge may be circumvented by application of the technology to more refined gasoline and diesel substrates, where aqueous-hydrocarbon emulsions are more easily generated. Molecular approaches are being used to broaden the substrate specificity and increase the rates and extents of desulfurization. Bacterial processes are being commercialized for removal of H(2)S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments. [Abstract/Link to Full Text]

Strobel G, Daisy B
Bioprospecting for microbial endophytes and their natural products.
Microbiol Mol Biol Rev. 2003 Dec;67(4):491-502.
Endophytic microorganisms are to be found in virtually every plant on earth. These organisms reside in the living tissues of the host plant and do so in a variety of relationships, ranging from symbiotic to slightly pathogenic. Because of what appears to be their contribution to the host plant, the endophytes may produce a plethora of substances of potential use to modern medicine, agriculture, and industry. Novel antibiotics, antimycotics, immunosuppressants, and anticancer compounds are only a few examples of what has been found after the isolation, culture, purification, and characterization of some choice endophytes in the recent past. The potential prospects of finding new drugs that may be effective candidates for treating newly developing diseases in humans, plants, and animals are great. [Abstract/Link to Full Text]

Warner JB, Lolkema JS
CcpA-dependent carbon catabolite repression in bacteria.
Microbiol Mol Biol Rev. 2003 Dec;67(4):475-90.
Carbon catabolite repression (CCR) by transcriptional regulators follows different mechanisms in gram-positive and gram-negative bacteria. In gram-positive bacteria, CcpA-dependent CCR is mediated by phosphorylation of the phosphoenolpyruvate:sugar phosphotransferase system intermediate HPr at a serine residue at the expense of ATP. The reaction is catalyzed by HPr kinase, which is activated by glycolytic intermediates. In this review, the distribution of CcpA-dependent CCR among bacteria is investigated by searching the public databases for homologues of HPr kinase and HPr-like proteins throughout the bacterial kingdom and by analyzing their properties. Homologues of HPr kinase are commonly observed in the phylum Firmicutes but are also found in the phyla Proteobacteria, Fusobacteria, Spirochaetes, and Chlorobi, suggesting that CcpA-dependent CCR is not restricted to gram-positive bacteria. In the alpha and beta subdivisions of the Proteobacteria, the presence of HPr kinase appears to be common, while in the gamma subdivision it is more of an exception. The genes coding for the HPr kinase homologues of the Proteobacteria are in a gene cluster together with an HPr-like protein, termed XPr, suggesting a functional relationship. Moreover, the XPr proteins contain the serine phosphorylation sequence motif. Remarkably, the analysis suggests a possible relation between CcpA-dependent gene regulation and the nitrogen regulation system (Ntr) found in the gamma subdivision of the Proteobacteria. The relation is suggested by the clustering of CCR and Ntr components on the genome of members of the Proteobacteria and by the close phylogenetic relationship between XPr and NPr, the HPr-like protein in the Ntr system. In bacteria in the phylum Proteobacteria that contain HPr kinase and XPr, the latter may be at the center of a complex regulatory network involving both CCR and the Ntr system. [Abstract/Link to Full Text]

Black PN, DiRusso CC
Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification.
Microbiol Mol Biol Rev. 2003 Sep;67(3):454-72, table of contents.
The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids. These differences stem from the unique physical and chemical properties of long-chain fatty acids. To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane. More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered. Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics. In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase [AMP forming] [EC 6.2.1.3]). FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional. This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids. [Abstract/Link to Full Text]

Cotter PD, Hill C
Surviving the acid test: responses of gram-positive bacteria to low pH.
Microbiol Mol Biol Rev. 2003 Sep;67(3):429-53, table of contents.
Gram-positive bacteria possess a myriad of acid resistance systems that can help them to overcome the challenge posed by different acidic environments. In this review the most common mechanisms are described: i.e., the use of proton pumps, the protection or repair of macromolecules, cell membrane changes, production of alkali, induction of pathways by transcriptional regulators, alteration of metabolism, and the role of cell density and cell signaling. We also discuss the responses of Listeria monocytogenes, Rhodococcus, Mycobacterium, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, oral streptococci, and lactic acid bacteria to acidic environments and outline ways in which this knowledge has been or may be used to either aid or prevent bacterial survival in low-pH environments. [Abstract/Link to Full Text]

Naglik JR, Challacombe SJ, Hube B
Candida albicans secreted aspartyl proteinases in virulence and pathogenesis.
Microbiol Mol Biol Rev. 2003 Sep;67(3):400-28, table of contents.
Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis. [Abstract/Link to Full Text]

Bitterman KJ, Medvedik O, Sinclair DA
Longevity regulation in Saccharomyces cerevisiae: linking metabolism, genome stability, and heterochromatin.
Microbiol Mol Biol Rev. 2003 Sep;67(3):376-99, table of contents.
When it was first proposed that the budding yeast Saccharomyces cerevisiae might serve as a model for human aging in 1959, the suggestion was met with considerable skepticism. Although yeast had proved a valuable model for understanding basic cellular processes in humans, it was difficult to accept that such a simple unicellular organism could provide information about human aging, one of the most complex of biological phenomena. While it is true that causes of aging are likely to be multifarious, there is a growing realization that all eukaryotes possess surprisingly conserved longevity pathways that govern the pace of aging. This realization has come, in part, from studies of S. cerevisiae, which has emerged as a highly informative and respected model for the study of life span regulation. Genomic instability has been identified as a major cause of aging, and over a dozen longevity genes have now been identified that suppress it. Here we present the key discoveries in the yeast-aging field, regarding both the replicative and chronological measures of life span in this organism. We discuss the implications of these findings not only for mammalian longevity but also for other key aspects of cell biology, including cell survival, the relationship between chromatin structure and genome stability, and the effect of internal and external environments on cellular defense pathways. We focus on the regulation of replicative life span, since recent findings have shed considerable light on the mechanisms controlling this process. We also present the specific methods used to study aging and longevity regulation in S. cerevisiae. [Abstract/Link to Full Text]

Wickstead B, Ersfeld K, Gull K
Repetitive elements in genomes of parasitic protozoa.
Microbiol Mol Biol Rev. 2003 Sep;67(3):360-75, table of contents.
Repetitive DNA elements have been a part of the genomic fauna of eukaryotes perhaps since their very beginnings. Millions of years of coevolution have given repeats central roles in chromosome maintenance and genetic modulation. Here we review the genomes of parasitic protozoa in the context of the current understanding of repetitive elements. Particular reference is made to repeats in five medically important species with ongoing or completed genome sequencing projects: Plasmodium falciparum, Leishmania major, Trypanosoma brucei, Trypanosoma cruzi, and Giardia lamblia. These organisms are used to illustrate five thematic classes of repeats with different structures and genomic locations. We discuss how these repeat classes may interact with parasitic life-style and also how they can be used as experimental tools. The story which emerges is one of opportunism and upheaval which have been employed to add genetic diversity and genomic flexibility. [Abstract/Link to Full Text]

Penalva LO, Sánchez L
RNA binding protein sex-lethal (Sxl) and control of Drosophila sex determination and dosage compensation.
Microbiol Mol Biol Rev. 2003 Sep;67(3):343-59, table of contents.
In the past two decades, scientists have elucidated the molecular mechanisms behind Drosophila sex determination and dosage compensation. These two processes are controlled essentially by two different sets of genes, which have in common a master regulatory gene, Sex-lethal (Sxl). Sxl encodes one of the best-characterized members of the family of RNA binding proteins. The analysis of different mechanisms involved in the regulation of the three identified Sxl target genes (Sex-lethal itself, transformer, and male specific lethal-2) has contributed to a better understanding of translation repression, as well as constitutive and alternative splicing. Studies using the Drosophila system have identified the features of the protein that contribute to its target specificity and regulatory functions. In this article, we review the existing data concerning Sxl protein, its biological functions, and the regulation of its target genes. [Abstract/Link to Full Text]

Xie G, Keyhani NO, Bonner CA, Jensen RA
Ancient origin of the tryptophan operon and the dynamics of evolutionary change.
Microbiol Mol Biol Rev. 2003 Sep;67(3):303-42, table of contents.
The seven conserved enzymatic domains required for tryptophan (Trp) biosynthesis are encoded in seven genetic regions that are organized differently (whole-pathway operons, multiple partial-pathway operons, and dispersed genes) in prokaryotes. A comparative bioinformatics evaluation of the conservation and organization of the genes of Trp biosynthesis in prokaryotic operons should serve as an excellent model for assessing the feasibility of predicting the evolutionary histories of genes and operons associated with other biochemical pathways. These comparisons should provide a better understanding of possible explanations for differences in operon organization in different organisms at a genomics level. These analyses may also permit identification of some of the prevailing forces that dictated specific gene rearrangements during the course of evolution. Operons concerned with Trp biosynthesis in prokaryotes have been in a dynamic state of flux. Analysis of closely related organisms among the Bacteria at various phylogenetic nodes reveals many examples of operon scission, gene dispersal, gene fusion, gene scrambling, and gene loss from which the direction of evolutionary events can be deduced. Two milestone evolutionary events have been mapped to the 16S rRNA tree of Bacteria, one splitting the operon in two, and the other rejoining it by gene fusion. The Archaea, though less resolved due to a lesser genome representation, appear to exhibit more gene scrambling than the Bacteria. The trp operon appears to have been an ancient innovation; it was already present in the common ancestor of Bacteria and Archaea. Although the operon has been subjected, even in recent times, to dynamic changes in gene rearrangement, the ancestral gene order can be deduced with confidence. The evolutionary history of the genes of the pathway is discernible in rough outline as a vertical line of descent, with events of lateral gene transfer or paralogy enriching the analysis as interesting features that can be distinguished. As additional genomes are thoroughly analyzed, an increasingly refined resolution of the sequential evolutionary steps is clearly possible. These comparisons suggest that present-day trp operons that possess finely tuned regulatory features are under strong positive selection and are able to resist the disruptive evolutionary events that may be experienced by simpler, poorly regulated operons. [Abstract/Link to Full Text]

Grohmann E, Muth G, Espinosa M
Conjugative plasmid transfer in gram-positive bacteria.
Microbiol Mol Biol Rev. 2003 Jun;67(2):277-301, table of contents.
Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer. [Abstract/Link to Full Text]

Canchaya C, Proux C, Fournous G, Bruttin A, Brüssow H
Prophage genomics.
Microbiol Mol Biol Rev. 2003 Jun;67(2):238-76, table of contents.
The majority of the bacterial genome sequences deposited in the National Center for Biotechnology Information database contain prophage sequences. Analysis of the prophages suggested that after being integrated into bacterial genomes, they undergo a complex decay process consisting of inactivating point mutations, genome rearrangements, modular exchanges, invasion by further mobile DNA elements, and massive DNA deletion. We review the technical difficulties in defining such altered prophage sequences in bacterial genomes and discuss theoretical frameworks for the phage-bacterium interaction at the genomic level. The published genome sequences from three groups of eubacteria (low- and high-G+C gram-positive bacteria and gamma-proteobacteria) were screened for prophage sequences. The prophages from Streptococcus pyogenes served as test case for theoretical predictions of the role of prophages in the evolution of pathogenic bacteria. The genomes from further human, animal, and plant pathogens, as well as commensal and free-living bacteria, were included in the analysis to see whether the same principles of prophage genomics apply for bacteria living in different ecological niches and coming from distinct phylogenetical affinities. The effect of selection pressure on the host bacterium is apparently an important force shaping the prophage genomes in low-G+C gram-positive bacteria and gamma-proteobacteria. [Abstract/Link to Full Text]

Chazal N, Gerlier D
Virus entry, assembly, budding, and membrane rafts.
Microbiol Mol Biol Rev. 2003 Jun;67(2):226-37, table of contents.
As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth. [Abstract/Link to Full Text]

Ballicora MA, Iglesias AA, Preiss J
ADP-glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis.
Microbiol Mol Biol Rev. 2003 Jun;67(2):213-25, table of contents.
The accumulation of alpha-1,4-polyglucans is an important strategy to cope with transient starvation conditions in the environment. In bacteria and plants, the synthesis of glycogen and starch occurs by utilizing ADP-glucose as the glucosyl donor for elongation of the alpha-1,4-glucosidic chain. The main regulatory step takes place at the level of ADP-glucose synthesis, a reaction catalyzed by ADP-Glc pyrophosphorylase (PPase). Most of the ADP-Glc PPases are allosterically regulated by intermediates of the major carbon assimilatory pathway in the organism. Based on specificity for activator and inhibitor, classification of ADP-Glc PPases has been expanded into nine distinctive classes. According to predictions of the secondary structure of the ADP-Glc PPases, they seem to have a folding pattern common to other sugar nucleotide pyrophosphorylases. All the ADP-Glc PPases as well as other sugar nucleotide pyrophosphorylases appear to have evolved from a common ancestor, and later, ADP-Glc PPases developed specific regulatory properties, probably by addition of extra domains. Studies of different domains by construction of chimeric ADP-Glc PPases support this hypothesis. In addition to previous chemical modification experiments, the latest random and site-directed mutagenesis experiments with conserved amino acids revealed residues important for catalysis and regulation. [Abstract/Link to Full Text]

Dourmishev LA, Dourmishev AL, Palmeri D, Schwartz RA, Lukac DM
Molecular genetics of Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) epidemiology and pathogenesis.
Microbiol Mol Biol Rev. 2003 Jun;67(2):175-212, table of contents.
Kaposi's sarcoma had been recognized as unique human cancer for a century before it manifested as an AIDS-defining illness with a suspected infectious etiology. The discovery of Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, in 1994 by using representational difference analysis, a subtractive method previously employed for cloning differences in human genomic DNA, was a fitting harbinger for the powerful bioinformatic approaches since employed to understand its pathogenesis in KS. Indeed, the discovery of KSHV was rapidly followed by publication of its complete sequence, which revealed that the virus had coopted a wide armamentarium of human genes; in the short time since then, the functions of many of these viral gene variants in cell growth control, signaling apoptosis, angiogenesis, and immunomodulation have been characterized. This critical literature review explores the pathogenic potential of these genes within the framework of current knowledge of the basic herpesvirology of KSHV, including the relationships between viral genotypic variation and the four clinicoepidemiologic forms of Kaposi's sarcoma, current viral detection methods and their utility, primary infection by KSHV, tissue culture and animal models of latent- and lytic-cycle gene expression and pathogenesis, and viral reactivation from latency. Recent advances in models of de novo endothelial infection, microarray analyses of the host response to infection, receptor identification, and cloning of full-length, infectious KSHV genomic DNA promise to reveal key molecular mechanisms of the candidate pathogeneic genes when expressed in the context of viral infection. [Abstract/Link to Full Text]

Condon C
RNA processing and degradation in Bacillus subtilis.
Microbiol Mol Biol Rev. 2003 Jun;67(2):157-74, table of contents.
This review focuses on the enzymes and pathways of RNA processing and degradation in Bacillus subtilis, and compares them to those of its gram-negative counterpart, Escherichia coli. A comparison of the genomes from the two organisms reveals that B. subtilis has a very different selection of RNases available for RNA maturation. Of 17 characterized ribonuclease activities thus far identified in E. coli and B. subtilis, only 6 are shared, 3 exoribonucleases and 3 endoribonucleases. Some enzymes essential for cell viability in E. coli, such as RNase E and oligoribonuclease, do not have homologs in B. subtilis, and of those enzymes in common, some combinations are essential in one organism but not in the other. The degradation pathways and transcript half-lives have been examined to various degrees for a dozen or so B. subtilis mRNAs. The determinants of mRNA stability have been characterized for a number of these and point to a fundamentally different process in the initiation of mRNA decay. While RNase E binds to the 5' end and catalyzes the rate-limiting cleavage of the majority of E. coli RNAs by looping to internal sites, the equivalent nuclease in B. subtilis, although not yet identified, is predicted to scan or track from the 5' end. RNase E can also access cleavage sites directly, albeit less efficiently, while the enzyme responsible for initiating the decay of B. subtilis mRNAs appears incapable of direct entry. Thus, unlike E. coli, RNAs possessing stable secondary structures or sites for protein or ribosome binding near the 5' end can have very long half-lives even if the RNA is not protected by translation. [Abstract/Link to Full Text]

Miller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, Rüger W
Bacteriophage T4 genome.
Microbiol Mol Biol Rev. 2003 Mar;67(1):86-156, table of contents.
Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the "cell-puncturing device," combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages-the most abundant and among the most ancient biological entities on Earth. [Abstract/Link to Full Text]

Pivetti CD, Yen MR, Miller S, Busch W, Tseng YH, Booth IR, Saier MH
Two families of mechanosensitive channel proteins.
Microbiol Mol Biol Rev. 2003 Mar;67(1):66-85, table of contents.
Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin. [Abstract/Link to Full Text]

Errington J, Daniel RA, Scheffers DJ
Cytokinesis in bacteria.
Microbiol Mol Biol Rev. 2003 Mar;67(1):52-65, table of contents.
Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum. [Abstract/Link to Full Text]

Desai MJ, Armstrong DW
Separation, identification, and characterization of microorganisms by capillary electrophoresis.
Microbiol Mol Biol Rev. 2003 Mar;67(1):38-51, table of contents.
The use of capillary electrophoresis (CE) for the analysis, identification, and characterization of microorganisms has been gaining in popularity. The advantages of CE, such as small sample requirements, minimal sample preparation, rapid and simultaneous analysis, ease of quantitation and identification, and viability assessment, make it an attractive technique for the analysis of microbial analytes. As this instrumental method has evolved, higher peak efficiencies have been achieved by optimizing CE conditions, such as pH, ionic strength, and polymer additive concentration. Experimental improvements have allowed better quantitation and more accurate results. Many practical applications of this technique have been investigated. Viability and identification of microbes can be accomplished in a single analysis. This is useful for evaluation of microbial analytes in consumer products. Diagnosis of microbe-based diseases is now possible, in some cases, without the need for culture methods. Microbe-molecule, virus-antibody, or bacteria-antibiotic interactions can be monitored using CE, allowing for the screening of possible drug candidates. Fermentation can be monitored using this system. This instrumental approach can be adapted to many different applications, including assessing the viability of sperm cells. Progress has been made in the development of microelectrophoresis instrumentation. These advances will eventually allow the development of small, dedicated devices for the rapid, repetitive analyses of specific microbial samples. Although these methods may never fully replace traditional approaches, they are proving to be a valuable addition to the collection of techniques used to analyze, quantitate, and characterize microbes. This review outlines the recent developments in this rapidly growing field. [Abstract/Link to Full Text]

---

Recent Articles in Applied and Environmental Microbiology

---

Miyake R, Kawamoto J, Wei YL, Kitagawa M, Kato I, Kurihara T, Esaki N
Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host.
Appl Environ Microbiol. 2007 Aug;73(15):4849-56.
A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce beta-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system. [Abstract/Link to Full Text]

Nuccio SP, Chessa D, Weening EH, Raffatellu M, Clegg S, Bäumler AJ
SIMPLE approach for isolating mutants expressing fimbriae.
Appl Environ Microbiol. 2007 Jul;73(14):4455-62.
Genomes of members of the family Enterobacteriaceae contain large repertoires of putative fimbrial operons. Since many of these operons are poorly expressed in vitro, a convenient method for inducing elaboration of the encoded fimbriae would greatly facilitate their functional characterization. Here we describe a new technique for identifying fimbriated bacteria from a library of transposon mutants by screening with immunomagnetic particles for ligand expression (SIMPLE). The SIMPLE method was applied to identify the T-POP mutants of Salmonella enterica serotype Typhimurium carrying on their surfaces filaments composed of PefA, the major subunit product of a fimbrial operon (pef) that is not expressed during growth in Luria-Bertani broth. Four such mutants were identified from a library of 24,000 mutants, each of which carried a T-POP insertion within the hns gene, which encodes a global silencer of horizontally acquired genes. Our data suggest that the SIMPLE method is an effective approach for isolating fimbriated bacteria, which can be readily applied to fimbrial operons identified by whole-genome sequencing. [Abstract/Link to Full Text]

Fyfe JA, Lavender CJ, Johnson PD, Globan M, Sievers A, Azuolas J, Stinear TP
Development and application of two multiplex real-time PCR assays for the detection of Mycobacterium ulcerans in clinical and environmental samples.
Appl Environ Microbiol. 2007 Aug;73(15):4733-40.
Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen. [Abstract/Link to Full Text]

Kindaichi T, Tsushima I, Ogasawara Y, Shimokawa M, Ozaki N, Satoh H, Okabe S
In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms.
Appl Environ Microbiol. 2007 Aug;73(15):4931-9.
We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria. [Abstract/Link to Full Text]

Héry-Arnaud G, Bruant G, Lanotte P, Brun S, Picard B, Rosenau A, van der Mee-Marquet N, Rainard P, Quentin R, Mereghetti L
Mobile genetic elements provide evidence for a bovine origin of clonal complex 17 of Streptococcus agalactiae.
Appl Environ Microbiol. 2007 Jul;73(14):4668-72.
We sought an explanation for epidemiological changes in Streptococcus agalactiae infections by investigating the link between ecological niches of the bacterium by determining the prevalence of 11 mobile genetic elements. The prevalence of nine of these elements differed significantly according to the human or bovine origin of the isolate. Correlating this distribution with the phylogeny obtained by multilocus sequence analysis, we observed that human isolates harboring GBSi1, a clear marker of the bovine niche, clustered in clonal complex 17. Our results are thus consistent with the emergence of this virulent human clone from a bovine ancestor. [Abstract/Link to Full Text]

Snyder H, Stock SP, Kim SK, Flores-Lara Y, Forst S
New insights into the colonization and release processes of Xenorhabdus nematophila and the morphology and ultrastructure of the bacterial receptacle of its nematode host, Steinernema carpocapsae.
Appl Environ Microbiol. 2007 Aug;73(16):5338-46.
We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that Xenorhabdus nematophila colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of Steinernema carpocapsae. This region is connected to the esophagus at the esophagointestinal junction. The process by which X. nematophila leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that Xenorhabdus colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of Xenorhabdus down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of X. nematophila out of the receptacle and into the intestinal lumen. [Abstract/Link to Full Text]

Andam CP, Mondo SJ, Parker MA
Monophyly of nodA and nifH genes across Texan and Costa Rican populations of Cupriavidus nodule symbionts.
Appl Environ Microbiol. 2007 Jul;73(14):4686-90.
nodA and nifH phylogenies for Cupriavidus nodule bacteria from native legumes in Texas and Costa Rica grouped all strains into a single clade nested among neotropical Burkholderia strains. Thus, Cupriavidus symbiotic genes were not acquired independently in different regions and are derived from other Betaproteobacteria rather than from alpha-rhizobial donors. [Abstract/Link to Full Text]

Morono Y, Kitagawa W, Kimura N, Noda N, Nakamura K, Kamagata Y
"Mark the gene": a method for nondestructive introduction of marker sequences inside the gene frame of transgenes.
Appl Environ Microbiol. 2007 Aug;73(15):4915-21.
A specific marking and detection technique is a fundamental requirement for the safer use of genetically modified (GM) organisms. Here we propose a simple and effective method for directly marking functional transgenes in GM organisms. For that purpose, we introduced nucleotide substitutions (NS), based on the degeneracy of codons as markers (NS markers), into the bphC (2,3-dihydroxybiphenyl dioxygenase) and tomA3 (toluene-ortho-monooxygenase) gene frames using a PCR-based method. No change was observed in the enzyme activity of translated proteins, and alignments with homologous genes showed the uniqueness of the NS markers. Furthermore, we constructed tomA3 variations harboring NS markers in different positions. Although the translational products were identical, the constructed variation genes could be distinguished through their marker patterns by multiplex PCR, showing that NS markers could serve as product-specific tags for identifying individual GM organisms. This direct method of marking the functional transgene provides a simple, low-risk, and robust marking method without causing the gene functions to deteriorate. [Abstract/Link to Full Text]

Ashby MN, Rine J, Mongodin EF, Nelson KE, Dimster-Denk D
Serial analysis of rRNA genes and the unexpected dominance of rare members of microbial communities.
Appl Environ Microbiol. 2007 Jul;73(14):4532-42.
The accurate description of a microbial community is an important first step in understanding the roles of its components in ecosystem function. A method for surveying microbial communities termed serial analysis of rRNA genes (SARD) is described here. Through a series of molecular cloning steps, short DNA sequence tags are recovered from the fifth variable (V5) region of the prokaryotic 16S rRNA genes from microbial communities. These tags are ligated to form concatemers comprised of 20 to 40 tags which are cloned and identified by DNA sequencing. Four agricultural soil samples were profiled with SARD to assess the method's utility. A total of 37,008 SARD tags comprising 3,127 unique sequences were identified. A comparison of duplicate profiles from one soil genomic DNA preparation revealed that the method was highly reproducible. The large numbers of singleton tags, together with nonparametric richness estimates, indicated that a significant amount of sequence tag diversity remained undetected with this level of sampling. The abundance classes of the observed tags were scale-free and conformed to a power law distribution. Numerically, the majority of the total tags observed belonged to abundance classes that were each present at less than 1% of the community. Over 99% of the unique tags individually made up less than 1% of the community. Therefore, from either a numerical or diversity standpoint, taxa with low abundance comprised a significant proportion of the microbial communities examined and could potentially make a large contribution to ecosystem function. SARD may provide a means to explore the ecological roles of these rare members of microbial communities in qualitative and quantitative terms. [Abstract/Link to Full Text]

Lee JH, Halgerson JS, Kim JH, O'Sullivan DJ
Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector.
Appl Environ Microbiol. 2007 Jul;73(14):4417-24.
While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three-a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria. [Abstract/Link to Full Text]

Kawarai T, Furukawa S, Ogihara H, Yamasaki M
Mixed-species biofilm formation by lactic acid bacteria and rice wine yeasts.
Appl Environ Microbiol. 2007 Jul;73(14):4673-6.
We found that species combinations such as Lactobacillus casei subsp. rhamnosus IFO3831 and Saccharomyces cerevisiae Kyokai-10 can form a mixed-species biofilm in coculture. Moreover, the Kyokai-10 yeast strain can form a biofilm in monoculture in the presence of conditioned medium (CM) from L. casei IFO3831. The active substance(s) in bacterial CM is heat sensitive and has a molecular mass of between 3 and 5 kDa. In biofilms from cocultures or CM monocultures, yeast cells had a distinct morphology, with many hill-like protrusions on the cell surface. [Abstract/Link to Full Text]

Muller JF, Stevens AM, Craig J, Love NG
Transcriptome analysis reveals that multidrug efflux genes are upregulated to protect Pseudomonas aeruginosa from pentachlorophenol stress.
Appl Environ Microbiol. 2007 Jul;73(14):4550-8.
Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole-genome responses to this contaminant stress is limited. This study examined the transcriptome response of a common soil bacterium, Pseudomonas aeruginosa, to the common environmental contaminant pentachlorophenol (PCP). Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole-cell acetate uptake rates (WAURs) and cell viability and for gene expression changes by Affymetrix GeneChip technology and real-time reverse transcriptase PCR. Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP. In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes. In particular, genes coding for multidrug efflux pumps, including MexAB-OprM, were strongly upregulated. The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant. [Abstract/Link to Full Text]

Martinez LR, Casadevall A
Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light.
Appl Environ Microbiol. 2007 Jul;73(14):4592-601.
The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. We describe the characteristics of C. neoformans biofilm development using a microtiter plate model, microscopic examinations, and a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay to observe the metabolic activity of cryptococci within a biofilm. A strong correlation between XTT and CFU assays was demonstrated. Chemical analysis of the exopolymeric material revealed sugar composition consisting predominantly of xylose, mannose, and glucose, indicating the presence of other polysaccharides in addition to glucurunoxylomannan. Biofilm formation was affected by surface support differences, conditioning films on the surface, characteristics of the medium, and properties of the microbial cell. A specific antibody to the capsular polysaccharide of this fungus was used to stain the extracellular polysaccharide matrix of the fungal biofilms using light and confocal microscopy. Additionally, the susceptibility of C. neoformans biofilms and planktonic cells to environmental stress was investigated using XTT reduction and CFU assays. Biofilms were less susceptible to heat, cold, and UV light exposition than their planktonic counterparts. Our findings demonstrate that fungal biofilm formation is dependent on support surface characteristics and that growth in the biofilm state makes fungal cells less susceptible to potential environmental stresses. [Abstract/Link to Full Text]

Gerlach RG, Hölzer SU, Jäckel D, Hensel M
Rapid engineering of bacterial reporter gene fusions by using Red recombination.
Appl Environ Microbiol. 2007 Jul;73(13):4234-42.
Reporter gene fusions are essential tools for the investigation of gene regulation. Such fusions are traditionally generated by transposon mutagenesis and identified by a suitable selection procedure. Alternatively, specific reporter fusions can be generated by cloning of DNA fragments containing promoters or other regulatory elements in reporter plasmids. Here, we describe a novel approach for the rapid generation of reporter gene fusions in single copies at defined positions in bacterial genomes. This technique utilizes the Red recombinase for the homologous recombination of PCR-generated cassettes containing various currently used reporter genes, such as those for beta-galactosidase, luciferase, and green fluorescent protein. The approach allows the generation of transcriptional or translational reporter fusions in a single step without the requirement for recombinant DNA constructs and is applicable to various enterobacterial species. Generation of reporter fusions by Red recombination is rapid, overcomes the current limitations of transposon mutagenesis or reporter plasmids, and offers new options for the study of bacterial gene regulation. [Abstract/Link to Full Text]

Felip M, Andreatta S, Sommaruga R, Straskrábová V, Catalan J
Suitability of flow cytometry for estimating bacterial biovolume in natural plankton samples: comparison with microscopy data.
Appl Environ Microbiol. 2007 Jul;73(14):4508-14.
The relationship between flow cytometry data and epifluorescence microscopy measurements was assessed in bacterioplankton samples from 80 lakes to estimate bacterial biovolume and cell size distribution. The total counts of 4',6'-diamidino-2-phenylindole-stained cells estimated by both methods were significantly related, and the slope of their linear regression was not significantly different from 1, indicating that both methods produce very similar estimates of bacterial abundance. The relationships between side scatter (SSC) and 4',6'-diamidino-2-phenylindole fluorescence and cell volume (microscopy values) were improved by binning of the data in three frequency classes for each, but further increases in the number of classes did not improve these relationships. Side scatter was the best cell volume predictor, and significant relationships were observed between the SSC classes and the smallest (R2 = 0.545, P < 0.001, n = 80) and the largest (R2 = 0.544, P < 0.001, n = 80) microscopy bacterial-size classes. Based on these relationships, a reliable bacterial biomass estimation was obtained from the SSC frequency classes. Our study indicates that flow cytometry can be used to properly estimate bacterioplankton biovolume, with an accuracy similar to those of more time-consuming microscopy methods. [Abstract/Link to Full Text]

Miyauchi R, Oki K, Aoi Y, Tsuneda S
Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting.
Appl Environ Microbiol. 2007 Aug;73(16):5331-7.
"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake. [Abstract/Link to Full Text]

Song Y, Choi MH, Park JN, Kim MW, Kim EJ, Kang HA, Kim JY
Engineering of the yeast Yarrowia lipolytica for the production of glycoproteins lacking the outer-chain mannose residues of N-glycans.
Appl Environ Microbiol. 2007 Jul;73(14):4446-54.
In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative alpha-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man8GlcNAc2, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8GlcNAc2 to Man12GlcNAc2. Digestion with alpha-1,2- and alpha-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first alpha-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides. [Abstract/Link to Full Text]

Girlich D, Poirel L, Carattoli A, Kempf I, Lartigue MF, Bertini A, Nordmann P
Extended-spectrum beta-lactamase CTX-M-1 in Escherichia coli isolates from healthy poultry in France.
Appl Environ Microbiol. 2007 Jul;73(14):4681-5.
Genes encoding extended-spectrum beta-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a bla(CTX-M-1) gene located on transferable plasmids of different sizes and structures. [Abstract/Link to Full Text]

Baertsch C, Paez-Rubio T, Viau E, Peccia J
Source tracking aerosols released from land-applied class B biosolids during high-wind events.
Appl Environ Microbiol. 2007 Jul;73(14):4522-31.
DNA-based microbial source tracking (MST) methods were developed and used to specifically and sensitively track the unintended aerosolization of land-applied, anaerobically digested sewage sludge (biosolids) during high-wind events. Culture and phylogenetic analyses of bulk biosolids provided a basis for the development of three different MST methods. They included (i) culture- and 16S rRNA gene-based identification of Clostridium bifermentans, (ii) direct PCR amplification and sequencing of the 16S rRNA gene for an uncultured bacterium of the class Chloroflexi that is commonly present in anaerobically digested biosolids, and (iii) direct PCR amplification of a 16S rRNA gene of the phylum Euryarchaeota coupled with terminal restriction fragment length polymorphism to distinguish terminal fragments that are unique to biosolid-specific microorganisms. Each method was first validated with a broad group of bulk biosolids and soil samples to confirm the target's exclusive presence in biosolids and absence in soils. Positive responses were observed in 100% of bulk biosolid samples and in less than 11% of the bulk soils tested. Next, a sampling campaign was conducted in which all three methods were applied to aerosol samples taken upwind and downwind of fields that had recently been land applied with biosolids. When average wind speeds were greater than 5 m/s, source tracking results confirmed the presence of biosolids in 56% of the downwind samples versus 3% of the upwind samples. During these high-wind events, the biosolid concentration in downwind aerosols was between 0.1 and 2 microg/m3. The application of DNA-based source tracking to aerosol samples has confirmed that wind is a possible mechanism for the aerosolization and off-site transport of land-applied biosolids. [Abstract/Link to Full Text]

Bachmann H, Santos F, Kleerebezem M, van Hylckama Vlieg JE
Luciferase detection during stationary phase in Lactococcus lactis.
Appl Environ Microbiol. 2007 Jul;73(14):4704-6.
The luminescence signal of luxAB-encoded bacterial luciferase strongly depends on the metabolic state of the host cell, which restricts the use of this reporter system to metabolically active bacteria. Here we show that in stationary-phase cells of Lactococcus lactis, detection of luciferase is significantly improved by the addition of riboflavin or flavin mononucleotide to whole-cell assay systems. [Abstract/Link to Full Text]

Fung JM, Morris RM, Adrian L, Zinder SH
Expression of reductive dehalogenase genes in Dehalococcoides ethenogenes strain 195 growing on tetrachloroethene, trichloroethene, or 2,3-dichlorophenol.
Appl Environ Microbiol. 2007 Jul;73(14):4439-45.
Reductive dehalogenase (RD) gene transcript levels in Dehalococcoides ethenogenes strain 195 were investigated using reverse transcriptase quantitative PCR during growth and reductive dechlorination of tetrachloroethene (PCE), trichloroethene (TCE), or 2,3-dichlorophenol (2,3-DCP). Cells grown with PCE or TCE had high transcript levels (greater than that for rpoB) for tceA, which encodes the TCE RD, pceA, which encodes the PCE RD, and DET0162, which contains a predicted stop codon and is considered nonfunctional. In cells grown with 2,3-DCP, tceA mRNA was less than 1% of that for rpoB, indicating that its transcription was regulated. pceA and DET0162 were the only RD genes with high transcript levels in cells grown with 2,3-DCP. Proteomic analysis of PCE-grown cells detected both PceA and TceA with high peptide coverage but not DET0162, and analysis of 2,3-DCP-grown cells detected PceA with high coverage but not TceA, DET0162, or any other potential RD. Cells grown with PCE or 2,3-DCP were tested for the ability to dechlorinate PCE, TCE, or 2,3-DCP with H2 as the electron donor. 2,3-DCP-grown cells were unable to dechlorinate TCE but dechlorinated PCE to TCE without a lag, and PCE-grown cells dechlorinated 2,3-DCP without a lag. These results show that 2,3-DCP-grown cells do not produce TceA and that DET0162 is transcribed but its translation product is not detectable in cells and are consistent with PceA's being bifunctional, also serving as the 2,3-DCP RD. Chlorophenols naturally occur in soils and are good candidates for the original substrates for PceA. [Abstract/Link to Full Text]

Hua Q, Joyce AR, Palsson BŘ, Fong SS
Metabolic characterization of Escherichia coli strains adapted to growth on lactate.
Appl Environ Microbiol. 2007 Jul;73(14):4639-47.
In comparison with intensive studies of genetic mechanisms related to biological evolutionary systems, much less analysis has been conducted on metabolic network responses to adaptive evolution that are directly associated with evolved metabolic phenotypes. Metabolic mechanisms involved in laboratory evolution of Escherichia coli on gluconeogenic carbon sources, such as lactate, were studied based on intracellular flux states determined from 13C tracer experiments and 13C-constrained flux analysis. At the end point of laboratory evolution, strains exhibited a more than doubling of the average growth rate and a 50% increase in the average biomass yield. Despite different evolutionary trajectories among parallel evolved populations, most improvements were obtained within the first 250 generations of evolution and were generally characterized by a significant increase in pathway capacity. Partitioning between gluconeogenic and pyruvate catabolic flux at the pyruvate node remained almost unchanged, while flux distributions around the key metabolites phosphoenolpyruvate, oxaloacetate, and acetyl-coenzyme A were relatively flexible over the course of evolution on lactate to meet energetic and anabolic demands during rapid growth on this gluconeogenic carbon substrate. There were no clear qualitative correlations between most transcriptional expression and metabolic flux changes, suggesting complex regulatory mechanisms at multiple levels of genetics and molecular biology. Moreover, higher fitness gains for cell growth on both evolutionary and alternative carbon sources were found for strains that adaptively evolved on gluconeogenic carbon sources compared to those that evolved on glucose. These results provide a novel systematic view of the mechanisms underlying microbial adaptation to growth on a gluconeogenic substrate. [Abstract/Link to Full Text]

Beliën T, Van Campenhout S, Van Acker M, Robben J, Courtin CM, Delcour JA, Volckaert G
Mutational analysis of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum reveals comprehensive insights into their inhibitor insensitivity.
Appl Environ Microbiol. 2007 Jul;73(14):4602-8.
Endo-beta-1,4-xylanases (EC 3.2.1.8; endoxylanases), key enzymes in the degradation of xylan, are considered to play an important role in phytopathogenesis, as they occupy a prominent position in the arsenal of hydrolytic enzymes secreted by phytopathogens to breach the cell wall and invade the plant tissue. Plant endoxylanase inhibitors are increasingly being pinpointed as part of a counterattack mechanism. To understand the surprising XIP-type endoxylanase inhibitor insensitivity of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum, an extensive mutational study of these enzymes was performed. Using combinatorial and site-directed mutagenesis, the XIP insensitivity of XylA as well as XylB was proven to be solely due to amino acid sequence adaptations in the "thumb" structural region. While XylB residues Cys141, Asp148, and Cys149 were shown to prevent XIP interaction, the XIP insensitivity of XylA could be ascribed to the occurrence of only one aberrant residue, i.e., Val151. This study, in addition to providing a thorough explanation for the XIP insensitivity of both F. graminearum endoxylanases at the molecular level, generated XylA and XylB mutants with altered inhibition specificities and pH optima. As this is the first experimental elucidation of the molecular determinants dictating the specificity of the interaction between endoxylanases of phytopathogenic origin and a plant inhibitor, this work sheds more light on the ongoing evolutionary arms race between plants and phytopathogenic fungi involving recognition of endoxylanases. [Abstract/Link to Full Text]

Ohene-Adjei S, Teather RM, Ivan M, Forster RJ
Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen.
Appl Environ Microbiol. 2007 Jul;73(14):4609-18.
Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen. [Abstract/Link to Full Text]

Klerks MM, van Gent-Pelzer M, Franz E, Zijlstra C, van Bruggen AH
Physiological and molecular responses of Lactuca sativa to colonization by Salmonella enterica serovar Dublin.
Appl Environ Microbiol. 2007 Aug;73(15):4905-14.
This paper describes the physiological and molecular interactions between the human-pathogenic organism Salmonella enterica serovar Dublin and the commercially available mini Roman lettuce cv. Tamburo. The association of S. enterica serovar Dublin with lettuce plants was first determined, which indicated the presence of significant populations outside and inside the plants. The latter was evidenced from significant residual concentrations after highly efficient surface disinfection (99.81%) and fluorescence microscopy of S. enterica serovar Dublin in cross sections of lettuce at the root-shoot transition region. The plant biomass was reduced significantly compared to that of noncolonized plants upon colonization with S. enterica serovar Dublin. In addition to the physiological response, transcriptome analysis by cDNA amplified fragment length polymorphism analysis also provided clear differential gene expression profiles between noncolonized and colonized lettuce plants. From these, generally and differentially expressed genes were selected and identified by sequence analysis, followed by reverse transcription-PCR displaying the specific gene expression profiles in time. Functional grouping of the expressed genes indicated a correlation between colonization of the plants and an increase in expressed pathogenicity-related genes. This study indicates that lettuce plants respond to the presence of S. enterica serovar Dublin at physiological and molecular levels, as shown by the reduction in growth and the concurrent expression of pathogenicity-related genes. In addition, it was confirmed that Salmonella spp. can colonize the interior of lettuce plants, thus potentially imposing a human health risk when processed and consumed. [Abstract/Link to Full Text]

Ventura M, Canchaya C, Zhang Z, Fitzgerald GF, van Sinderen D
Molecular characterization of hsp20, encoding a small heat shock protein of bifidobacterium breve UCC2003.
Appl Environ Microbiol. 2007 Jul;73(14):4695-703.
Small heat shock proteins (sHSPs) are members of a diverse family of stress proteins that are important in cells to protect proteins under stressful conditions. Genome analysis of Bifidobacterium breve UCC2003 revealed a single sHSP-encoding gene, which was classified as a hsp20 gene by comparative analyses. Genomic surveillance of available genome sequences indicated that hsp20 homologs are not widely distributed in bacteria. In members of the genus Bifidobacterium, this gene appears to be present in only 7 of the 30 currently described species. Moreover, phylogenetic analysis using all available bacterial and eukaryotic sHSP sequences revealed a close relationship between bifidobacterial HSP20 and the class B sHSPs found in members of the division Firmicutes. The results of this comparative analysis and variation in codon usage content suggest that hsp20 was acquired by certain bifidobacteria through horizontal gene transfer. Analysis by slot blot, Northern blot, and primer extension experiments showed that transcription of hsp20 is strongly induced in response to severe heat shock regimens and by osmotic shock. [Abstract/Link to Full Text]

Nakamura J, Hirano S, Ito H, Wachi M
Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production.
Appl Environ Microbiol. 2007 Jul;73(14):4491-8.
Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants and penicillin also induce L-glutamic acid secretion, even in the presence of biotin. However, the mechanism of L-glutamic acid secretion remains unclear. It was recently reported that disruption of odhA, encoding a subunit of the 2-oxoglutarate dehydrogenase complex, resulted in L-glutamic acid secretion without induction. In this study, we analyzed odhA disruptants and found that those which exhibited constitutive L-glutamic acid secretion carried additional mutations in the NCgl1221 gene, which encodes a mechanosensitive channel homolog. These NCgl1221 gene mutations lead to constitutive L-glutamic acid secretion even in the absence of odhA disruption and also render cells resistant to an L-glutamic acid analog, 4-fluoroglutamic acid. Disruption of the NCgl1221 gene essentially abolishes L-glutamic acid secretion, causing an increase in the intracellular L-glutamic acid pool under biotin-limiting conditions, while amplification of the wild-type NCgl1221 gene increased L-glutamate secretion, although only in response to induction. These results suggest that the NCgl1221 gene encodes an L-glutamic acid exporter. We propose that treatments that induce L-glutamic acid secretion alter membrane tension and trigger a structural transformation of the NCgl1221 protein, enabling it to export L-glutamic acid. [Abstract/Link to Full Text]

Flament D, Barbeyron T, Jam M, Potin P, Czjzek M, Kloareg B, Michel G
Alpha-agarases define a new family of glycoside hydrolases, distinct from beta-agarase families.
Appl Environ Microbiol. 2007 Jul;73(14):4691-4.
The gene encoding the alpha-agarase from "Alteromonas agarilytica" (proposed name) has been cloned and sequenced. The gene product (154 kDa) is unrelated to beta-agarases and instead belongs to a new family of glycoside hydrolases (GH96). The alpha-agarase also displays a complex modularity, with the presence of five thrombospondin type 3 repeats and three carbohydrate-binding modules. [Abstract/Link to Full Text]

Du C, Zhang Y, Li Y, Cao Z
Novel redox potential-based screening strategy for rapid isolation of Klebsiella pneumoniae mutants with enhanced 1,3-propanediol-producing capability.
Appl Environ Microbiol. 2007 Jul;73(14):4515-21.
This report describes a novel redox potential (oxidoreduction potential [ORP])-based screening strategy for the isolation of mutants of Klebsiella pneumoniae which have an increased ability to produce 1,3-propanediol (1,3-PD). This method can be described as follows: first, to determine an ORP range which is preferred for the wild-type strain to grow and to produce 1,3-PD; second, to subject a chemically mutagenized culture to a reduced ORP level which is deleterious for the wild-type strain. Colonies that showed high specific growth rates at deleterious ORP levels were selected, and their abilities to produce 1,3-PD were investigated. In an ORP-based screening experiment where the ORP was controlled at -280 mV, 4 out of 11 isolated strains were recognized as positive mutant strains. A mutant which is capable of producing higher concentrations of 1,3-PD was subjected to fed-batch fermentations for further characterization. Its preferred ORP level (-280 mV) was significantly lower than that of its parent (-190 mV). The highest 1,3-PD concentration of the mutant was 915 mmol liter(-1), which was 63.1% higher than that of the parent. Metabolic-flux analysis suggested that the intracellular reductive branch of the mutant was strengthened, which improved 1,3-PD biosynthesis. The procedure and results presented here provide a novel method of screening for strains with improved product formation. [Abstract/Link to Full Text]

Rizzello CG, De Angelis M, Di Cagno R, Camarca A, Silano M, Losito I, De Vincenzi M, De Bari MD, Palmisano F, Maurano F, Gianfrani C, Gobbetti M
Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease.
Appl Environ Microbiol. 2007 Jul;73(14):4499-507.
Presently, the only effective treatment for celiac disease is a life-long gluten-free diet. In this work, we used a new mixture of selected sourdough lactobacilli and fungal proteases to eliminate the toxicity of wheat flour during long-time fermentation. Immunological (R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay [ELISA] and R5 antibody-based Western blot), two-dimensional electrophoresis, and mass spectrometry (matrix-assisted laser desorption ionization-time of flight, strong-cation-exchange-liquid chromatography/capillary liquid chromatography-electrospray ionization-quadrupole-time of flight [SCX-LC/CapLC-ESI-Q-TOF], and high-pressure liquid chromatography-electrospray ionization-ion trap mass spectrometry) analyses were used to determine the gluten concentration. Assays based on the proliferation of peripheral blood mononuclear cells (PBMCs) and gamma interferon production by PBMCs and intestinal T-cell lines (iTCLs) from 12 celiac disease patients were used to determine the protein toxicity of the pepsin-trypsin digests from fermented wheat dough (sourdough). As determined by R5-based sandwich and competitive ELISAs, the residual concentration of gluten in sourdough was 12 ppm. Albumins, globulins, and gliadins were completely hydrolyzed, while ca. 20% of glutenins persisted. Low-molecular-weight epitopes were not detectable by SCX-LC/CapLC-ESI-Q-TOF mass spectrometry and R5-based Western blot analyses. The kinetics of the hydrolysis of the 33-mer by lactobacilli were highly efficient. All proteins extracted from sourdough activated PBMCs and induced gamma interferon production at levels comparable to the negative control. None of the iTCLs demonstrated immunoreactivity towards pepsin-trypsin digests. Bread making was standardized to show the suitability of the detoxified wheat flour. Food processing by selected sourdough lactobacilli and fungal proteases may be considered an efficient approach to eliminate gluten toxicity. [Abstract/Link to Full Text]

Kienesberger S, Gorkiewicz G, Joainig MM, Scheicher SR, Leitner E, Zechner EL
Development of experimental genetic tools for Campylobacter fetus.
Appl Environ Microbiol. 2007 Jul;73(14):4619-30.
Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus. [Abstract/Link to Full Text]

---

Recent Articles in Applied Microbiology

---

Schroeder HW, Kelton WH
Production of sterigmatocystin by some species of the genus Aspergillus and its toxicity to chicken embryos.
Appl Microbiol. 1975 Oct;30(4):589-91.
Sterigmatocystin was produced by 59% of Aspergillus flavus cultures and by 16% of A. parasiticus cultures. All sterigmatocystin-producing cultures of the A. flavus group also simultaneously produced aflatoxin or O-methylsterigmatocystin. Sterigmatocystin was produced by A. chevalieri, A. ruber, and A. amstelodami, species not previously reported to produce the compound. In 5-day-old chicken embryos, the no-effect level of toxicity of sterigmatocystin was between 1 and 2 mug/egg; the mean lethal dose was 5 to 7 mug; and 90 to 100% of the embryos were killed with 10 mug. Teratogenic effects and weight reduction were generally associated with nonlethal doses. [Abstract/Link to Full Text]

Koehler PE, Hanlin RT, Beraha L
Production of aflatoxins B1 and G1 by Aspergillus flavus and Aspergillus parasiticus isolated from market pecans.
Appl Microbiol. 1975 Oct;30(4):581-3.
One hundred and forty-eight isolates of Aspergillus flavus and A. parasiticus were isolated from 5,608 pecans obtained from Chicago and Georgia markets. The percentage of internal contamination by these species was 7.3% in the Chicago market pecans and 1.7% in those from markets in Georgia. Of the 148 isolates, 93% of the A. parasiticus, but only 54% of the A. flavus, were capable of producing aflatoxin. Overall, 57% of the isolates were potentially aflatoxigenic. A. parasiticus isolates generally produced a greater amount of aflatoxins than A. flavus. [Abstract/Link to Full Text]

Gilliland SE, Speck ML, Morgan CG
Detection of Lactobacillus acidophilus in feces of humans, pigs, and chickens.
Appl Microbiol. 1975 Oct;30(4):541-5.
Lactobacilli in fecal material from humans, pigs, and chickens were enumerated on lactobacillus selective agar (LBS). In all samples, higher numbers of lac-tobacilli were detected when plates were incubated in a system flushed with CO2 rather than in air. Much higher numbers of bacteria from human feces were detected when the LBS agar plates were incubated anaerobically in a hydrogen-carbon dioxide atmosphere (GasPak) than when incubated in CO2. The bacteria from human feces isolated on LBS agar incubated anaerobically were predominately bifidobacteria. Cultures from all three sources isolated on LBS agar incubated under CO2 were lactobacilli, including Lactobacillus acidophilus. Differences were observed in biochemical characteristics of some of the L. acidophilus isolated from all three sources. Guanine plus cytosine base ratios of deoxyribonucleic acid isolated from L. acidophilus cultures from humans were lower, in most cases, than those from pigs and chickens. [Abstract/Link to Full Text]

Munnecke DM, Hsieh DP
Microbial metabolism of a parathion-xylene pesticide formulation.
Appl Microbiol. 1975 Oct;30(4):575-80.
A mixed bacterial culture was adapted to growth on a mixed carbon substrate consisting of the pesticide parathion and its xylene-based formulation. The environmental growth parameters of temperature, pH, and dissolved oxygen concentration were optimized to obtain complete metabolism of parathion from this mixed carbon substrate. This adapted culture grew rapidly (mu = 0.7 per h) on the pesticide formulation at high parathion suspensions (3,000 mg/liter). Carbon utilization from this mixed substrate was strongly dependent on pH. At slightly acidic pH, xylene was preferentially metabolized, whereas at slightly alkaline pH, parathion was preferentially metabolized. Diethylthiophosphoric acid, a metabolite from parathion, and toluic acid, a metabolite from xylene, also influenced the selection of the primary carbon source. [Abstract/Link to Full Text]

Kundu AK, Manna S
Purification and characterization of extracellular proteinases of Aspergillus oryzae.
Appl Microbiol. 1975 Oct;30(4):507-13.
The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes. [Abstract/Link to Full Text]

Macris BJ
Mechanism of benzoic acid uptake by Saccharomyces cerevisiae.
Appl Microbiol. 1975 Oct;30(4):503-6.
A fast uptake of the preservative benzoic acid was observed in Saccharomyces cerevisiae, reaching saturation in about two min and then remaining constant at this level. The strong dependence of benzoic acid uptake on pH was due to the relative distribution of molecular and ionic forms in solution and not to the pH itself. The molecular form was the only one taken up by the cells. The specificity of the uptake mechanism was evidenced by the pattern of irreversible heat inactivation of the uptake system resembling protein denaturation by heat. Furthermore, the effect of temperature on the uptake was similar to that observed in enzymic reactions, whereas the kinetic data of uptake conformed to the Michaelis-Menten curve of saturation with a Km of 1.54 X 10(-2) M and Vmax of 3 X 10(-3) M/10s. The evidence presented in this paper indicates that compounds of protein nature are involved in the uptake of this preservative. [Abstract/Link to Full Text]

Liras P, Kasparian SS, Umbreit WW
Enzymatic transformation of morphine by hydroxysteroid dehydrogenase from Pseudomonas testosteroni.
Appl Microbiol. 1975 Oct;30(4):650-6.
Eznyme preparations from Pseudomonas testosteroni containing alpha- and beta- hydroxysteroid dehydrogenases catalyzed the oxidation of morphine and codeine by nicotinamide adenine dinucleotide. Morphine was converted in relatively low yield into 14-hydroxymorphinone probably via morphinone as an intermediate. Codeine was converted to codeinone and 14-hydroxycodeinone. Only the conversions at the 6-position were carred out by the hydroxysteroid dehydrogenase. Hydroxylation at the 14-position did occur spontaneously (or enzymatically with a contaminating enzyme) ater oxidation at the 6-position. [Abstract/Link to Full Text]

Sobsey MD, Wallis C, Melnick JL
Studies on the survival and fate of enteroviruses in an experimental model of a municipal solid waste landfill and leachate.
Appl Microbiol. 1975 Oct;30(4):565-74.
In laboratory scale municipal solid waste lysimeters containing simulated refuse, and seeded with either laboratory or field strains of poliovirus type 1 and echovirus type 7, viruses were not detected in the lysimeter leachate produced over a 4-month period. In addition, viruses were detected in the lysimeter refuse contents after termination of lysimeter operation. These results appeared to be due to virus retention in the lysimeter caused by virus adsorption and virus inactivation. Evidence for virus inactivation was provided by the results of experiments on virus inactivation in composite leachate samples. Evidence for virus adsorption was supported by the rapid adsorption of viruses to various municipal solid waste components in the presence of a salt similar in composition to the major inorganic salts of leachates. [Abstract/Link to Full Text]

MacDonald IA, Bishop JM, Mahony DE, Williams CN
Convenient non-chromatographic assays for the microbial deconjugation and 7alpha-OH bioconversion of taurocholate.
Appl Microbiol. 1975 Oct;30(4):530-5.
We described two convenient assay methods to estimate bile acid deconjugation and bile acid bioconversion at the 7alpha-OH position by individual microorganisms grown in media containing taurocholic acid. The methods are based on (i) a selective chemical assay for taurine conjugates previously described and (ii) the use of a cell-free preparation of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli to directly quantify 7alpha-OH groups. These non-chromatographic approaches have been applied to the study of three model strains of intestinal organisms, E. coli, Bacteroides fragilis, and Clostridium perfringens, grown in standard media in the presence of purified tritiated taurocholate. Assay results were confirmed by thin-layer chromatography solvent systems designed to separate conjugated from unconjugated bile acid and unmodified cholic acid nucleus from 7alpha-OH bioconversion product(s) (primarily 3alpha, 12alpha dihydroxy, 7-keto-cholanoic acid). In addition, 7alpha-hydroxysteroid dehydrogenase activity was demonstrated in cell-free extracts of all three organisms. Of the three organisms, only C. perfringens was demonstrated to (i) deconjugate taurocholic acid, (ii) contain 3alpha-hydroxysteroid dehydrogenase activity, (iii) convert cholic acid into at least five labeled metabolites visible on thin-layer chromatography, and (iv) catalyze significant tritium exchange with water in the medium. [Abstract/Link to Full Text]

Bernáth S
Solid-phase radioimmunoassays for quantitative antibody determination of Clostridium perfringens type D epsilon toxin.
Appl Microbiol. 1975 Oct;30(4):499-502.
Two reversed solid-phase radioimmunoassays were developed for quantitative determination of antibodies against Clostridium perfringens type D epsilon toxin. 125I-labeled prototoxin was used in the bromoacetylcellulose-bound antibody method and in the antibody-coated tube method. The antibody values which can be detected by the assays are in the range of 0.004 IU/ml of investigated serum. The methods allow the screening investigation of large groups of vaccinated sheep in a rapid and inexpensive way, and are very suitable for measuring small amounts of C. perfringens D epsilon antibodies with a small experimental error. [Abstract/Link to Full Text]

Chai TJ, Levin RE
Characteristics of Heavily Mucoid Bacterial Isolated from Fish Pen Slime.
Appl Microbiol. 1975 Sep;30(3):450-455.
Weakly gram-positive pleomorphic rods isolated from the slime on the storage pen surfaces of fishing trawlers were found to produce extensive capsular material and intensely mucoid colonies. The isolates studied produced highly pleomorphic club-shaped cells indicative of coryneforms. Their DNA base composition ranged from 64.2 to 68.2 mol% guanine plus cytosine on the basis of melting point determinations. [Abstract/Link to Full Text]

Lee BH, Blackburn TH
Cellulase Production by a Thermophilic Clostridium Species.
Appl Microbiol. 1975 Sep;30(3):346-353.
Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 mum) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C(1) cellulase (degrades native cellulose) and C(x) cellulase (beta-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl(2) precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C(x) cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature. [Abstract/Link to Full Text]

Schenck S, Pramer D
The effects of volatile compounds from nematodes on trap formation by a nematode-trapping fungus.
Appl Microbiol. 1975 Sep;30(3):496-7.
Although the presence of prey induced trap formation by nematode-trapping fungi, it is improbable that the activity was associated with volatile compounds of nematode origin. [Abstract/Link to Full Text]

Guskey LE, Jenkin HM
Adaptation of BHK-21 cells to growth in shaker culture and subsequent challenge by Japanese encephalitis virus.
Appl Microbiol. 1975 Sep;30(3):433-8.
Baby hamster kidney (BHK-21) cells were adapted to grow in shaker culture using Waymouth medium 752/1 containing 20 mM N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid buffer and supplemented with 2.5% (vol/vol) calf serum, 0.002% (wt/vol) sodium oleate, and 0.2% fatty acid-free bovine serum albumin (WO2.5). Infectivity of Japanese encephalitis virus grown in the cells adapted to WO2.5 approached 2 x 10(8) plaque-forming units per ml. The culture volume of infected cells was reduced fivefold 12 h after infection. This step resulted in a 10-fold increase in infectivity over that obtained from infected cultures not subjected to volume reduction. [Abstract/Link to Full Text]

Cowman RA, Perrella MM, Adams BO, Fitzgerald RJ
Amino acid requirements and proteolytic activity of Streptococcus sanguis.
Appl Microbiol. 1975 Sep;30(3):374-80.
The growth response of Streptococcus sanguis groups 1:A and 1:B in a complete chemically defined medium was not influenced by the oxygen concentration of the growth atmosphere. All of the cultures required cysteine and arginine; tyrosine and branched-chain amino acids were frequently required. Proteolysis of casein, mucin, and the anionic proteins of germfree rat saliva by S. sanguis was demonstrated. Hydrolytic activity toward casein was found in the soluble contents of the cells and in the cellular debris after disruption of the cells, with the soluble fractions exhibiting greater proteolytic activity toward casein. The soluble fractions from S. sanguis did not hydrolyze mucin, but this substrate was hydrolyzed by the cell debris fraction. When the amino acid requirements and proteolytic activity of S. sanguis and S. mutans were compared, these two oral streptococcal species exhibited distinct and characteristic differences. [Abstract/Link to Full Text]

Kotsonis FN, Smalley EB, Ellison RA, Gale CM
Feed refusal factors in pure cultures of Fusarium roseum 'graminearum'.
Appl Microbiol. 1975 Sep;30(3):362-8.
Isolations from 1972 Wisconsin feed refusal corn yielded predominantly cultures of Fusarium roseum 'graminearum.' With one possible exception, none of the selected isolates of this fungus induced emesis in pigeons, whereas six of nine isolates produced feed refusal responses in all test animals. A single isolate of F. roseum 'equiseti' also induced a severe refusal response and possibly slight emesis. None of the other fungi isolated from this corn (F. moniliforme, Acremoniella atra) or controls caused either emesis or feed refusal. Zearalenone was detected in all isolates and was shown to be partially responsible for refusal activity. The remaining activity was ascribed to one or more nonvolatile, neutral, relatively polar molecules. T-2 toxin, although not detected in these isolates, was shown to have dramatic refusal activity in rats. [Abstract/Link to Full Text]

Willetts A
Fungal growth on C1 compounds: a study of the amino acid composition of a methanol-utilizing strain of Trichoderma lignorum.
Appl Microbiol. 1975 Sep;30(3):343-5.
The amino acid composition of the C1-utilizing fungus Trichoderma lignorum, growing at the expense of methanol as the sole source of carbon, was determined. With the exception of an insufficient content of methionine, the essential amino acid composition of this novel protein source appears adequate in terms of the Food and Agricultural Organization Reference Protein for both direct and indirect use in the human diet as a food or animal feed, respectively. [Abstract/Link to Full Text]

Kotsonis FN, Ellison RA, Smalley EB
Isolation of acetyl T-2 toxin from Fusarium poae.
Appl Microbiol. 1975 Sep;30(3):493-5.
Acetyl T-2 toxin (3,4,15-triacetoxy-8-isovaleroxy-12,13-epoxy-delta9-trichothecene) was isolated and characterized as a naturally occurring emetic trichothecene from liquid cultures of Fusarium poae (NRRL 3287). Acetyl T-2 toxin was shown to be much less toxic than T-2 toxin in pigeon assays. [Abstract/Link to Full Text]

Aronson JN, Borris DP, Doerner JF, Akers E
Gamma-aminobutyric acid pathway and modified tricarboxylic acid cycle activity during growth and sporulation of Bacillus thuringiensis.
Appl Microbiol. 1975 Sep;30(3):489-92.
Enzymatic analyses of Bacillus thuringiensis extracts suggest that a modified Krebs tricarboxylic acid cycle (without alpha-ketoglutarate dehydrogenase) can operate during sporulation in conjunction with the glyoxylic acid cycle and the gamma-aminobutyric acid pathway. [Abstract/Link to Full Text]

Furuya A, Sato A
Fermentative accumulation of guanosine polyphosphates by Brevibacterium ammoniagenes.
Appl Microbiol. 1975 Sep;30(3):480-2.
Guanosine-3'-diphosphate-5'-monophosphate (3.35 mg/ml), guanosine-3'-diphosphate-5'-diphosphate (MSI) (5.21 mg/ml), and guanosine-3'-diphosphate-5'-triphosphate (MSII) (0.82 mg/ml), in addition to guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate, were accumulated by microbial conversion of 5'-xanthylic acid with a mutant of Brevibacterium ammoniagenes. [Abstract/Link to Full Text]

Toraya T, Yongsmith B, Tanaka A, Fukui S
Vitamin B12 production by a methanol-utilizing bacterium.
Appl Microbiol. 1975 Sep;30(3):477-9.
Vitamin B(12) production by a newly isolated strain of a methanol-utilizing bacterium was studied. The maximal yield of the vitamin, 2.6 mg/liter of medium was attained by optimization. [Abstract/Link to Full Text]

Hamdy MK, Noyes OR
Formation of methyl mercury by bacteria.
Appl Microbiol. 1975 Sep;30(3):424-32.
Twenty-three Hg2+-resistant cultures were isolated from sediment of the Savannah River in Georgia; of these, 14 were gram-negative short rods belonging to the genera Escherichia and Enterobacter, six were gram-positive cocci (three Staphylococcus sp. and three Streptococcus sp.) and three were Bacillus sp. All the Escherichia, Enterobacter, and the Bacillus strain were more resistant to Hg2+ than the strains of staphylococci and streptococci. Adaptation using serial dilutions and concentration gradient agar plate techniques showed that it was possible to select a Hg2+-resistant strain from a parent culture identified as Enterobacter aerogenes. This culture resisted 1,200 mug of Hg2+ per ml of medium and produced methyl mercury from HgCl2, but was unable to convert Hg2+ to volatile elemental mercury (Hg0). Under constant aeration (i.e., submerged culture), slightly more methyl mercury was formed than in the absence of aeration. Production of methyl mercury was cyclic in nature and slightly decreased if DL-homocysteine was present in media, but increased with methylcobalamine. It is concluded that the bacterial production of methyl mercury may be a means of resistance and detoxification against mercurials in which inorganic Hg2+ is converted to organic form and secreted into the environment. [Abstract/Link to Full Text]

Sugiyama H, Brenner SL, Dasgupta BR
Detection of Clostridium botulinum toxin by local paralysis elicited with intramuscular challenge.
Appl Microbiol. 1975 Sep;30(3):420-3.
Clostridium botulinum toxin can be identified by a characteristic, acute local paralysis that follows its injection into the gastrocnemius ("calf" muscle) of mice. The local botulism can be elicited with slightly less than one-tenth the toxin amount that is needed to kill mice by the intraperitoneal (i.p.) challenge route. The practical sensitivities of the intramuscular (i.m.) versus i.p. tests are about equal, however, because maximum sample volume injectable i.m. is 0.1 ml as compared to the 0.5-ml range that can be given i.p. i.m. injection of 10 or more mouse i.p. mean lethal doses causes paralysis in about 1 h, and an i.m. injection of about 0.5 i.p. mean lethal doses causes paralysis in 3 to 4 h. Toxin neutralization by homologous type of antitoxin only can be demonstrated with an incubated mixture of toxin and antitoxin. Although not as convenient as the i.p. method for routine use to detect botulinum toxin, the i.m. method has characteristics which could make it a useful supplement to the presently accepted i.p. procedure. [Abstract/Link to Full Text]

Grubb JA, Dehority BA
Effects of an abrupt change in ration from all roughage to high concentrate upon rumen microbial numbers in sheep.
Appl Microbiol. 1975 Sep;30(3):404-12.
When three sheep were abruptly changed from a ration of 100% orchardgrass hay to 60% cracked corn-40% orchardgrass hay, fed at equal dry-matter intakes, significant increases in concentration were observed in the rumen microbial population. Bacterial numbers (colony counts) per gram of rumen contents did not appear to have stabilized within 21 days after the ration change; however, protozoan numbers per milliliter plateaued after 5 days. The concentration of cellulose-digesting bacteria varied considerably between animals and decreased in all animals with the change. Changes were observed in total and molar percentages of volatile fatty acids, which were typical for the two types of rations. Although the concentration of protozoa increased after the ration change, only minor differences were observed in their percent generic distribution. A significant decrease in rumen volume was measured in two of the three sheep with the change in ration; however, fluid turnover rates were not significantly affected. Rates of rumen dry-matter turnover were slower with the concentrate ration, although rumen dry-matter digestion was increased. Calculation of total bacterial numbers based on total rumen volume completely negated the effect of ration change in one animal, whereas total numbers in the other two animals were still significantly different between rations and very similar between animals. Adjustment of total protozoa numbers did not alter the trends seen previously with concentration values. [Abstract/Link to Full Text]

Atlas RM
Effects of temperature and crude oil composition on petroleum biodegradation.
Appl Microbiol. 1975 Sep;30(3):396-403.
The biodegradability of seven different crude oils was found to be highly dependent on their composition and on incubation temperature. At 20 C lighter oils had greater abiotic losses and were more susceptible to biodegradation than heavier oils. These light crude oils, however, possessed toxic volatile components which evaporated only slowly and inhibited microbial degradation of these oils at 10 C. No volatile toxic fraction was associated with the heavier oils tested. Rates of oil mineralization for the heavier oils were significantly lower at 20 C than for the lighter ones. Similar relative degradation rates were found with a mixed microbial community, using CO2 evolution as the measure, and with a Pseudomonas isolate from the Arctic, using O2 consumption as the measure. The paraffinic, aromatic, and asphaltic fractions were subject to biodegradation. Some preference was shown for paraffin degradation, especially at low temperatures. Branched paraffins, such as pristane, were degraded at both 10 and 20 C. At best, a 20% residue still remained after 42 days of incubation. Oil residues generally had a lower relative percentage of paraffins and higher percentage of asphaltics than fresh or weathered oil. [Abstract/Link to Full Text]

Chalfan Y, Levy R, Mateles RI
Detection of mannitol formation by bacteria.
Appl Microbiol. 1975 Sep;30(3):476.
A test is described by means of which formation of mannitol from fructose by lactic acid bacteria can be readily detected. The test is based on removal of interference of residual fructose by dehydration with hydrochloric acid followed by thin-layer chromatography. [Abstract/Link to Full Text]

Hart A, Ng SN
Effect of temperature on the sterilization of isopropyl alcohol by liquid propylene oxide.
Appl Microbiol. 1975 Sep;30(3):483-4.
Liquid propylene oxide added to a solution of isopropyl alcohol and incubated at different temperatures markedly reduced the time required to sterilize the alcohol solution. [Abstract/Link to Full Text]

Weiss E, Coolbaugh JC, Williams JC
Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation.
Appl Microbiol. 1975 Sep;30(3):456-63.
Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity. [Abstract/Link to Full Text]

Smucker RA, Pfister RM
Liquid nitrogen cryo-impacting: a new concept for cell disruption.
Appl Microbiol. 1975 Sep;30(3):445-9.
High-efficiency disruption of bacteria can be accomplished in 2 or more min by the new procedure of liquid nitrogen cryo-impacting. Release of the dipicolinic acid-Ca2+ chelate paralleled the breakage of Bacillus megaterium endospores. Lactate dehydrogenase activity was much better in supernates from liquid nitrogen cryo-impacting-broken Escherichia coli cells than in those from sonically treated and broken E. coli cells. [Abstract/Link to Full Text]

Crawford RL
Degradation of 3-hydroxybenzoate by bacteria of the genus Bacillus.
Appl Microbiol. 1975 Sep;30(3):439-44.
The pathway whereby certain bacterial strains of the genus Bacillus degrade m-hydroxybenzoate is delineated. Of 12 strains examined, nine were tentatively classified as representatives of the species Bacillus brevis, two of Bacillus sphaericus and one of Bacillus megaterium. All strains degraded m-hydroxybenzoate via the same pathway. m-Hydroxybenzoate was hydroxylated to 2,5-dihydroxybenzoate (gentisate), which was oxidized by a gentisate 1,2-deoxygenase yielding maleylpyruvate. Maleylpyruvate was hydrolyzed without prior cis, cis to cis, trans isomerization yielding pyruvate and maleic acid. Numerous soils were examined by plate-count procedures and found to contain 10(4) to 10(6) aerobic sporeformers able to grow on m-hydroxybenzoate per g of dry soil. [Abstract/Link to Full Text]

---

Recent Articles in Bacteriological Reviews

---

Henriksen SD
Moraxella, Acinetobacter, and the Mimeae.
Bacteriol Rev. 1973 Dec;37(4):522-61. [Abstract/Link to Full Text]

London J, Kline K
Aldolase of lactic acid bacteria: a case history in the use of an enzyme as an evolutionary marker.
Bacteriol Rev. 1973 Dec;37(4):453-78. [Abstract/Link to Full Text]

Payne WJ
Reduction of nitrogenous oxides by microorganisms.
Bacteriol Rev. 1973 Dec;37(4):409-52. [Abstract/Link to Full Text]

Kingsley VV, Hoeniger JF
Growth, structure, and classification of Selenomonas.
Bacteriol Rev. 1973 Dec;37(4):479-521. [Abstract/Link to Full Text]


Chemistry and Biology of the Polyene Macrolide Antibiotics.
Bacteriol Rev. 1973 Sep;37(3):407.
[This corrects the article on p. 166 in vol. 37, PMID: 4578757.]. [Abstract/Link to Full Text]

Hopwood DA, Chater KF, Dowding JE, Vivian A
Advances in Streptomyces coelicolor genetics.
Bacteriol Rev. 1973 Sep;37(3):371-405. [Abstract/Link to Full Text]

Porter JR
Agostino Bassi bicentennial (1773-1973).
Bacteriol Rev. 1973 Sep;37(3):284-8. [Abstract/Link to Full Text]

Weiss E
Growth and physiology of rickettsiae.
Bacteriol Rev. 1973 Sep;37(3):259-83. [Abstract/Link to Full Text]

Singleton R, Amelunxen RE
Proteins from thermophilic microorganisms.
Bacteriol Rev. 1973 Sep;37(3):320-42. [Abstract/Link to Full Text]

McFadden BA
Autotrophic CO2 assimilation and the evolution of ribulose diphosphate carboxylase.
Bacteriol Rev. 1973 Sep;37(3):289-319. [Abstract/Link to Full Text]

Padan E, Shilo M
Cyanophages-viruses attacking blue-green algae.
Bacteriol Rev. 1973 Sep;37(3):343-70. [Abstract/Link to Full Text]

Hamilton-Miller JM
Chemistry and biology of the polyene macrolide antibiotics.
Bacteriol Rev. 1973 Sep;37(3):166-96. [Abstract/Link to Full Text]


Peptidoglycan Types of Bacterial Cell Walls and Their Taxonomic Implications.
Bacteriol Rev. 1973 Jun;37(2):258.
[This corrects the article on p. 409 in vol. 36.]. [Abstract/Link to Full Text]

Davis FM, Adelberg EA
Use of somatic cell hybrids for analysis of the differentiated state.
Bacteriol Rev. 1973 Jun;37(2):197-214. [Abstract/Link to Full Text]

Knox KW, Wicken AJ
Immunological properties of teichoic acids.
Bacteriol Rev. 1973 Jun;37(2):215-57. [Abstract/Link to Full Text]

Hamilton-Miller JM
Chemistry and biology of the polyene macrolide antibiotics.
Bacteriol Rev. 1973 Jun;37(2):166-96. [Abstract/Link to Full Text]

Poupard JA, Husain I, Norris RF
Biology of the bifidobacteria.
Bacteriol Rev. 1973 Jun;37(2):136-65. [Abstract/Link to Full Text]

Dales S
Early events in cell-animal virus interactions.
Bacteriol Rev. 1973 Jun;37(2):103-35. [Abstract/Link to Full Text]

Wolk CP
Physiology and cytological chemistry blue-green algae.
Bacteriol Rev. 1973 Mar;37(1):32-101. [Abstract/Link to Full Text]

Fuccillo DA, Sever JL
Viral teratology.
Bacteriol Rev. 1973 Mar;37(1):19-31. [Abstract/Link to Full Text]

Cho CT, Wenner HA
Monkeypox virus.
Bacteriol Rev. 1973 Mar;37(1):1-18. [Abstract/Link to Full Text]

Henrichsen J
Bacterial surface translocation: a survey and a classification.
Bacteriol Rev. 1972 Dec;36(4):478-503. [Abstract/Link to Full Text]

Sanderson KE, Ross H, Ziegler L, Mäkelä PH
F + , Hfr, and F' strains of Salmonella typhimurium and Salmonella abony.
Bacteriol Rev. 1972 Dec;36(4):608-37. [Abstract/Link to Full Text]

Low KB
Escherichia coli K-12 F-prime factors, old and new.
Bacteriol Rev. 1972 Dec;36(4):587-607. [Abstract/Link to Full Text]

Sanderson KE
Linkage map of Salmonella typhimurium, edition IV.
Bacteriol Rev. 1972 Dec;36(4):558-86. [Abstract/Link to Full Text]

Bachmann BJ
Pedigrees of some mutant strains of Escherichia coli K-12.
Bacteriol Rev. 1972 Dec;36(4):525-57. [Abstract/Link to Full Text]

Taylor AL, Trotter CD
Linkage map of Escherichia coli strain K-12.
Bacteriol Rev. 1972 Dec;36(4):504-24. [Abstract/Link to Full Text]

Schleifer KH, Kandler O
Peptidoglycan types of bacterial cell walls and their taxonomic implications.
Bacteriol Rev. 1972 Dec;36(4):407-77. [Abstract/Link to Full Text]

Flick JA
Human reagins: appraisal of the properties of the antibody of immediate-type hypersensitivity.
Bacteriol Rev. 1972 Sep;36(3):311-60. [Abstract/Link to Full Text]

Smith H
Mechanisms of virus pathogenicity.
Bacteriol Rev. 1972 Sep;36(3):291-310. [Abstract/Link to Full Text]

---

Recent Articles in BMC Immunology

---

Marodon G, Klatzmann D
In situ transduction of stromal cells and thymocytes upon intrathymic injection of lentiviral vectors.
BMC Immunol. 2004 Aug 19;518.
BACKGROUND: The thymus is the primary site for T-cell development and induction of self-tolerance. Previous approaches towards manipulation of T-cell differentiation have used intrathymic injection of antigens, as proteins, cells or adenoviruses, leading to transient expression of the foreign protein. Lentiviral vectors, due to their unique ability to integrate into the genome of quiescent cells, may be best suited for long-term expression of a transgene in the thymus. RESULTS: Young adult mice were injected in the thymus with lentiviral vectors expressing eGFP or the hemaglutinin of the Influenza virus under the control of the ubiquitous phospho glycerate kinase promoter. Thymi were examined 5 to 90 days thereafter directly under a UV-light microscope and by flow cytometry. Intrathymic injection of lentiviral vectors predominantly results in infection of stromal cells that could be detected for at least 3 months. Importantly, hemaglutinin expression by thymic stromal cells mediated negative selection of thymocytes expressing the cognate T-cell receptor. In addition and despite the low multiplicity of infection, transduced thymocytes were also detected, even 30 days after injection. CONCLUSIONS: Our results demonstrate that intrathymic delivery of a lentiviral vector is an efficient means for stable expression of a foreign gene in the thymus. This new method of gene delivery may prove useful for induction of tolerance to a specific antigen and for gene therapy of severe combined immunodeficiencies. [Abstract/Link to Full Text]

Nagel JE, Smith RJ, Shaw L, Bertak D, Dixit VD, Schaffer EM, Taub DD
Identification of genes differentially expressed in T cells following stimulation with the chemokines CXCL12 and CXCL10.
BMC Immunol. 2004 Aug 5;517.
BACKGROUND: Chemokines are involved in many biological activities ranging from leukocyte differentiation to neuronal morphogenesis. Despite numerous reports describing chemokine function, little is known about the molecular changes induced by cytokines. METHODS: We have isolated and identified by differential display analysis 182 differentially expressed cDNAs from CXCR3-transfected Jurkat T cells following treatment with CXCL12 or CXCL10. These chemokine-modulated genes were further verified using quantitative RT-PCR and Western blot analysis. RESULTS: One hundred and forty-six of the cDNAs were successfully cloned, sequenced, and identified by BLAST. Following removal of redundant and non-informative clones, seventeen mRNAs were found to be differentially expressed post treatment with either chemokine ligand with several representing known genes with established functions. Twenty-one genes were upregulated in these transfected Jurkat cells following both CXCL12 and CXCL10, four genes displayed a discordant response and seven genes were downregulated upon treatment with either chemokine. Identified genes include geminin (GEM), thioredoxin (TXN), DEAD/H box polypeptide 1 (DDX1), growth hormone inducible transmembrane protein (GHITM), and transcription elongation regulator 1 (TCERG1). Subsequent analysis of several of these genes using semi-quantitative PCR and western blot analysis confirmed their differential expression post ligand treatment. CONCLUSIONS: Together, these results provide insight into chemokine-induced gene activation and identify potentially novel functions for known genes in chemokine biology. [Abstract/Link to Full Text]

Wang L, Jacobsen SE, Bengtsson A, Erlinge D
P2 receptor mRNA expression profiles in human lymphocytes, monocytes and CD34+ stem and progenitor cells.
BMC Immunol. 2004 Aug 3;516.
BACKGROUND: Extracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors. Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack of truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors. In addition, we describe for the first time the expression of P2 receptors in CD34+ stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function. RESULTS: Using a quantitative mRNA assay, we assessed the hypothesis that there are specific P2 receptor profiles in inflammatory cells. The P2X4 receptor had the highest expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y12 and P2Y2 had highest expression in lymphocytes, while the P2Y2 and P2Y13 had highest expression in monocytes. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4) in CD34+ stem and progenitor cells. CONCLUSIONS: The most interesting findings were the high mRNA expression of P2Y12 receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y13 receptors in monocytes and a previously unrecognised expression of P2X4 in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns was studied in CD34+ stem and progenitor cells. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes. [Abstract/Link to Full Text]

Hu L, Dixit VD, de Mello-Coelho V, Taub DD
Age-associated alterations in CXCL1 chemokine expression by murine B cells.
BMC Immunol. 2004 Jul 26;515.
BACKGROUND: The CXCL1 chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), have been shown to play a role in a number of pathophysiological disease states including endotoxin-induced inflammation and bacterial meningitis. While the expression of these chemokines has been identified in a variety of cell types in the mouse, little is known about their expression with murine B-lymphocytes. RESULTS: Here, we demonstrate that highly purified murine splenic B cells are capable of expressing both MIP-2 and KC protein and mRNA upon activation with lipopolysaccharide (LPS) but not in response to anti-micro and anti-CD40 in combination with interleukin-4 (IL-4) stimulation. Moreover, these chemokines are expressed at higher levels in B cells derived from young (4 m) compared to old (24-29 m) mice. Upon fractionation into distinct B-cell subsets, we found that the expression of MIP-2 and KC by aged follicular (FO) B cells is significantly decreased when compared to the same cells from younger mice, while only MIP-2 production was found to be diminished in aged marginal zone (MZ) B cells. Interestingly, MIP-2 and KC production by newly formed (NF) B cells did not significantly differ with age. Moreover, the potential relevance of these findings is supported by the poor ability of LPS-activated aged B cells to specifically mediate CXCL1-dependent leukocyte recruitment when compared to younger B cells. CONCLUSION: Overall, the decreased expression of CXCL1 chemokines by aged B cells in response to LPS may have potential implications on the secondary recruitment of leukocytes to sites of microbial infections and inflammation possibly contributing to the increased susceptibility of older subjects to pathogen challenge. [Abstract/Link to Full Text]

McClelland EE, Damjanovich K, Gardner K, Groesbeck ZJ, Ma MS, Nibley M, Richardson KS, Wilkinson M, Morrison LC, Bernhardt P, Potts WK
Infection-dependent phenotypes in MHC-congenic mice are not due to MHC: can we trust congenic animals?
BMC Immunol. 2004 Jul 9;514.
BACKGROUND: Congenic strains of mice are assumed to differ only at a single gene or region of the genome. These mice have great importance in evaluating the function of genes. However, their utility depends on the maintenance of this true congenic nature. Although, accumulating evidence suggests that congenic strains suffer genetic divergence that could compromise interpretation of experimental results, this problem is usually ignored. During coinfection studies with Salmonella typhimurium and Theiler's murine encephalomyelitis virus (TMEV) in major histocompatibility complex (MHC)-congenic mice, we conducted the proper F2 controls and discovered significant differences between these F2 animals and MHC-genotype-matched P0 and F1 animals in weight gain and pathogen load. To systematically evaluate the apparent non-MHC differences in these mice, we infected all three generations (P0, F1 and F2) for 5 MHC genotypes (b/b, b/q and q/q as well as d/d, d/q, and q/q) with Salmonella and TMEV. RESULTS: Infected P0 MHC q/q congenic homozygotes lost significantly more weight (p = 0.02) and had significantly higher Salmonella (p < 0.01) and TMEV (p = 0.02) titers than the infected F2 q/q homozygotes. Neither weight nor pathogen load differences were present in sham-infected controls. CONCLUSIONS: These data suggest that these strains differ for genes other than those in the MHC congenic region. The most likely explanation is that deleterious recessive mutations affecting response to infection have accumulated in the more than 40 years that this B10.Q-H-2q MHC-congenic strain has been separated from its B10-H-2b parental strain. During typical experiments with congenic strains, the phenotypes of these accumulated mutations will be falsely ascribed to the congenic gene(s). This problem likely affects any strains separated for appreciable time and while usually ignored, can be avoided with the use of F2 segregants. [Abstract/Link to Full Text]

Rodriguez MW, Paquet AC, Yang YH, Erle DJ
Differential gene expression by integrin beta 7+ and beta 7- memory T helper cells.
BMC Immunol. 2004 Jul 5;513.
BACKGROUND: The cell adhesion molecule integrin alpha 4 beta 7 helps direct the migration of blood lymphocytes to the intestine and associated lymphoid tissues. We hypothesized that beta 7+ and beta 7- blood memory T helper cells differ in their expression of genes that play a role in the adhesion or migration of T cells. RESULTS: RNA was prepared from beta 7+ and beta 7- CD4+ CD45RA- blood T cells from nine normal human subjects and analyzed using oligonucleotide microarrays. Of 21357 genes represented on the arrays, 16 were more highly expressed in beta 7+ cells and 18 were more highly expressed in beta 7- cells (>/=1.5 fold difference and adjusted P < 0.05). Several of the differentially expressed transcripts encode proteins with established or putative roles in lymphocyte adhesion and chemotaxis, including the chemokine receptors CCR9 and CCR10, the integrin alpha 4 subunit, L-selectin, KLRB1 (CD161), NT5E (CD73), LGALS1 and LGALS2 (galectin-1 and -2), and RGS1. Flow cytometry was used to determine whether differences in levels of transcripts encoding cell surface proteins were associated with differential expression of those proteins. Using this approach, we found that surface expression of KLRB1, LAIR1, and NT5E proteins was higher on beta 7+ memory/effector T cells than on beta 7- cells. CONCLUSIONS: Memory/effector T cells that express integrin beta 7 have a distinct pattern of expression of a set of gene transcripts. Several of these molecules can affect cell adhesion or chemotaxis and are therefore likely to modulate the complex multistep process that regulates trafficking of CD4+ memory T cell subsets with different homing behaviors. [Abstract/Link to Full Text]

Patil NS, Wong DL, Collier KD, McDonald HC
Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines.
BMC Immunol. 2004 Jun 28;512.
BACKGROUND: We have devised a simple and efficient fluorescence-based method to track antigen uptake and processing in human B lymphoblastoid cells (B-LCL). Fluorescein labelled subtilisin was used to optimize antigen uptake conditions and identify processed peptides from human cell lines. RESULTS: Fluorescein labelled subtilisin conjugates had 0.06 to 2 moles of fluorescein per subtilisin molecule. High performance liquid chromatography and mass spectrometry (NanoESI-LC/MS/MS) analysis identified fluorescein conjugated to K141, K256, and the N terminus. Conjugates retained antigenic specificity to subtilisin specific antibodies and could be processed by whole cell extracts into low molecular weight fragments at pH 5.2. Maximal antigen uptake and processing occurred when PMSF (phenylmethylsulfonyl fluoride) inhibited subtilisin conjugate was incubated with cells at 100-200 microg/ml for 16 to 24 hr. Once optimal uptake conditions were established, processed subtilisin peptides were isolated and identified from human cell lines. CONCLUSION: Our studies show that FITC-conjugation provides an efficient tool to track the uptake and processing of this protease antigen and to facilitate identification of processed antigenic peptides from human cell lines. [Abstract/Link to Full Text]

Conlon TM, Meyer KB
Cloning and functional characterisation of avian transcription factor E2A.
BMC Immunol. 2004 Jun 14;511.
BACKGROUND: During B lymphocyte development the E2A gene is a critical regulator of cell proliferation and differentiation. With regards to the immunoglobulin genes the E2A proteins contribute to the regulation of gene rearrangement, expression and class switch recombination. We are now using the chicken cell line DT40 as a model system to further analyse the function of E2A. RESULTS: Here we report the cloning and functional analysis of the transcription factor E2A from chicken. Using RACE PCR on the chicken lymphoma cell line DT40 we have isolated full-length clones for the two E2A splice variants E12 and E47. Sequence conservation between the human and chicken proteins is extensive: the basic-helix-loop-helix DNA binding domain of human and chicken E47 and E12 are 93% and 92% identical, respectively. In addition high levels of conservation are seen in activation domain I, the potential NLS and the ubiquitin ligase interaction domain. E2A is expressed in a variety of tissues in chicken, with higher levels of expression in organs rich in immune cells. We demonstrate that chicken E12 and E47 proteins are strong transcriptional activators whose function depends on the presence of activation domain I. As in mammals, the dominant negative proteins Id1 and Id3 can inhibit the function of chicken E47. CONCLUSIONS: The potential for homologous recombination in DT40 allows the genetic dissection of biochemical pathways in somatic cells. With the cloning of avian E2A and the recent description of an in vitro somatic hypermutation assay in this cell line, it should now be possible to dissect the potential role of E2A in the regulation of somatic hypermutation and gene conversion. [Abstract/Link to Full Text]

Mizgerd JP, Lupa MM, Spieker MS
NF-kappaB p50 facilitates neutrophil accumulation during LPS-induced pulmonary inflammation.
BMC Immunol. 2004 Jun 9;510.
BACKGROUND: Transcription factors have distinct functions in regulating immune responses. During Escherichia coli pneumonia, deficiency of NF-kappaB p50 increases gene expression and neutrophil recruitment, suggesting that p50 normally limits these innate immune responses. p50-deficient mice were used to determine how p50 regulates responses to a simpler, non-viable bacterial stimulus in the lungs, E. coli lipopolysaccharide (LPS). RESULTS: In contrast to previous results with living E. coli, neutrophil accumulation elicited by E. coli LPS in the lungs was decreased by p50 deficiency, to approximately 30% of wild type levels. Heat-killed E. coli induced neutrophil accumulation which was not decreased by p50 deficiency, demonstrating that bacterial growth and metabolism were not responsible for the different responses to bacteria and LPS. p50 deficiency increased the LPS-induced expression of kappaB-regulated genes essential to neutrophil recruitment, including KC, MIP-2, ICAM-1, and TNF-alpha suggesting that p50 normally limited this gene expression and that decreased neutrophil recruitment did not result from insufficient expression of these genes. Neutrophils were responsive to the chemokine KC in the peripheral blood of p50-deficient mice with or without LPS-induced pulmonary inflammation. Interleukin-6 (IL-6), previously demonstrated to decrease LPS-induced neutrophil recruitment in the lungs, was increased by p50 deficiency, but LPS-induced neutrophil recruitment was decreased by p50 deficiency even in IL-6 deficient mice. CONCLUSION: p50 makes essential contributions to neutrophil accumulation elicited by LPS in the lungs. This p50-dependent pathway for neutrophil accumulation can be overcome by bacterial products other than LPS and does not require IL-6. [Abstract/Link to Full Text]

Munthe E, Finne EF, Aasheim HC
Expression and functional effects of Eph receptor tyrosine kinase A family members on Langerhans like dendritic cells.
BMC Immunol. 2004 Jun 3;59.
BACKGROUND: The Eph receptors are the largest receptor tyrosine kinase family. Several family members are expressed in hematopoietic cells. Previously, the expression of a member of this family, EphA2, was identified on dendritic like cells in tonsils. We therefore specifically examined the expression of EphA2 on in vitro generated dendritic cells. RESULTS: In this study, expression of the EphA2 receptor was identified on in vitro generated Langerhans like dendritic cells compared to in vitro generated dendritic cells. We show that ligand induced engagement of the EphA2 receptor leads to receptor autophosphorylation indicating a functional receptor signaling pathway in these cells. We also observe phosphorylation and dephosphorylation of distinct proteins following ligand activation of EphA receptors. In co-stimulation assays, receptor-ligand interaction reduces the capacity of the Langerhans like dendritic cells to stimulate resting CD4+ T cells. CONCLUSION: Engagement of EphA receptor tyrosine kinases on Langerhans like dendritic cells induces signaling as shown by tyrosine phosphorylation and dephosphorylation of distinct proteins. Furthermore this engagement renders the cells less capable of stimulating CD4+ T cells. [Abstract/Link to Full Text]

Rumfelt LL, Lohr RL, Dooley H, Flajnik MF
Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark.
BMC Immunol. 2004 May 6;5(1):8.
BACKGROUND: Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families. RESULTS: IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species. CONCLUSION: As in ratfish, sandbar and horn sharks, most nurse shark IgM VH genes are from one family with multiple, heterogeneous loci. Their IgW VH genes have diversified, forming at least three families. The neonatal shark Ig VH CDR3 repertoire, diversified via N-region addition, is shorter than the adult VDJ junction, suggesting one means of postnatal repertoire diversification is expression of longer CDR3 junctions. [Abstract/Link to Full Text]

Rothermel AL, Wang Y, Schechner J, Mook-Kanamori B, Aird WC, Pober JS, Tellides G, Johnson DR
Endothelial cells present antigens in vivo.
BMC Immunol. 2004 Mar 16;55.
BACKGROUND: Immune recognition of vascular endothelial cells (EC) has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli beta-galactosidase (beta-gal) in two lines of transgenic mice that express beta-gal exclusively in their EC. TIE2-lacZ mice express beta-gal in all EC and VWF-lacZ mice express beta-gal in heart and brain microvascular EC. RESULTS: Transgenic and congenic wild type FVB mice immunized with beta-gal expression vector DNA or beta-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express beta-gal, and their blood vessels showed no histological abnormalities. In response to beta-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-gamma. Infection with recombinant vaccinia virus encoding beta-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express beta-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost beta-gal+ EC and the hosts developed beta-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained beta-gal+ EC and no antisera developed, suggesting a tolerant host immune system. CONCLUSION: Resting, beta-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against beta-gal expressed by EC within transplanted skin. We conclude that EC effectively present intracellular "self" proteins to the immune system. However, antigen presentation by EC does not delete or anergize a large population of specific lymphocytes that respond to the same protein following conventional immunization with protein or expression vector DNA. These results clearly demonstrate striking context sensitivity in the immune recognition of EC, a subtlety that must be better understood in order to treat immune diseases and complications involving the vasculature. [Abstract/Link to Full Text]

Eberhard Y, Ortiz S, Ruiz Lascano A, Kuznitzky R, Serra HM
Up-regulation of the chemokine CCL21 in the skin of subjects exposed to irritants.
BMC Immunol. 2004 Apr 26;5(1):7.
BACKGROUND: Expression of murine CCL21 by dermal lymphatic endothelial cells (LEC) has been demonstrated to be one of the most important steps in Langerhans cell emigration from skin. Previously, our group and others have found that this chemokine is up-regulated in different human inflammatory skin diseases mediated by diverse specific immune responses. This study was carried out to investigate the involvement of CCL21 in human skin after challenge with irritant agents responsible for inducing Irritant Contact Dermatitis (ICD). RESULTS: Eleven normal individuals were challenged with different chemical or physical irritants. Two patients with Allergic Contact Dermatitis (ACD) were also challenged with the relevant antigen in order to have a positive control for CCL21 expression. Macroscopic as well as microscopic responses were evaluated. We observed typical ICD responses with mostly mononuclear cells in perivascular areas, but a predominance of polymorphonuclear cells away from the inflamed blood vessels and in the epidermis at 24 hours. Immunohistochemical studies showed up-regulation of CCL21 by lymphatic endothelial cells in all the biopsies taken from ICD and ACD lesions compared to normal skin. Kinetic study at 10, 48, 96 and 168 hours after contact with a classical irritant (sodium lauryl sulphate) showed that the expression of CCL21 was increased in lymphatic vessels at 10 hours, peaked at 48 hours, and then gradually declined. There was a strong correlation between CCL21 expression and the macroscopic response (r = 0.69; p = 0.0008), but not between CCL21 and the number of infiltrating cells in the lesions. CONCLUSIONS: These results provide new evidence for the role of CCL21 in inflammatory processes. Since the up-regulation of this chemokine was observed in ICD and ACD, it is tempting to speculate that this mechanism operates independently of the type of dermal insult, facilitating the emigration of CCR7+ cells. [Abstract/Link to Full Text]

Satti MZ, Cahen P, Skov PS, Joseph S, Jones FM, Fitzsimmons C, Hoffmann KF, Reimert C, Kariuki HC, Kazibwe F, Mwatha JK, Kimani G, Vennervald BJ, Ouma JH, Kabatereine NB, Dunne DW
Changes in IgE- and antigen-dependent histamine-release in peripheral blood of Schistosoma mansoni-infected Ugandan fishermen after treatment with praziquantel.
BMC Immunol. 2004 Apr 21;5(1):6.
BACKGROUND: Parasite-specific IgE levels correlate with human resistance to reinfection with Schistosoma spp. after chemotherapy. Although the role of eosinophils in schistosomiasis has been the focus of a great deal of important research, the involvement of other Fcepsilon receptor-bearing cells, such as mast cells and basophils, has not been investigated in relation to human immunity to schistosomes. Chemotherapy with praziquantel (PZQ) kills schistosomes living in an in vivo blood environment rich in IgE, eosinophils and basophils. This releases parasite Ags that have the potential to cross-link cell-bound IgE. However, systemic hypersensitivity reactions are not induced by treatment. Here, we describe the effects of schistosomiasis, and its treatment, on human basophil function by following changes in total cellular histamine and in vitro histamine-release induced by schistosome Ags or anti-IgE, in blood samples from infected Ugandan fishermen, who are continuously exposed to S. mansoni infection, before and 1-day and 21-days after PZQ treatment. RESULTS: There was a significant increase in the total cellular histamine in blood samples at 1-day post-treatment, followed by a very significant further increase by 21-days post-treatment. In vitro histamine-release induced by S. mansoni egg (SEA) or worm (SWA) Ags or anti-IgE antibody, was significantly reduced 1-day post-treatment. The degree of this reduction correlated with pre-treatment infection intensity. Twenty-1-days post-treatment, SEA-induced histamine-release was still significantly lower than at pretreatment. Histamine-release was not correlated to plasma concentrations of total or parasite-specific IgE, nor to specific IgG4 plasma concentrations. CONCLUSION: The biology of human blood basophils is modulated by S. mansoni infection and praziquantel treatment. Infection intensity-dependent suppression of basophil histamine-release, histamine-dependent resistance to infection, and similarities with allergen desensitisation are discussed as possible explanations of these observations. [Abstract/Link to Full Text]

Peinnequin A, Mouret C, Birot O, Alonso A, Mathieu J, Clarençon D, Agay D, Chancerelle Y, Multon E
Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green.
BMC Immunol. 2004 Feb 5;53.
BACKGROUND: Cytokine mRNA quantification is widely used to investigate cytokine profiles, particularly in small samples. Real-time polymerase chain reaction is currently the most reliable method of quantifying low-level transcripts such as cytokine and cytokine receptor mRNAs. This accurate technique allows the quantification of a larger pattern of cytokines than quantification at the protein level, which is limited to a smaller number of proteins. RESULTS: Although fluorogenic probes are considered more sensitive than fluorescent dyes, we have developed SYBR Green real-time RT-PCR protocols to assay pro-inflammatory cytokines (IL1a, IL1b and IL6, TNFa), cytokine receptors (IL1-r1, IL1-r2, IL6-r, TNF-r2) and related molecules (IL1-RA, SOCS3) mRNA in rats. This method enables normalisation against several housekeeping genes (beta-actin, GAPDH, CypA, HPRT) dependent on the specific experimental treatments and tissues using either standard curve, or comparative CT quantification method. PCR efficiency and sensitivity allow the assessment of; i) basal mRNA levels in many tissues and even decreases in mRNA levels, ii) mRNA levels from very small samples. CONCLUSION: Real-time RT-PCR is currently the best way to investigate cytokine networks. The investigations should be completed by the analysis of genes regulated by cytokines or involved in cytokine signalling, providing indirect information on cytokine protein expression. [Abstract/Link to Full Text]

de Mello Coelho V, Nguyen D, Giri B, Bunbury A, Schaffer E, Taub DD
Quantitative differences in lipid raft components between murine CD4+ and CD8+ T cells.
BMC Immunol. 2004 Jan 30;52.
BACKGROUND: Lipid rafts have been shown to play a role in T cell maturation, activation as well as in the formation of immunological synapses in CD4+ helper and CD8+ cytotoxic T cells. However, the differential expression of lipid raft components between CD4+ and CD8+ T cells is still poorly defined. To examine this question, we analyzed the expression of GM1 in T cells from young and aged mice as well as the expression of the glycosylphosphatidylinositol (GPI)-linked protein Thy-1 and cholesterol in murine CD4+ and CD8+ T cell subpopulations. RESULTS: We found that CD4+CD8- and CD8+CD4- thymocytes at different stages of maturation display distinct GM1 surface expression. This phenomenon did not change with progressive aging, as these findings were consistent over the lifespan of the mouse. In the periphery, CD8+ T cells express significantly higher levels of GM1 than CD4+ T cells. In addition, we observed that GM1 levels increase over aging on CD8+ T cells but not in CD4+ T cells. We also verified that naďve (CD44lo) and memory (CD44hi) CD8+ T cells as well as naďve and memory CD4+ T cells express similar levels of GM1 on their surface. Furthermore, we found that CD8+ T cells express higher levels of the GPI-anchored cell surface protein Thy-1 associated with lipid raft domains as compared to CD4+ T cells. Finally, we observed higher levels of total cellular cholesterol in CD8+ T cells than CD4+ T cells. CONCLUSION: These results demonstrate heterogeneity of lipid raft components between CD4+ and CD8+ T cells in young and aged mice. Such differences in lipid raft composition may contribute to the differential CD4 and CD8 molecule signaling pathways as well as possibly to the effector responses mediated by these T cell subsets following TCR activation. [Abstract/Link to Full Text]

Schountz T, Green R, Davenport B, Buniger A, Richens T, Root JJ, Davidson F, Calisher CH, Beaty BJ
Cloning and characterization of deer mouse (Peromyscus maniculatus) cytokine and chemokine cDNAs.
BMC Immunol. 2004 Jan 13;51.
BACKGROUND: Sin Nombre virus (SNV) establishes a persistent infection in the deer mouse, Peromyscus maniculatus. A strong antibody response occurs in response to SNV infection, but the role of the innate immune response is unclear. To address this issue, we have initiated an effort to identify and characterize deer mouse cytokine and chemokine genes. Such cytokines and chemokines are involved in various aspects of immunity, including the transition from innate to adaptive responses, type I and type II responses, recruitment of leukocytes to sites of infection, and production of mature cells from bone marrow progenitors. RESULTS: We established a colony of SNV antibody-negative deer mice and cloned 11 cytokine and chemokine partial cDNA sequences using directed PCR. Most of the deer mouse sequences were highly conserved with orthologous sequences from other rodent species and functional domains were identified in each putative polypeptide. CONCLUSIONS: The availability of these sequences will allow the examination of the role of these cytokines in deer mouse responses to infection with Sin Nombre virus. [Abstract/Link to Full Text]

Suni MA, Dunn HS, Orr PL, de Laat R, Sinclair E, Ghanekar SA, Bredt BM, Dunne JF, Maino VC, Maecker HT
Performance of plate-based cytokine flow cytometry with automated data analysis.
BMC Immunol. 2003 Sep 2;49.
BACKGROUND: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates. RESULTS: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis. CONCLUSION: When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses. [Abstract/Link to Full Text]

Kato A, Homma T, Batchelor J, Hashimoto N, Imai S, Wakiguchi H, Saito H, Matsumoto K
Interferon-alpha/beta receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006.
BMC Immunol. 2003 Jul 30;48.
BACKGROUND: Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported. RESULTS: This study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1) Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-kappaB. (2) Interferon (IFN)-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-alpha/beta receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN. CONCLUSION: This study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-alpha/beta receptor-mediated paracrine pathway. [Abstract/Link to Full Text]

Lau P, Amadou C, Brun H, Rouillon V, McLaren F, Le Rolle AF, Graham M, Butcher GW, Joly E
Characterisation of RT1-E2, a multigenic family of highly conserved rat non-classical MHC class I molecules initially identified in cells from immunoprivileged sites.
BMC Immunol. 2003 Jul 1;47.
BACKGROUND: So-called "immunoprivileged sites" are tissues or organs where slow allograft rejection correlates with low levels of expression of MHC class I molecules. Whilst classical class I molecules are recognised by cytotoxic T lymphocytes (CTL), some MHC class I molecules are called "non-classical" because they exhibit low polymorphism and are not widely expressed. These last years, several studies have shown that these can play different, more specialised roles than their classical counterparts. In the course of efforts to characterise MHC class I expression in rat cells obtained from immunoprivileged sites such as the central nervous system or the placenta, a new family of non-classical MHC class I molecules, which we have named RT1-E2, has been uncovered. RESULTS: Members of the RT1-E2 family are all highly homologous to one another, and the number of RT1-E2 loci varies from one to four per MHC haplotype among the six rat strains studied so far, with some loci predicted to give rise to soluble molecules. The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes. We present evidence that: i) RT1-E2 molecules can be detected at the surface of transfected mouse L cells and simian COS-7 cells, albeit at low levels; ii) their transport to the cell surface is dependent on a functional TAP transporter. In L cells, their transport is also hindered by protease inhibitors, brefeldin A and monensin. CONCLUSIONS: These findings suggest that RT1-E2 molecules probably associate with ligands of peptidic nature. The high homology between the RT1-E2 molecules isolated from divergent rat MHC haplotypes is particularly striking at the level of their extra-cellular portions. Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands. Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules. [Abstract/Link to Full Text]

Rodríguez-Burgos A
Detection in chick embryo of fetoproteins not recognized by the dam's immune system and of soluble alloantigens. Presumptive teratogenic and abortogenic capacity of their specific IgY.
BMC Immunol. 2003 Jun 27;46.
BACKGROUND: The aim of this work was to detect antigens, non-self to the dam, potentially present in chick embryo prior to organogenesis with a view to establishing the consequences of their neutralization on chick development. To this end, hens were immunized with the extract from embryos incubated for 53 h. Their eggs were either used to isolate immunoglobulins for dot and blot tests or incubated for variable lengths of time. RESULTS: Immunoblot tests, using adsorbed primary and secondary antibodies against paternal serum, revealed the presence of at least four antigens of 32, 34, 70 and 200 kDa that can be classified as soluble alloantigens. The same antibodies against chick embryo extracts (between 53 h and 9) showed at least five aged antigens of 34, 52, 90, 200 and 250 kDa, not detected in cock serum, that can thus be considered as soluble, foreign to the immunized hens and transitory antigens. The abnormalities observed included arrested development and fetal death, as well as minor functional damage in the few chicks that were born alive. The ratio of abnormal to normal embryos was 2.85 in the experimental group and 0.43 in the control group. With regard to congenital anomalies it must be said that of the 81 eggs incubated only four chicks were born alive, and of these, only one had a healthy birth and subsequent growth. The other three showed a transitory ataxia and one of them presented adult lumbar scoliosis and asymmetric pelvis. CONCLUSIONS: The problem of recurrent spontaneous abortions is revisited in the light of these results. Some recent data suggest that soluble alloantigens may be candidates for a new etiological entity in recurrent spontaneous abortions. They can also be the cause of some congenital anomalies. The soluble, foreign, transitory antigens may have a similar effect although there is no supportive data in the literature. [Abstract/Link to Full Text]

Wells CA, Ravasi T, Faulkner GJ, Carninci P, Okazaki Y, Hayashizaki Y, Sweet M, Wainwright BJ, Hume DA
Genetic control of the innate immune response.
BMC Immunol. 2003 Jun 26;45.
BACKGROUND: Susceptibility to infectious diseases is directed, in part, by the interaction between the invading pathogen and host macrophages. This study examines the influence of genetic background on host-pathogen interactions, by assessing the transcriptional responses of macrophages from five inbred mouse strains to lipopolysaccharide (LPS), a major determinant of responses to gram-negative microorganisms. RESULTS: The mouse strains examined varied greatly in the number, amplitude and rate of induction of genes expressed in response to LPS. The response was attenuated in the C3H/HeJlpsd strain, which has a mutation in the LPS receptor Toll-like receptor 4 (TLR4). Variation between mouse strains allowed clustering into early (C57Bl/6J and DBA/2J) and delayed (BALB/c and C3H/ARC) transcriptional phenotypes. There was no clear correlation between gene induction patterns and variation at the Bcg locus (Slc11A1) or propensity to bias Th1 versus Th2 T cell activation responses. CONCLUSION: Macrophages from each strain responded to LPS with unique gene expression profiles. The variation apparent between genetic backgrounds provides insights into the breadth of possible inflammatory responses, and paradoxically, this divergence was used to identify a common transcriptional program that responds to TLR4 signalling, irrespective of genetic background. Our data indicates that many additional genetic loci control the nature and the extent of transcriptional responses promoted by a single pathogen-associated molecular pattern (PAMP), such as LPS. [Abstract/Link to Full Text]

Valés-Gómez M, Browne H, Reyburn HT
Expression of the UL16 glycoprotein of Human Cytomegalovirus protects the virus-infected cell from attack by natural killer cells.
BMC Immunol. 2003 Mar 14;44.
BACKGROUND: Human Cytomegalovirus (HCMV) has acquired through evolution a number of genes to try to evade immune recognition of the virus-infected cell. Many of these mechanisms act to inhibit the MHC class I antigen presentation pathway, but any virus-infected cell which has down-regulated cell surface expression of MHC class I proteins, to avoid CTL attack, would be expected to become susceptible to lysis by Natural Killer cells. Surprisingly, however, HCMV infected fibroblasts were found to be resistant to NK cell mediated cytotoxicity. Expression of the UL16 glycoprotein could represent one mechanism to help the virus to escape from NK cell attack, as it has been shown to bind, in vitro, some of the ligands for NKG2D, the NK cell activating receptor. Here, we explored the role of UL16, in the context of a viral infection, by comparing the susceptibility to NK lysis of cells infected with HCMV and cells infected with a UL16 deletion mutant of this virus. RESULTS: Cells infected with the UL16 knockout virus were killed at substantially higher levels than cells infected with the wild-type virus. This increased killing could be correlated with a UL16-dependent reduction in surface expression of ligands for the NK cell activating receptor NKG2D. CONCLUSIONS: Expression of the UL16 glycoprotein was associated with protection of HCMV-infected cells from NK cell attack. This observation could be correlated with the downregulation of cell surface expression of NKG2D ligands. These data represent a first step towards understanding the mechanism(s) of action of the UL16 protein. [Abstract/Link to Full Text]

Mathison RD, Befus AD, Davison JS, Woodman RC
Modulation of neutrophil function by the tripeptide feG.
BMC Immunol. 2003 Mar 4;43.
BACKGROUND: Neutrophils are critical in the defense against potentially harmful microorganisms, but their excessive and inappropriate activation can contribute significantly to tissue damage and a worsening pathology. Through the release of endocrine factors submandibular glands contribute to achieving a balance in neutrophil function by modulating the state of activation and migratory potential of circulating neutrophils. A putative hormonal candidate for these effects on neutrophils was identified as a heptapeptide named submandibular gland peptide T (SGP-T; sequence = TDIFEGG). Since the tripeptide FEG, derived from SGP-T, and its D-amino acid analogue feG had similar inhibitory effects on inflammatory reactions, we investigated the effects of feG on human and rat neutrophil function. RESULTS: With human neutrophils feG had no discernible effect on oxidative burst or phagocytosis, but in picomolar amounts it reduced PAF-induced neutrophil movement and adhesion, and the binding of CD11b by 34% and that of CD16b close to control values. In the rat feG (10-11M) reduced the binding of CD11b and CD16 antibodies to PAF-stimulated circulating neutrophils by 35% and 43%, respectively, and at 100 micrograms/kilograms intraperitoneally feG reduced neutrophil in vivo migration by 40%. With ovalbumin-sensitized rats that were challenged with antigen, feG inhibited binding of antibodies against CD16b but not CD11b, on peritoneal leukocytes. CONCLUSIONS: The inhibitory effect of feG on neutrophil movement may be mediated by alterations in the co-stimulatory molecules CD11b and CD16. [Abstract/Link to Full Text]

Maecker HT
Human CD81 directly enhances Th1 and Th2 cell activation, but preferentially induces proliferation of Th2 cells upon long-term stimulation.
BMC Immunol. 2003 Feb 19;41.
BACKGROUND: CD81, a cell-surface protein of the tetraspanin superfamily, has been shown to costimulate T cell activation in murine T cells, and is involved in development of Th2 immune responses in mice. RESULTS: Here it is shown that stimulation of CD81 on human T cells can enhance T cell activation by antigen or superantigen, causing an increase in the early activation marker CD69, and increasing the number of cytokine-producing and proliferating T cells. Interestingly, CD81 costimulates cytokine production by T cells producing both Th1 and Th2 cytokines. Although human CD81 is highly expressed on non-T as well as T cells, CD81 costimulation appears to act directly on T cells. Pre-incubation of purified T cells with anti-CD81 antibody is sufficient to increase T cell activation, while pre-incubation of non-T cells is not. However, long-term polyclonal stimulation of T cells by anti-CD3 antibody, in the presence of CD81 costimulation, biases T cells towards the production of IL-4 and not IFNgamma. This is accomplished by a preferential proliferation of IL-4-producing cells. CONCLUSION: Thus, signalling through CD81 on T cells costimulates both Th1 and Th2 cells, but increases the number of Th2 cells during long-term activation. [Abstract/Link to Full Text]

Pedotti R, Sanna M, Tsai M, DeVoss J, Steinman L, McDevitt H, Galli SJ
Severe anaphylactic reactions to glutamic acid decarboxylase (GAD) self peptides in NOD mice that spontaneously develop autoimmune type 1 diabetes mellitus.
BMC Immunol. 2003 Feb 22;42.
BACKGROUND: Insulin dependent (i.e., "type 1") diabetes mellitus (T1DM) is considered to be a T cell mediated disease in which TH1 and Tc autoreactive cells attack the pancreatic islets. Among the beta-cell antigens implicated in T1DM, glutamic acid decarboxylase (GAD) 65 appears to play a key role in the development of T1DM in humans as well as in non-obese diabetic (NOD) mice, the experimental model for this disease. It has been shown that shifting the immune response to this antigen from TH1 towards TH2, via the administration of GAD65 peptides to young NOD mice, can suppress the progression to overt T1DM. Accordingly, various protocols of "peptide immunotherapy" of T1DM are under investigation. However, in mice with experimental autoimmune encephalomyelitis (EAE), another autoimmune TH1 mediated disease that mimics human multiple sclerosis, anaphylactic shock can occur when the mice are challenged with certain myelin self peptides that initially were administered with adjuvant to induce the disease. RESULTS: Here we show that NOD mice, that spontaneously develop T1DM, can develop fatal anaphylactic reactions upon challenge with preparations of immunodominant GAD65 self peptides after immunization with these peptides to modify the development of T1DM. CONCLUSIONS: These findings document severe anaphylaxis to self peptide preparations used in an attempt to devise immunotherapy for a spontaneous autoimmune disease. Taken together with the findings in EAE, these results suggest that peptide therapies designed to induce a TH1 to TH2 shift carry a risk for the development of anaphylactic reactivity to the therapeutic peptides. [Abstract/Link to Full Text]

Hsueh RC, Hammill AM, Lee JA, Uhr JW, Scheuermann RH
Activation of the Syk tyrosine kinase is insufficient for downstream signal transduction in B lymphocytes.
BMC Immunol. 2002 Dec 6;316.
BACKGROUND: Immature B lymphocytes and certain B cell lymphomas undergo apoptotic cell death following activation of the B cell antigen receptor (BCR) signal transduction pathway. Several biochemical changes occur in response to BCR engagement, including activation of the Syk tyrosine kinase. Although Syk activation appears to be necessary for some downstream biochemical and cellular responses, the signaling events that precede Syk activation remain ill defined. In addition, the requirements for complete activation of the Syk-dependent signaling step remain to be elucidated. RESULTS: A mutant form of Syk carrying a combination of a K395A substitution in the kinase domain and substitutions of three phenylalanines (3F) for the three C-terminal tyrosines was expressed in a murine B cell lymphoma cell line, BCL1.3B3 to interfere with normal Syk regulation as a means to examine the Syk activation step in BCR signaling. Introduction of this kinase-inactive mutant led to the constitutive activation of the endogenous wildtype Syk enzyme in the absence of receptor engagement through a 'dominant-positive' effect. Under these conditions, Syk kinase activation occurred in the absence of phosphorylation on Syk tyrosine residues. Although Syk appears to be required for BCR-induced apoptosis in several systems, no increase in spontaneous cell death was observed in these cells. Surprisingly, although the endogenous Syk kinase was enzymatically active, no enhancement in the phosphorylation of cytoplasmic proteins, including phospholipase Cgamma2 (PLCgamma2), a direct Syk target, was observed. CONCLUSION: These data indicate that activation of Syk kinase enzymatic activity is insufficient for Syk-dependent signal transduction. This observation suggests that other events are required for efficient signaling. We speculate that localization of the active enzyme to a receptor complex specifically assembled for signal transduction may be the missing event. [Abstract/Link to Full Text]

Servet-Delprat C, Arnaud S, Jurdic P, Nataf S, Grasset MF, Soulas C, Domenget C, Destaing O, Rivollier A, Perret M, Dumontel C, Hanau D, Gilmore GL, Belin MF, Rabourdin-Combe C, Mouchiroud G
Flt3+ macrophage precursors commit sequentially to osteoclasts, dendritic cells and microglia.
BMC Immunol. 2002 Oct 24;315.
BACKGROUND: Macrophages, osteoclasts, dendritic cells, and microglia are highly specialized cells that belong to the mononuclear phagocyte system. Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but developmental pathways leading to such diversity are still unclear. RESULTS: Mouse bone marrow cells were expanded in vitro in the presence of Flt3-ligand (FL), yielding high numbers of non-adherent cells exhibiting immature monocyte characteristics. Cells expanded for 6 days, 8 days, or 11 days (day 6-FL, day 8-FL, and day 11-FL cells, respectively) exhibited constitutive potential towards macrophage differentiation. In contrast, they showed time-dependent potential towards osteoclast, dendritic, and microglia differentiation that was detected in day 6-, day 8-, and day 11-FL cells, in response to M-CSF and receptor activator of NFkappaB ligand (RANKL), granulocyte-macrophage colony stimulating-factor (GM-CSF) and tumor necrosis factor-alpha (TNFalpha), and glial cell-conditioned medium (GCCM), respectively. Analysis of cell proliferation using the vital dye CFSE revealed homogenous growth in FL-stimulated cultures of bone marrow cells, demonstrating that changes in differential potential did not result from sequential outgrowth of specific precursors. CONCLUSIONS: We propose that macrophages, osteoclasts, dendritic cells, and microglia may arise from expansion of common progenitors undergoing sequential differentiation commitment. This study also emphasizes differentiation plasticity within the mononuclear phagocyte system. Furthermore, selective massive cell production, as shown here, would greatly facilitate investigation of the clinical potential of dendritic cells and microglia. [Abstract/Link to Full Text]

Wesa A, Galy A
Increased production of pro-inflammatory cytokines and enhanced T cell responses after activation of human dendritic cells with IL-1 and CD40 ligand.
BMC Immunol. 2002 Oct 18;314.
BACKGROUND: Various microbial, inflammatory and immune signals regulate the activation of dendritic cells (DC), determining their ability to interact with naďve T cells and to produce cytokines that direct T cell development. In particular, CD40L and IL-1 cooperatively activate DC to secrete high levels of IL-12. The immuno-stimulatory capacity of such DC is otherwise not well-defined prompting further characterization of the effects of IL-1 and family members on DC activation in comparison with other pro-inflammatory stimuli. RESULTS: Human DC co-activated in vitro by CD40L and IL-1beta expressed numerous cytokine genes including IL-12beta, IL-23 p19, IL-1beta, IL-1alpha, IL-1Ra, IL-10, IL-6, IL-18 and IFN-gamma. These DC produced high levels of IL-12 protein and appeared capable of producing IFN-gamma. Potent CD4+ and CD8+ T cell-stimulatory properties were acquired by DC under conditions that also induced IL-12. Notably, these DC induced rapid differentiation of fluMP-specific CD8+ T cells. Molecules related to IL-1beta, like IL-1alpha, co-induced IL-12 secretion whereas IL-18 did not. Conversely, the inhibitor IL-1Ra, produced endogenously by DC curtailed IL-12 production in response to CD40L. CONCLUSIONS: IL-1 and IL-1Ra play a biologically-relevant role in the positive and negative regulation of DC activation. In conjunction with CD40L, IL-1 sends a powerful activation signal to DC that could be distinguished from other modes of activation. This signal enables the production of pro-inflammatory cytokines by DC, and enhances the differentiation of naďve T cells into effectors of type-1 cellular immune responses. [Abstract/Link to Full Text]

Ares MP, Stollenwerk M, Olsson A, Kallin B, Jovinge S, Nilsson J
Decreased inducibility of TNF expression in lipid-loaded macrophages.
BMC Immunol. 2002 Oct 6;313.
BACKGROUND: Inflammation and immune responses are considered to be very important in the pathogenesis of atherosclerosis. Lipid accumulation in macrophages of the arterial intima is a characteristic feature of atherosclerosis which can influence the inflammatory potential of macrophages. We studied the effects of lipid loading on the regulation of TNF expression in human monocyte-derived macrophages. RESULTS: In macrophages incubated with acetylated low density lipoprotein (ac-LDL) for 2 days, mRNA expression of TNF in cells stimulated with TNF decreased by 75%. In cell cultures stimulated over night with IL-1beta, lipid loading decreased secretion of TNF into culture medium by 48%. These results suggest that lipid accumulation in macrophages makes them less responsive to inflammatory stimuli. Decreased basal activity and inducibility of transcription factor AP-1 was observed in lipid-loaded cells, suggesting a mechanism for the suppression of cytokine expression. NF-kappaB binding activity and inducibility were only marginally affected by ac-LDL. LDL and ac-LDL did not activate PPARgamma. In contrast, oxidized LDL stimulated AP-1 and PPARgamma but inhibited NF-kappaB, indicating that the effects of lipid loading with ac-LDL were not due to oxidation of lipids. CONCLUSIONS: Accumulation of lipid, mainly cholesterol, results in down-regulation of TNF expression in macrophages. Since monocytes are known to be activated by cell adhesion, these results suggest that foam cells in atherosclerotic plaques may contribute less potently to an inflammatory reaction than newly arrived monocytes/macrophages. [Abstract/Link to Full Text]

---

Recent Articles in BMC Infectious Diseases

---

Larru B, de Mendoza C, Bellón JM, de José MI, Mellado MJ, Soriano V, Muńoz-Fernandez MA, Ramos JT
Predictive factors of virological success to salvage regimens containing protease inhibitors in HIV-1 infected children.
BMC Infect Dis. 2007;755.
BACKGROUND: The impact of HIV drug resistance mutations in salvage therapy has been widely investigated in adults. By contrast, data available of predictive value of resistance mutations in pediatric population is scarce. METHODS: A multicenter, retrospective, observational study was conducted in children who received rescue salvage antiretroviral therapy after virologic failure. CD4 counts and viral load were determined at baseline and 6 months after rescue intervention. Genotypic HIV-1 resistance test and virtual phenotype were assessed at baseline. RESULTS: A total of 33 children met the inclusion criteria and were included in the analysis. The median viral load (VL) and median percentage of CD4+ at baseline was 4.0 HIV-RNA log copies/ml and 23.0% respectively. The median duration that children were taking the new rescue regimen was 24.3 weeks (23.8-30.6). Overall, 47% of the 33 children achieved virological response at 24 weeks. When we compared the group of children who achieved virological response with those who did not, we found out that mean number of PI related mutations among the group of responders was 3.8 vs. 5.4 (p = 0.115). Moreover, the mean number of susceptible drugs according to virtual phenotype clinical cut-off for maximal virologic response was 1.7 vs. 0.8 and mean number of susceptible drugs according to virtual phenotype cut-off for minimal virlologic response was 2.7 vs. 1.3 (p < 0.01 in all cases). Eighteen children were rescued with a regimen containing a boosted-PI and virological response was significantly higher in those subjects compared with the others (61.1% vs. 28.6%, p < 0.01). CONCLUSION: Salvage treatment containing ritonavir boosted-PIs in children with virological failure was very efficient. The use of new tools as virtual phenotype could help to improve virologic success in pediatric population. [Abstract/Link to Full Text]

Midilli K, Gargili A, Ergonul O, Sengöz G, Ozturk R, Bakar M, Jongejan F
Imported Crimean-Congo hemorrhagic fever cases in Istanbul.
BMC Infect Dis. 2007;754.
We described a series of imported cases of Crimean-Congo Hemorrhagic Fever (CCHF) in Istanbul and investigated the genetic diversity of the virus. All the suspected cases of CCHF, who were applied to the health centers in Istanbul, were screened for CCHF virus (CCHFv) infection by using semi-nested Polymerase Chain Reaction (PCR) following RT-PCR. Simultaneous blood samples were also sent to the national reference laboratory in Ankara for serologic investigation. In 10 out of 91 patients, CCHFv was detected by PCR, and among 9 out of 10, anti-CCHFv IgM antibodies were also positive. Clinical features were characterized by fever, myalgia, and hemorrhage. The levels of liver enzymes, creatinine phosphokinase, and lactate dehydrogenase were elevated, and bleeding markers were prolonged. All the cases were treated with ribavirin. There was no fatal case. All the strains clustered within the same group as other Europe/Turkey isolates. [Abstract/Link to Full Text]

Mertens PL, Stals FS, Steyerberg EW, Richardus JH
Sensitivity and specificity of single IgA and IgG antibody concentrations for early diagnosis of pertussis in adults: an evaluation for outbreak management in public health practice.
BMC Infect Dis. 2007;753.
BACKGROUND: An accurate, practical laboratory test is needed to confirm clinical diagnosis of pertussis in adults during the first 3 symptomatic weeks, when treatment is effective and transmission can be interrupted. METHODS: The sensitivity and specificity of single IgA and IgG levels were assessed in a cohort study of a pertussis epidemic in 99 adults in a closed community. Sensitivities were assessed in the sera of 46 laboratory confirmed clinical pertussis cases during the first 3 weeks. Specificities were calculated in sera of 35 asymptomatic controls without clinical symptoms or laboratory confirmed infections from the same community (internal controls). We compared these specificities with the specificities of single IgA and IgG levels in 4275 external controls from a cross-section of the general Dutch population aged 21-79 years who had not coughed for more than 2 weeks in the past year, and without pertussis diagnoses. The study was done in the Netherlands when whole-cell pertussis vaccine was used in the national vaccination programme. RESULTS: Levels of 24 U/ml for IgA and 27 U/ml for IgG gave sensitivities of 100% and 75%, respectively, in the first 2 weeks, 100% in the third week, and 97% after the fourth week. The levels were reached within 2 days after onset of increase, and remained above these levels for roughly 7.2 and 5.1 months, respectively. Specificity was 82% for IgA and 89% for IgG in the internal controls and 90% in the external controls, respectively. CONCLUSION: We suggest levels of 24 U/ml for IgA level and 27 U/ml (= 27 International Units (IU)/ml) for IgG as sensitive, specific, and practical for laboratory confirmation of clinical pertussis in adults in the first 3 weeks of outbreak management. [Abstract/Link to Full Text]

Assadian O, Askarian M, Stadler M, Shaghaghian S
Prevalence of vancomycin-resistant enterococci colonization and its risk factors in chronic hemodialysis patients in Shiraz, Iran.
BMC Infect Dis. 2007;752.
BACKGROUND: Vancomycin-resistant entrococci (VRE) are increasing in prevalence at many institutions, and are often reported in dialysis patients. The aim of this cross-sectional prevalence study was to determine the prevalence and risk factors of VRE colonization in chronic hemodialysis patients in two hemodialysis centers in Shiraz, Iran. METHODS: Rectal swabs were obtained from all consenting patients and were streaked on the surface of Cephalexin-aztreonam-arabinose agar (CAA) and incubated at 37 degrees C in air for 24 h. The vancomycin susceptibility of each isolate was confirmed by disk susceptibility testing. The MICs of vancomycin and teicoplanin were confirmed by the E test. To identify risk factors, a questionnaire was completed for all the studied patients and the data of VRE positive and negative groups were compared using Man-Withney U test for continues data and the Fisher exact test for categorical data. RESULTS: Of 146 patients investigated, 9 (6.2%) were positive for VRE. All VRE strains were genotypically distinguishable. Risk factors for a VRE-positive culture were "antimicrobial receipt within 2 months before culture" (P = 0.003) and "hospitalization during previous year" (P = 0.016). CONCLUSION: VRE colonization is an under-recognized problem among chronic dialysis patients in Iran. VRE colonization is associated with antibiotic consumption and hospitalization. [Abstract/Link to Full Text]

Lo WT, Lin WJ, Tseng MH, Lu JJ, Lee SY, Chu ML, Wang CC
Nasal carriage of a single clone of community-acquired methicillin-resistant Staphylococcus aureus among kindergarten attendees in northern Taiwan.
BMC Infect Dis. 2007;751.
BACKGROUND: To evaluate the prevalence and microbiological characterization of community-acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage in a kindergarten. METHODS: Point prevalence study. Nasal swabs were collected from healthy children younger than 7 years of age who were attending a kindergarten in Taipei, Taiwan. A parent questionnaire regarding MRSA risk factors was administered simultaneously. All CA-MRSA colonization isolates were archived for subsequent antimicrobial susceptibility and molecular typing. RESULTS: Of the 68 children who participated in the study, 17 (25%) had S. aureus isolated from nasal swabs. Nine (13.2%) of the 68 children had CA-MRSA carriage, and none of them had any identified risk factors. Antimicrobial susceptibility testing revealed all of the 9 CA-MRSA colonization isolates had uniformly high resistance (100%) to both clindamycin and erythromycin, the macrolide-lincosamide-streptogramin-constitutive phenotype and the ermB gene. Pulsed-field gel electrophoresis revealed 8 (88.9%) of 9 CA-MRSA colonization isolates were genetically related and multilocus sequence typing revealed all isolates had sequence type 59. All of the colonization isolates carried the staphylococcal cassette chromosome mec type IV, but none were positive for the Panton-Valentine leukocidin genes. CONCLUSION: The results of this study suggest that a single predominant CA-MRSA colonization strain featuring high clindamycin resistance circulated in this kindergarten. Additionally, due to the established transmissibility of colonization isolates, the high prevalence of nasal carriage of CA-MRSA among healthy attendees in kindergartens may indicate the accelerated spread of CA-MRSA in the community. [Abstract/Link to Full Text]

Ng MW, Zhou G, Chong WP, Lee LW, Law HK, Zhang H, Wong WH, Fok SF, Zhai Y, Yung RW, Chow EY, Au KL, Chan EY, Lim W, Peiris JS, He F, Lau YL
The association of RANTES polymorphism with severe acute respiratory syndrome in Hong Kong and Beijing Chinese.
BMC Infect Dis. 2007;750.
BACKGROUND: Chemokines play important roles in inflammation and antiviral action. We examined whether polymorphisms of RANTES, IP-10 and Mig affect the susceptibility to and outcome of severe acute respiratory syndrome (SARS). METHODS: We tested the polymorphisms of RANTES, IP-10 and Mig for their associations with SARS in 495 Hong Kong Chinese SARS patients and 578 controls. Then we tried to confirm the results in 356 Beijing Chinese SARS patients and 367 controls. RESULTS: RANTES -28 G allele was associated with SARS susceptibility in Hong Kong Chinese (P < 0.0001, OR = 2.80, 95%CI:2.11-3.71). Individuals with RANTES -28 CG and GG genotypes had a 3.28-fold (95%CI:2.32-4.64) and 3.06-fold (95%CI:1.47-6.39) increased risk of developing SARS respectively (P < 0.0001). This -28 G allele conferred risk of death in a gene-dosage dependent manner (P = 0.014) with CG and GG individuals having a 2.12-fold (95% CI: 1.11-4.06) and 4.01-fold (95% CI: 1.30-12.4) increased risk. For the replication of RANTES data in Beijing Chinese, the -28 G allele was not associated with susceptibility to SARS. However, -28 CG (OR = 4.27, 95%CI:1.64-11.1) and GG (OR = 3.34, 95%CI:0.37-30.7) were associated with admission to intensive care units or death due to SARS (P = 0.011). CONCLUSION: RANTES -28 G allele plays a role in the pathogenesis of SARS. [Abstract/Link to Full Text]

Smedley RC, Patterson JS, Miller R, Massey JP, Wise AG, Maes RK, Wu P, Kaneene JB, Kiupel M
Sensitivity and specificity of monoclonal and polyclonal immunohistochemical staining for West Nile virus in various organs from American crows (Corvus brachyrhynchos).
BMC Infect Dis. 2007;749.
BACKGROUND: Based on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV) detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos). Our objectives were to determine 1) the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC) diagnosis of WNV infection in free-ranging American crows, 2) which organ(s) is/are most suitable for IHC-based diagnosis of WNV, and 3) how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection. METHODS: Various combinations, depending on tissue availability, of sections of heart, kidney, brain, liver, lung, spleen, and small intestine from 85 free-ranging American crows were stained using a rabbit-polyclonal anti-WNV antibody as well as a monoclonal antibody directed against an epitope on Domain III of the E protein of WNV. The staining intensity and the extent of staining were determined for each organ using both antibodies. Real-time RT-PCR on formalin-fixed paraffin-embedded tissues from all 85 crows was performed. RESULTS: Forty-three crows were IHC-positive in at least one of the examined organs with the polyclonal antibody, and of these, only 31 were positive when IHC was performed with the monoclonal antibody. Real-time RT-PCR amplified WNV-specific sequences from tissue extracts of the same 43 crows that were IHC-positive using the polyclonal antibody. All other 42 crows tested negative for WNV with real-time PCR and IHC staining. Both antibodies had a test specificity of 100% when compared to PCR results. The test sensitivity of monoclonal antibody-based IHC staining was only 72%, compared to 100% when using the polyclonal antibody. CONCLUSION: The most sensitive, readily identified, positively staining organs for IHC are the kidney, liver, lung, spleen, and small intestine. Real-time RT-PCR and IHC staining using a polyclonal antibody on sections of these tissues are highly sensitive diagnostic tests for the detection of WNV in formalin-fixed tissues of American crows. [Abstract/Link to Full Text]

Jha HC, Vardhan H, Gupta R, Varma R, Prasad J, Mittal A
Higher incidence of persistent chronic infection of Chlamydia pneumoniae among coronary artery disease patients in India is a cause of concern.
BMC Infect Dis. 2007;748.
BACKGROUND: There is growing evidence that Chlamydia pneumoniae may be involved in the pathogenesis of atherosclerosis, as several studies have demonstrated the presence of the organism in atherosclerotic lesions. C. pneumoniae infections, which are especially persistent infections, have been difficult to diagnose either by serological methods or isolation of the organism from the tissue. Nucleic Acid Amplification tests (NAATs) has emerged as an important method for detecting C. pneumoniae. Inspite of high prevalence of C. pneumoniae specific antibodies in coronary heart disease patients, direct detection of C. pneumoniae in circulating blood of coronary artery disease (CAD) patients by sensitive nucleic acid amplification tests nested PCR (nPCR), multiplex PCR (mPCR) has not been carried out is required. Further correlation of the presence of C. pneumoniae in blood of CAD patients with C. pneumoniae specific IgA and IgG antibodies, which may indicative of the status of infection with the progression of atherosclerosis. This will help in order to prepare strategies for the antibiotic intervention to avoid the progression towards CAD. METHODS: Venous blood was obtained from 91 CAD patients and 46 healthy controls. Nucleic acid amplification tests viz. nested-, semi-nested- and multiplex PCR were used for detection of C. pneumoniae. ELISA carried out prevalence of C. pneumoniae specific IgG and IgA antibodies. RESULTS: 29.67% (27/91) patients were positive for C. pneumoniae using nested PCR. The sensitivity and specificity of semi-nested and multiplex PCR were 37.03%, 96.96% and 22.22%, 100% with respect to nested PCR. Positive nPCR patients were compared with presence of C. pneumoniae specific IgA, IgA+IgG and IgG antibodies. Among 27 (29.67%) nPCR C. pneumoniae positive CAD patients, 11(12%) were IgA positive, 13(14.2%) were IgA+IgG positive and only1 (1.1%) was IgG positive. A significant presence of C. pneumoniae was detected in heavy smokers, non-alcoholics and with family histories of diabetes and blood pressure group of CAD patients by nPCR. CONCLUSION: The results indicate synergistic association of C. pneumoniae infection and development of CAD with other risk factors. We also detected increased positivity for C. pneumoniae IgA than IgG in nPCR positive CAD patients. Positive nPCR findings in conjunction with persisting high C. pneumoniae specific antibody strongly suggest an ongoing infection. [Abstract/Link to Full Text]

Zuckerman IH, Perencevich EN, Harris AD
Concurrent acute illness and comorbid conditions poorly predict antibiotic use in upper respiratory tract infections: a cross-sectional analysis.
BMC Infect Dis. 2007;747.
BACKGROUND: Inappropriate antibiotic use promotes resistance. Antibiotics are generally not indicated for upper respiratory infections (URIs). Our objectives were to describe patterns of URI treatment and to identify patient and provider factors associated with antibiotic use for URIs. METHODS: This study was a cross-sectional analysis of medical and pharmacy claims data from the Pennsylvania Medicaid fee-for-service program database. We identified Pennsylvania Medicaid recipients with a URI office visit over a one-year period. Our outcome variable was antibiotic use within seven days after the URI visit. Study variables included URI type and presence of concurrent acute illnesses and chronic conditions. We considered the associations of each study variable with antibiotic use in a logistic regression model, stratifying by age group and adjusting for confounders. RESULTS: Among 69,936 recipients with URI, 35,786 (51.2%) received an antibiotic. In all age groups, acute sinusitis, chronic sinusitis, otitis, URI type and season were associated with antibiotic use. Except for the oldest group, physician specialty and streptococcal pharyngitis were associated with antibiotic use. History of chronic conditions was not associated with antibiotic use in any age group. In all age groups, concurrent acute illnesses and history of chronic conditions had only had fair to poor ability to distinguish patients who received an antibiotic from patients who did not. CONCLUSION: Antibiotic prevalence for URIs was high, indicating that potentially inappropriate antibiotic utilization is occurring. Our data suggest that demographic and clinical factors are associated with antibiotic use, but additional reasons remain unexplained. Insight regarding reasons for antibiotic prescribing is needed to develop interventions to address the growing problem of antibiotic resistance. [Abstract/Link to Full Text]

Belfiori R, Terenzi A, Marchesini L, Repetto A
Absidia Corymbifera in an immune competent accident victim with multiple abdominal injuries: case report.
BMC Infect Dis. 2007;746.
BACKGROUND: We report a case of mucormycosis in a healthy 17-year-old accident victim with multiple abdominal injuries which was caused by infection with Absidia Corymbifera, a ubiquitous saphrophyte in the ground. CASE PRESENTATION: The patient was admitted to hospital with massive abdominal trauma. During an 8-hour emergency operation he received transfusions of compacted red blood cells, plasma, platelets and hemagel. He developed a crush syndrome with acute renal failure, resolved with extra-corporeal dialysis and had to undergo splenectomy because of spleen hematoma. As wound secretion and central venous catheter (CVC) blood cultures and drainage fluid were positive for Enterococcus Faecium, Providentia Rettgeri, Hafnia Alvei and Candida Albicans, tecoplanin, metronidazole, imipenem, and flucanozole were administered.Although the CVC was changed high fever persisted and discharge continued from the large abdominal wound. Repeated tampons in different sections and wound secretion smears were positive for A. corymbifera. Flucanozole was stopped and liposomal amphotericin (Ambisome; 5 mg/Kg i.v.) given for over 3 months.The patient improved; fever gradually disappeared. After 8 days, tampons and wound secretion smears were negative for A. corymbifera. No other fungal infections developed. Drainage fluid was later positive for tecoplanin-resistant E. faecium and Pseudomonas Aeroginosa responding only to meropenem and ciprofloxacin. Abdominal computerized tomography visualized fluid accumulation around the iliac-femoral bypass. Abcess was ruled out when scintigraphy showed no tracer uptake. The lesion was drained. Drainage fluid cultures were negative for bacteria and fungi. Fluid accumulation gradually disappeared with prolonged antibiotic and antifungal therapy.One year after the accident the patient is in good health, with normal quality of life. CONCLUSION: Successful outcome was due to early, specific antifungal therapy, at sufficiently high dosage which was prolonged for an adequate period of time. Early diagnosis of mucormycosis is essential for efficacious anti-fungal treatment and prevention of irreversible spread of mucormycosis to vital organs. It presupposes awareness that A. corymbifera infection can develop in healthy individuals who are stressed and traumatized through skin-ground contact in accidents. [Abstract/Link to Full Text]

Daeschlein G, Krüger WH, Selepko C, Rochow M, Dölken G, Kramer A
Hygienic safety of reusable tap water filters (Germlyser) with an operating time of 4 or 8 weeks in a haematological oncology transplantation unit.
BMC Infect Dis. 2007;745.
BACKGROUND: Microbial safe tap water is crucial for the safety of immunosuppressed patients. METHODS: To evaluate the suitability of new, reusable point-of-use filters (Germlyser, Aquafree GmbH, Hamburg, Germany), three variations of a reusable filter with the same filter principle but with different outlets (with and without silver) and inner surface coating of the filter encasements (with and without nano-crystalline silver) were tested. The filter efficacy was monitored over 1, 4 and 8 weeks operating time in a haematological oncology transplantation unit equipped with 18 water outlets (12 taps, 6 showers). RESULTS: The filtered water fulfilled the requirements of absence of pathogens over time. From 348 samples, 8 samples (2.3%) exceeded 100 cfu/ml (no sample > or = 500 cfu/ml). As no reprocessed filter exhibited 100% filter efficacy in the final quality control after each reprocessing, these contaminations could be explained by retrograde contamination during use. CONCLUSION: As a consequence of the study, the manufacturer recommends changing filters after 4 weeks in high risk areas and after 8 weeks in moderate infectious risk areas, together with routine weekly alcohol-based surface disinfection and additionally in case of visible contamination. The filter efficacy of the 3 filters types did not differ significantly regarding total bacterial counts. Manual reprocessing proved to be insufficient. Using a validated reprocessing in a washer/disinfector with alkaline, acid treatment and thermic disinfection, the filters were effectively reprocessable and now provide tap water meeting the German drinking water regulations as well as the WHO guidelines, including absence of pathogens. [Abstract/Link to Full Text]

Forgie SE, Robinson JL
Pediatric malignancies presenting as a possible infectious disease.
BMC Infect Dis. 2007;744.
BACKGROUND: The clinical, laboratory, and radiological features of malignancy can overlap with those of infection. The purpose of this study was to determine the findings in children who were initially thought to have an infectious disease but ultimately proved to have a malignancy. METHODS: The database of patients diagnosed with a malignancy in the Northern Alberta Children's Cancer Program (NACCP) January 1, 1993 to December 31, 2003 was merged with the database of inpatients referred to the infectious diseases service at the Stollery Children's Hospital and charts were reviewed on all patients referred to the infectious diseases consult service prior to the diagnosis of malignancy. RESULTS: An infectious diseases consultation for diagnosis was requested in 21 of 561 patients prior to the confirmation of malignancy, and 3 of these 21 patients had both infection and malignancy (leukemia (N = 13), lymphoma (N = 3), rhabdomyosarcoma (N = 1), Langerhan's cell histiocytosis (N = 1), fibrous histicocytosis (N = 1), ependymoma (N = 1), and neuroblastoma (N = 1). The most common reason for infectious diseases consultation was suspected muskuloskeletal infection (N = 9). A palpable or radiographically enlarged spleen was noted in 11 patients (52%). All but 2 patients had abnormal hematologic parameters while an elevated lactate dehydrogenase (LDH) occurred in 10 patients (48%). Delay of diagnosis because of investigation or therapy for an infectious disease occurred in only 2 patients. CONCLUSION: It is not common for treatment of pediatric malignancies to be delayed because infection is thought to be the primary diagnosis. However, pediatric infectious diseases physicians should consider malignancy in the differential diagnosis when they see patients with fever and bone pain, unexplained splenomegaly or abnormal complete blood cell counts. Other clues may include hepatomegaly or elevated LDH. [Abstract/Link to Full Text]

Blomberg B, Manji KP, Urassa WK, Tamim BS, Mwakagile DS, Jureen R, Msangi V, Tellevik MG, Holberg-Petersen M, Harthug S, Maselle SY, Langeland N
Antimicrobial resistance predicts death in Tanzanian children with bloodstream infections: a prospective cohort study.
BMC Infect Dis. 2007;743.
BACKGROUND: Bloodstream infection is a common cause of hospitalization, morbidity and death in children. The impact of antimicrobial resistance and HIV infection on outcome is not firmly established. METHODS: We assessed the incidence of bloodstream infection and risk factors for fatal outcome in a prospective cohort study of 1828 consecutive admissions of children aged zero to seven years with signs of systemic infection. Blood was obtained for culture, malaria microscopy, HIV antibody test and, when necessary, HIV PCR. We recorded data on clinical features, underlying diseases, antimicrobial drug use and patients' outcome. RESULTS: The incidence of laboratory-confirmed bloodstream infection was 13.9% (255/1828) of admissions, despite two thirds of the study population having received antimicrobial therapy prior to blood culture. The most frequent isolates were klebsiella, salmonellae, Escherichia coli, enterococci and Staphylococcus aureus. Furthermore, 21.6% had malaria and 16.8% HIV infection. One third (34.9%) of the children with laboratory-confirmed bloodstream infection died. The mortality rate from Gram-negative bloodstream infection (43.5%) was more than double that of malaria (20.2%) and Gram-positive bloodstream infection (16.7%). Significant risk factors for death by logistic regression modeling were inappropriate treatment due to antimicrobial resistance, HIV infection, other underlying infectious diseases, malnutrition and bloodstream infection caused by Enterobacteriaceae, other Gram-negatives and candida. CONCLUSION: Bloodstream infection was less common than malaria, but caused more deaths. The frequent use of antimicrobials prior to blood culture may have hampered the detection of organisms susceptible to commonly used antimicrobials, including pneumococci, and thus the study probably underestimates the incidence of bloodstream infection. The finding that antimicrobial resistance, HIV-infection and malnutrition predict fatal outcome calls for renewed efforts to curb the further emergence of resistance, improve HIV care and nutrition for children. [Abstract/Link to Full Text]

Marra AR, Edmond MB, Wenzel RP, Bearman GM
Hospital-acquired Clostridium difficile-associated disease in the intensive care unit setting: epidemiology, clinical course and outcome.
BMC Infect Dis. 2007;742.
BACKGROUND: Clostridium difficile-associated disease (CDAD) is a serious nosocomial infection, however few studies have assessed CDAD outcome in the intensive care unit (ICU). We evaluated the epidemiology, clinical course and outcome of hospital-acquired CDAD in the critical care setting. METHODS: We performed a historical cohort study on 58 adults with a positive C. difficile cytotoxin assay result occurring in intensive care units. RESULTS: Sixty-two percent of patients had concurrent infections, 50% of which were bloodstream infections. The most frequently prescribed antimicrobials prior to CDAD were anti-anaerobic agents (60.3%). Septic shock occurred in 32.8% of CDAD patients. The in-hospital mortality was 27.6%. Univariate analysis revealed that SOFA score, at least one organ failure and age were predictors of mortality. Charlson score >/=3, gender, concurrent infection, and number of days with diarrhea before a positive C. difficile toxin assay were not significant predictors of mortality on univariate analysis. Independent predictors for death were SOFA score at infection onset (per 1-point increment, OR 1.40; CI95 1.13-1.75) and age (per 1-year increment, OR 1.10; CI95 1.02-1.19). CONCLUSION: In ICU patients with CDAD, advanced age and increased severity of illness at the onset of infection, as measured by the SOFA score, are independent predictors of death. [Abstract/Link to Full Text]

Lawn SD, Myer L, Bangani N, Vogt M, Wood R
Plasma levels of soluble urokinase-type plasminogen activator receptor (suPAR) and early mortality risk among patients enrolling for antiretroviral treatment in South Africa.
BMC Infect Dis. 2007;741.
BACKGROUND: Serum concentrations of soluble urokinase-type plasminogen activator receptor (suPAR) have a strong independent association with HIV-1-related mortality. The practical utility of plasma suPAR in assessing short-term all-cause mortality risk was evaluated in patients with advanced immunodeficiency enrolling in an antiretroviral treatment (ART) programme in South Africa. METHODS: An enzyme-linked immunosorbent assay (ELISA) was used to measure plasma concentrations of suPAR in patients at the time of enrollment to the ART programme. The association between plasma suPAR concentrations, baseline patient characteristics and cohort outcomes after 4 months of ART were determined. RESULTS: Patients (n = 293, 70% female) had a median age of 33 years and were followed up for a median of 5 months from enrollment. The median CD4 cell count was 47 cells/mul (IQR = 22-72) and 38% of patients had WHO stage 4 disease. 218 (74%) patients remained alive after 4 months of ART; 39 (13%) died and 36 (12%) were lost to the programme for other reasons. Patients who died had significantly higher plasma suPAR concentrations compared to those who either survived (P < 0.01) or left the programme for other reasons (P < 0.043). In multivariate analysis, higher log10 suPAR concentrations were significantly associated with lower CD4 cell counts, WHO clinical stage 4 disease and male sex. In multivariate analysis to identify factors associated with death, log10 suPAR concentration was the most strongly associated variable (P < 0.001). However, examination of sensitivity and specificity characteristics using receiver operating characteristic (ROC) analysis revealed that results from this assay did not have a discriminatory cut-point to provide clinically useful information. CONCLUSION: Plasma suPAR concentration was the strongest independent predictor of short-term mortality risk among patients with advanced immunodeficiency enrolling in this ART programme. However, lack of a discriminatory threshold did not permit this marker to be used to triage patients according to short-term mortality risk. [Abstract/Link to Full Text]

Vagace JM, Sanz-Rodriguez C, Casado MS, Alonso N, Garcia-Dominguez M, de la Llana FG, Zarallo L, Fajardo M, Bajo R
Resolution of disseminated fusariosis in a child with acute leukemia treated with combined antifungal therapy: a case report.
BMC Infect Dis. 2007;740.
BACKGROUND: Fusarium spp. is being isolated with increasing frequency as a pathogen in oncohematologic patients. Caspofungin and amphotericin B have been reported to have synergistic activity against Fusarium spp. CASE PRESENTATION: We herein report a case of disseminated fusariosis diagnosed by chest CT scan and positive blood cultures to Fusarium spp. Because the patient's clinical condition deteriorated, CRP levels increased, and blood cultures continued to yield Fusarium spp. despite liposomal amphotericin B monotherapy up to 5 mg/kg daily, treatment with caspofungin was added. Within 2 weeks of onset of combined antifungal therapy, the chest CT scan demonstrated a progressive resolution of the pulmonary lesions. Upon discontinuation of intravenous antifungals, the patient received suppressive therapy with oral voriconazole. Three months later, a chest CT scan showed no abnormalities. Twenty-five months after discontinuation of all antifungal therapy, the patient remains in complete remission of her neoplastic disease with no signs of clinical activity of the Fusarium infection. CONCLUSION: This is the first description of successful treatment of disseminated fusariosis in a pediatric patient with acute lymphoblastic leukemia with caspofungin and amphotericin B followed by oral suppressive therapy with voriconazole. [Abstract/Link to Full Text]

Kisenge PR, Hawkins AT, Maro VP, McHele JP, Swai NS, Mueller A, Houpt ER
Low CD4 count plus coma predicts cryptococcal meningitis in Tanzania.
BMC Infect Dis. 2007;739.
BACKGROUND: Largely due to the lack of diagnostic reagents, the prevalence and clinical presentation of cryptococcal meningitis in Tanzania is poorly understood. This in turn is limiting the impact of increased fluconazole availability. METHODS: We evaluated a cohort of 149 consecutive HIV-infected adult inpatients presenting with headache or altered mental status for clinical features, CD4 count, cryptococcal infection, and outcome. Cryptococcal meningitis was diagnosed via India ink and latex agglutination assay of CSF (n = 24 and 40 positive, respectively). Associations between cryptococcal meningitis and clinical features were evaluated by t-test. The sensitivity, specificity, and positive likelihood ratio of such features were determined. RESULTS: Cryptococcal meningitis was associated with confusion, social withdrawal, seizures, fever, tachycardia, meningismus, oral candidiasis, and low Glasgow coma scales and CD4 count. CD4 count < 100/mul provided the highest sensitivity for the diagnosis (93%), coma (Glasgow coma scale < or = 8) provided the highest specificity (84%), and the combination provided the highest positive likelihood ratio (3.8). All cryptococcal meningitis patients were initiated on 800 milligrams of fluconazole daily and 50% survived to discharge, however no clinical or laboratory findings correlated with prognosis. CONCLUSION: Cryptococcal meningitis is common among Tanzanian HIV inpatients presenting with headache or altered mental status. Purely clinical features are insensitive for establishing the diagnosis or prognosis. We advocate expanding laboratory capacity for cryptococcal antigen testing to maximize survival. [Abstract/Link to Full Text]

Ritz R, Roser F, Morgalla M, Dietz K, Tatagiba M, Will BE
Do antibiotic-impregnated shunts in hydrocephalus therapy reduce the risk of infection? An observational study in 258 patients.
BMC Infect Dis. 2007;738.
BACKGROUND: Shunt infection in hydrocephalus patients is a severe, even life-threatening complication. Antibiotic-impregnated shunts (AIS) have been developed in an attempt to reduce rate of shunt infection. The study was performed to analyze if AIS can diminish the rate of shunt infection. The pathogenic nature of shunt infection in patients with AIS systems and those without antibiotic impregnated shunts (non-AIS) was compared. METHODS: Over a period of 24 months in the Department of Neurosurgery at University Hospital of Tübingen shunt surgery was performed in 258 patients. In 86 patients AIS systems were implanted. Shunt catheters were commercially impregnated with clindamycin and rifampicin. Analysis of the clinical data included sex, age, classification of hydrocephalus, shunt types and risk factors for shunt infection [age (< 1 year and > 80 years), prematurely born patients, external ventricular drainage, former shunt infection, former systemic infection, disturbance of consciousness, former radiation-/chemotherapy]. Infection rates and underlying bacterial pathogens of patients with AIS were compared to patients with implanted non-AIS systems (172 patients). RESULTS: AIS and non-AIS patients did not differ in sex, etiology of hydrocephalus and the shunt type. In the AIS group 72 out of 86 patients had at least one risk factor (83.7 %), compared to 126 patients in the non-AIS group (73.3 %). There was no significant difference between the two groups (p = 0.0629; Fisher's exact test). In patients with no risk factors, only one patient with non-AIS suffered from shunt infection. In patients with one or more risk factors the rate for shunt infection was 7.14 % in patients with non-AIS and 6.94 % in patients with AIS. Former shunt infection (p = 0.0124) was related to higher risk for shunt infection. The use of AIS had therefore no significant advantage (p = 0.8611; multiple logistic regression).Significantly related to a shunt infection was the number of shunt surgeries. 190 interventions in the AIS group (2.21 interventions per patient) and 408 in the non-AIS group (2.37 interventions per patient) had been performed (p = 0.3063; Wilcoxon). There was no shunt infection in the group of patients on whom only one shunt surgery was performed. In patients with at least two shunt surgeries the infection rate was 9%. The infection rate in AIS patients was 5/52 (9.6 %) and in the non-AIS 10/114 (8.77 %), (p = 1.0; Fisher's exact test). Staphylococcus epidermidis was the most frequent pathogen for shunt infection. Fourteen out of 15 infections occurred within the first 6 months of surgery. The most frequent pathogen for shunt infection was S. epidermidis. No toxic or allergic complications were seen using the AIS shunt systems. The presented data show a remarkably low infection rate of 5.8 % in the non-AIS group compared to other studies which demonstrated a significant decrease in the infection rate by AIS. CONCLUSION: AIS did not significantly reduce shunt infection in hydrocephalus patients in the presented study. In the AIS group three patients suffered from shunt infections caused by skin ulceration or neurosurgical procedures with exposure of the cerebrospinal liquor after shunt implantation. AIS was not developed to prevent infection in such cases, therefore an advantage of AIS can not be excluded. In view of the presented data and the small number of reported studies a prospective randomized multicenter study is required. [Abstract/Link to Full Text]

Marra CM, Maxwell CL, Collier AC, Robertson KR, Imrie A
Interpreting cerebrospinal fluid pleocytosis in HIV in the era of potent antiretroviral therapy.
BMC Infect Dis. 2007;737.
BACKGROUND: Cerebrospinal fluid (CSF) pleocytosis may be seen in asymptomatic HIV-infected individuals. This finding complicates interpretation of CSF abnormalities when such individuals are evaluated for other central nervous system infections. The goal of this study was to determine the relationship between CSF pleocytosis, central nervous system (CNS) antiretroviral penetration, adherence to antiretroviral medication regimens, neurological symptoms and performance on neuropsychological tests. METHODS: Clinically stable HIV-infected individuals at any peripheral blood CD4+ T cell count or any plasma viral load were asked to attend study visits at entry and every 6 months thereafter for at least one year. At each visit, they underwent a standardized neurological and medication history; neurological examination; a brief neuropsychological test battery: venipuncture; lumbar puncture; and assessment of medication adherence. Generalized estimating equations (GEE) were used to assess the relationships between CSF pleocytosis and other variables. RESULTS: CSF pleocytosis was independently and significantly related to lack of current antiretroviral use (OR 5.9, 95% CI 1.8-18.6, p = 0.003), CD4 count > 200/ul (OR 23.4, 95% CI 3.1-177.3, p = 0.002) and detectable plasma HIV RNA (OR 3.3, 95% CI 1.1-9.4, p = 0.03). At visits where antiretrovirals were used, and taking into account detectable plasma HIV RNA, an antiretroviral regimen that contained two or more agents with good CNS penetration conferred a trend toward lower odds of CSF pleocytosis (OR 0.45, 95% CI 0.18-1.12, p = 0.087). CONCLUSION: CSF pleocytosis is a characteristic of HIV disease that varies significantly with easily identifiable clinical and laboratory features. Use of antiretroviral agents decreases the odds of pleocytosis. This association may be stronger when the regimen contains two or more agents with good CNS penetration. [Abstract/Link to Full Text]

Drapeau CM, Angeletti C, Festa A, Petrosillo N
Role of previous hospitalization in clinically-significant MRSA infection among HIV-infected inpatients: results of a case-control study.
BMC Infect Dis. 2007;736.
BACKGROUND: HIV-infected subjects have high incidence rates of Staphylococcus aureus infections, with both methicillin-susceptible and methicillin-resistant (MRSA) strains. Possible explanations could include the high burden of colonization, the behavioral risk factors, and the frequent exposures to health care facilities of HIV-infected patients. The purpose of the study was to assess the risk factors for clinically- significant methicillin-resistant Staphylococcus aureus (CS-MRSA) infections in HIV-infected patients admitted to Infectious Diseases Units. METHODS: From January 1, 2002 to December 31, 2005, we conducted a retrospective case-control (1:2) study. We identified all the cases of CS-MRSA infections in HIV-infected patients admitted to the National Institute for Infectious Diseases (INMI) "Lazzaro Spallanzani" in the 4-year study period. A conditional logistic regression model was used to identify risk factors for CS-MRSA infection. RESULTS: We found 27 CS-MRSA infections, i.e. 0.9 CS-MRSA infections per 100 HIV-infected individuals cared for in our Institute. At multivariate analysis, independent predictors of CS-MRSA infection were cumulative hospital stay, invasive procedures in the previous year, and low CD4 cell count. Particularly, the risk for CS-MRSA increased by 14% per an increase of 5 days hospitalization in the previous year. Finally, we identified a low frequency of community-acquired MRSA infections (only 1 of 27; 3.7%) among HIV-infected patients. CONCLUSION: Clinicians should be aware of the risk for CS-MRSA infection in the clinical management of HIV-infected patients, especially in those patients with a low CD4 cell count, longer previous hospital stay, and previous invasive procedures. [Abstract/Link to Full Text]

van Gageldonk-Lafeber AB, van der Sande MA, Heijnen ML, Peeters MF, Bartelds AI, Wilbrink B
Risk factors for acute respiratory tract infections in general practitioner patients in The Netherlands: a case-control study.
BMC Infect Dis. 2007;735.
BACKGROUND: Acute respiratory tract infections (ARTI) are an important public health problem. Improved identification of risk factors might enable targeted intervention. Therefore we carried out a case-control study with the aim of identifying environmental risk factors for ARTI consultations in the Dutch general population. METHODS: A subset of patients visiting their GP in the period of 2000-2003 with an ARTI (cases) and age-matched controls (visiting for other complaints) were included in a case-control study. They were asked to complete a questionnaire about potential risk factors. Conditional logistic regression was used to calculate odds ratio's (OR) and 95% confidence intervals (CI) to estimate the independent effect of potential risk factors. RESULTS: A total of 493 matched pairs of case and control subjects were enrolled. Exposure to persons with respiratory complaints, both inside and outside the household, was found to be an independent risk factor for visiting a GP with an ARTI (respectively ORadj = 1.9 and ORadj = 3.7). Participants exposed to dampness or mould at home (ORadj=0.5) were significantly less likely to visit their GP. In accordance with the general risk of consultations for ARTI, participants with a laboratory-confirmed ARTI who were exposed to persons with respiratory complaints outside the household were also significantly more likely to visit their GP (ORadj=2.5). CONCLUSION: This study confirmed that heterogeneity in the general population as well as in pathogens causing ARTI makes it complicated to detect associations between potential risk factors and respiratory infections. Whereas it may be difficult to intervene on the risk posed by exposure to persons with respiratory complaints, transmission of ARTI in the general population might be reduced by improved hygienic conditions. [Abstract/Link to Full Text]

Conen A, Weisser M, Tsakiris DA, Siegemund M
Failure of recombinant factor VIIa in a patient with severe polymicrobial sepsis and postoperative uncontrolled intraabdominal bleeding.
BMC Infect Dis. 2007;734.
BACKGROUND: This report discusses a case of unsuccessful treatment with recombinant factor VIIa (rFVIIa) in off-label use. The need for international guidelines concerning the off-label use of rFVIIa is outlined as well as the need for methods to control the efficacy of rFVIIa objectively. CASE PRESENTATION: 54 year old male with severe polymicrobial sepsis due to a perforated diverticulitis of the sigmoid colon and consecutive overt disseminated intravascular coagulation. He suffered severe intraabdominal bleeding after abdominal surgery despite conventional haemostatic support. Repeated applications of factor VIIa temporarily improved coagulation essays but did not stop clinical bleeding. The patient died in multiorgan failure due to septic and haemorrhagic shock. CONCLUSION: Off-label use of rFVIIa could result in more side effects than could be expected from literature because of a publication bias. However for most off-label applications large prospective, randomised and controlled trials to confirm the positive findings are missing. For the future, not only guidelines concerning the off-label use of rFVIIa are urgently needed but also guidelines for monitoring the efficacy of rFVIIa. [Abstract/Link to Full Text]

Matos GI, Covas Cde J, Bittar Rde C, Gomes-Silva A, Marques F, Maniero VC, Amato VS, Oliveira-Neto MP, Mattos Mda S, Pirmez C, Sampaio EP, Moraes MO, Da-Cruz AM
IFNG +874T/A polymorphism is not associated with American tegumentary leishmaniasis susceptibility but can influence Leishmania induced IFN-gamma production.
BMC Infect Dis. 2007;733.
BACKGROUND: Interferon-gamma is a key cytokine in the protective responses against intracellular pathogens. A single nucleotide polymorphism (SNP) located in the first intron of the human IFN-gamma gene can putatively influence the secretion of cytokine with an impact on infection outcome as demonstrated for tuberculosis and other complex diseases. Our aim was to investigate the putative association of IFNG+874T/A SNP with American tegumentary leishmaniasis (ATL) and also the influence of this SNP in the secretion of IFN-gamma in vitro. METHODS: Brazilian ATL patients (78 cutaneous, CL, and 58 mucosal leishmaniasis, ML) and 609 healthy volunteers were evaluated. The genotype of +874 region in the IFN-gamma gene was carried out by Amplification Refractory Mutational System (ARMS-PCR). Leishmania-induced IFN-gamma production on peripheral blood mononuclear cell (PBMC) culture supernatants was assessed by ELISA. RESULTS: There are no differences between +874T/A SNP frequency in cases and controls or in ML versus CL patients. Cutaneous leishmaniasis cases exhibiting AA genotype produced lower levels of IFN-gamma than TA/TT genotypes. In mucosal cases, high and low IFN-gamma producers were clearly demonstrated but no differences in the cytokine production was observed among the IFNG +874T or A carriers. CONCLUSION: Our results suggest that +874T/A polymorphism was not associated with either susceptibility or severity to leishmaniasis. Despite this, IFNG +874T/A SNP could be involved in the pathogenesis of leishmaniasis by influencing the amount of cytokine released by CL patients, although it could not prevent disease development. On the other hand, it is possible that in ML cases, other potential polymorphic regulatory genes such as TNF-alpha and IL-10 are also involved thus interfering with IFN-gamma secretion. [Abstract/Link to Full Text]

Amadoz A, González-Candelas F
epiPATH: an information system for the storage and management of molecular epidemiology data from infectious pathogens.
BMC Infect Dis. 2007;732.
BACKGROUND: Most research scientists working in the fields of molecular epidemiology, population and evolutionary genetics are confronted with the management of large volumes of data. Moreover, the data used in studies of infectious diseases are complex and usually derive from different institutions such as hospitals or laboratories. Since no public database scheme incorporating clinical and epidemiological information about patients and molecular information about pathogens is currently available, we have developed an information system, composed by a main database and a web-based interface, which integrates both types of data and satisfies requirements of good organization, simple accessibility, data security and multi-user support. RESULTS: From the moment a patient arrives to a hospital or health centre until the processing and analysis of molecular sequences obtained from infectious pathogens in the laboratory, lots of information is collected from different sources. We have divided the most relevant data into 12 conceptual modules around which we have organized the database schema. Our schema is very complete and it covers many aspects of sample sources, samples, laboratory processes, molecular sequences, phylogenetics results, clinical tests and results, clinical information, treatments, pathogens, transmissions, outbreaks and bibliographic information. Communication between end-users and the selected Relational Database Management System (RDMS) is carried out by default through a command-line window or through a user-friendly, web-based interface which provides access and management tools for the data. CONCLUSION: epiPATH is an information system for managing clinical and molecular information from infectious diseases. It facilitates daily work related to infectious pathogens and sequences obtained from them. This software is intended for local installation in order to safeguard private data and provides advanced SQL-users the flexibility to adapt it to their needs.The database schema, tool scripts and web-based interface are free software but data stored in our database server are not publicly available. epiPATH is distributed under the terms of GNU General Public License. More details about epiPATH can be found at http://genevo.uv.es/epipath. [Abstract/Link to Full Text]

Canuel M, De Serres G, Duval B, Gilca R, De Wals P, Gilca V
Trends of hepatitis A hospitalization and risk factors in Quebec, Canada, between 1990 and 2003.
BMC Infect Dis. 2007;731.
BACKGROUND: In Canada, targeted vaccination of at risk groups for hepatitis A (HA) is done since the mid 1990s resulting in declining incidence. This study estimated the year and age specific hospitalization rates and distribution of risk factors for HA in Quebec, Canada, between 1990 and 2003. METHODS: Records of patients hospitalized with HA-related diagnostic codes were retrieved from the provincial database. Hospital charts of all deceased cases and a random sample of all other records were reviewed. RESULTS: From 1503 hospitalization records, 573 charts were reviewed including 49 (91%) of the 54 deceased patients. Confirmed acute HA was present in 79% of records where HA was the primary diagnosis, and in 3%-8% of records where HA was a secondary diagnosis. From the total estimated number of hospitalizations, 96% had HA as the primary diagnosis. The hospitalization rate decreased from 1.06 per 100 000 person-years between 1990 and 1997 to 0.36 between 1998 and 2003. During the study period, 54% HA hospitalizations were in 20-39 year-olds. The overall case fatality ratio among hospitalized patients was 1.4%, increasing from 0.4% in those < 40 years old to 12.5% in those > or =60 years. By decreasing order, reported risk factors were travel to HA endemic countries (30%), MSM (18%) and household contacts (11%). CONCLUSION: HA hospitalization rates have been low since 1998 but the cause of this is unclear given the cyclical pattern of HA. Travel to endemic countries remains the most important risk factor and improved control of HA will require better strategies to vaccinate travelers. [Abstract/Link to Full Text]

Gouriet F, Lepidi H, Habib G, Collart F, Raoult D
From cat scratch disease to endocarditis, the possible natural history of Bartonella henselae infection.
BMC Infect Dis. 2007;730.
BACKGROUND: Most patients with infectious endocarditis (IE) due to Bartonella henselae have a history of exposure to cats and pre-existing heart valve lesions. To date, none of the reported patients have had a history of typical cat scratch disease (CSD) which is also a manifestation of infection with B. henselae. CASE PRESENTATION: Here we report the case of a patient who had CSD and six months later developed IE of the mitral valve caused by B. henselae. CONCLUSION: Based on this unique case, we speculate that CSD represents the primary-infection of B. henselae and that IE follows in patients with heart valve lesions. [Abstract/Link to Full Text]

Sader HS, Watters AA, Fritsche TR, Jones RN
Daptomycin antimicrobial activity tested against methicillin-resistant staphylococci and vancomycin-resistant enterococci isolated in European medical centers (2005).
BMC Infect Dis. 2007;729.
BACKGROUND: Daptomycin is a cyclic lipopeptide with potent activity and broad spectrum against Gram-positive bacteria currently used for the treatment of complicated skin and skin structure infections and bacteremia, including right sided endocarditis. We evaluated the in vitro activity of this compound and selected comparator agents tested against clinical strains of staphylococci and enterococci collected in European medical centers in 2005. METHODS: A total of 4,640 strains from 23 medical centers located in 10 European countries, Turkey and Israel (SENTRY Program platform) were tested for susceptibility by reference broth microdilution methods according to Clinical and Laboratory Standards Institute guidelines and interpretative criteria. Mueller-Hinton broth was supplemented to 50 mg/L Ca++ for testing daptomycin. Results for oxacillin (methicillin)-resistant staphylococci and vancomycin-resistant enterococci were analyzed separately. RESULTS: Oxacillin resistance rates among Staphylococcus aureus varied from 2.1% in Sweden to 42.5% in the United Kingdom (UK) and 54.7% in Ireland (29.1% overall), while vancomycin resistance rates varied from 0.0% in France, Sweden and Switzerland to 66.7% in the UK and 71.4% in Ireland among Enterococcus faecium (17.9% overall). All S. aureus strains were inhibited at daptomycin MIC of 1 mg/L (MIC50/90, 0.25/0.5 mg/L; 100.0% susceptible) and only one coagulase-negative staphylococci strain (0.1%) showed an elevated (>1 mg/L) daptomycin MIC value (4 mg/L). Among E. faecalis (MIC50/90, 0.5/1 mg/L; 100% susceptible) the highest daptomycin MIC value was 2 mg/L; while among E. faecium (MIC50/90, 2/4 mg/L; 100% susceptible) the highest MIC result was 4 mg/L. CONCLUSION: Daptomycin showed excellent in vitro activity against staphylococci and enterococci collected in European medical centers in 2005 and resistance to oxacillin, vancomycin or quinupristin/dalfopristin did not compromise its activity overall against these pathogens. Based on these results and those of previous publications, daptomycin appears to be an excellent therapeutic option for serious infections caused by oxacillin-resistant staphylococci and vancomycin-resistant enterococci in Europe. [Abstract/Link to Full Text]

Cesaro S, Giacchino M, Locatelli F, Spiller M, Buldini B, Castellini C, Caselli D, Giraldi E, Tucci F, Tridello G, Rossi MR, Castagnola E
Safety and efficacy of a caspofungin-based combination therapy for treatment of proven or probable aspergillosis in pediatric hematological patients.
BMC Infect Dis. 2007;728.
BACKGROUND: Fungal infections are diagnosed increasingly often in patients affected by hematological diseases and their mortality has remained high. The recent development of new antifungal drugs gives the clinician the possibility to assess the combination of antifungal drugs with in-vitro or in animal-model synergistic effect. METHODS: We analyzed retrospectively the safety and efficacy of caspofungin-based combination therapy in 40 children and adolescents, most of them were being treated for a malignant disease, who developed invasive aspergillosis (IA) between November 2002 and November 2005. RESULTS: Thirteen (32.5%) patients developed IA after hematopoietic stem cell transplantation (HSCT), 13 after primary diagnosis, usually during remission-induction chemotherapy, and 14 after relapse of disease. Severe neutropenia was present in 31 (78%) out of the 40 patients. IA was classified as probable in 20 (50%) and documented in 20 (50%) patients, respectively. A favorable response to antifungal therapy was obtained in 21 patients (53%) and the probability of 100-day survival was 70%. Different, though not significant, 100-day survival was observed according to the timing of diagnosis of IA: 51.9% after HSCT; 71.4% after relapse; and 84.6% after diagnosis of underlying disease, p 0.2. After a median follow-up of 0.7 years, 20 patients are alive (50%). Overall, the combination therapy was well tolerated. In multivariate analysis, the factors that were significantly associated to a better overall survival were favorable response to antifungal therapy, p 0.003, and the timing of IA in the patient course of underlying disease, p 0.04. CONCLUSION: This study showed that caspofungin-based combination antifungal therapy is an effective therapeutic option also for pediatric patients with IA. These data need to be confirmed by prospective, controlled studies. [Abstract/Link to Full Text]

Romoren M, Sundby J, Velauthapillai M, Rahman M, Klouman E, Hjortdahl P
Chlamydia and gonorrhoea in pregnant Batswana women: time to discard the syndromic approach?
BMC Infect Dis. 2007;727.
BACKGROUND: Chlamydia and gonorrhoea are major causes of morbidity among women in developing countries. Both infections have been associated with pregnancy-related complications, and case detection and treatment in pregnancy is essential. In countries without laboratory support, the diagnosis and treatment of cervical infections is based on the syndromic approach. In this study we measured the prevalence of chlamydia and gonorrhoea among antenatal care attendees in Botswana. We evaluated the syndromic approach for the detection of cervical infections in pregnancy, and determined if risk scores could improve the diagnostic accuracy. METHODS: In a cross-sectional study, 703 antenatal care attendees in Botswana were interviewed and examined, and specimens were collected for the identification of C trachomatis, N gonorrhoeae and other reproductive tract infections. Risk scores to identify attendees with cervical infections were computed based on identified risk factors, and their sensitivities, specificities, likelihood ratios and predictive values were calculated. RESULTS: The prevalence of chlamydia was 8%, and gonorrhoea was found in 3% of the attendees. Symptoms and signs of vaginal discharge did not predict cervical infection, and a syndromic approach failed to identify infected women. Age (youth) risk factor most strongly associated with cervical infection. A risk score with only sociodemographic factors had likelihood ratios equivalent to risk scores which incorporated clinical signs and microscopy results. However, all the evaluated risk scores were of limited value in the diagnosis of chlamydia and gonorrhoea. A cut-off set at an acceptable sensitivity to avoid infected antenatal care attendees who remained untreated would inevitably lead to considerable over-treatment. CONCLUSION: Although in extensive use, the syndromic approach is unsuitable for diagnosing cervical infections in antenatal care attendees in Botswana. None of the evaluated risk scores can replace this management. Without diagnostic tests, there are no adequate management strategies for C trachomatis and N gonorrhoeae in pregnant women in Botswana, a situation which is likely to apply to other countries in sub-Saharan Africa. Screening for cervical infections in pregnant women is an essential public health measure, and rapid tests will hopefully be available in developing countries within a few years. [Abstract/Link to Full Text]

Onozuka D, Hagihara A
Geographic prediction of tuberculosis clusters in Fukuoka, Japan, using the space-time scan statistic.
BMC Infect Dis. 2007;726.
BACKGROUND: Tuberculosis (TB) has reemerged as a global public health epidemic in recent years. Although evaluating local disease clusters leads to effective prevention and control of TB, there are few, if any, spatiotemporal comparisons for epidemic diseases. METHODS: TB cases among residents in Fukuoka Prefecture between 1999 and 2004 (n = 9,119) were geocoded at the census tract level (n = 109) based on residence at the time of diagnosis. The spatial and space-time scan statistics were then used to identify clusters of census tracts with elevated proportions of TB cases. RESULTS: In the purely spatial analyses, the most likely clusters were in the Chikuho coal mining area (in 1999, 2002, 2003, 2004), the Kita-Kyushu industrial area (in 2000), and the Fukuoka urban area (in 2001). In the space-time analysis, the most likely cluster was the Kita-Kyushu industrial area (in 2000). The north part of Fukuoka Prefecture was the most likely to have a cluster with a significantly high occurrence of TB. CONCLUSION: The spatial and space-time scan statistics are effective ways of describing circular disease clusters. Since, in reality, infectious diseases might form other cluster types, the effectiveness of the method may be limited under actual practice. The sophistication of the analytical methodology, however, is a topic for future study. [Abstract/Link to Full Text]

---

Recent Articles in BMC Microbiology

---

Perandin F, Cariani E, Pollara CP, Manca N
Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma.
BMC Microbiol. 2007;722.
BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy) and an in-house assay (EBV RQ-PCR). RESULTS: The range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R2 = 0.7789; p < 0.0001), with the systematic overestimation by EBV RQ-PCR possibly linked to different amplification efficiency in calibration standards. CONCLUSION: Both the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement. [Abstract/Link to Full Text]

Coldewey SM, Hartmann M, Schmidt DS, Engelking U, Ukena SN, Gunzer F
Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli.
BMC Microbiol. 2007;721.
BACKGROUND: Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor sigmaS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. METHODS: Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. RESULTS: First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. CONCLUSION: The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype. [Abstract/Link to Full Text]

Chen Y, Bystricky P, Adeyeye J, Panigrahi P, Ali A, Johnson JA, Bush CA, Morris JG, Stine OC
The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region.
BMC Microbiol. 2007;720.
BACKGROUND: In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. RESULTS: The structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified. CONCLUSION: The co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations. [Abstract/Link to Full Text]

Tiyawisutsri R, Holden MT, Tumapa S, Rengpipat S, Clarke SR, Foster SJ, Nierman WC, Day NP, Peacock SJ
Burkholderia Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis.
BMC Microbiol. 2007;719.
BACKGROUND: The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. RESULTS: Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. CONCLUSION: Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study. [Abstract/Link to Full Text]

Sevilla I, Garrido JM, Geijo M, Juste RA
Pulsed-field gel electrophoresis profile homogeneity of Mycobacterium avium subsp. paratuberculosis isolates from cattle and heterogeneity of those from sheep and goats.
BMC Microbiol. 2007;718.
BACKGROUND: Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI endonucleases. Differences in strain isolation upon various media conditions were also studied. RESULTS: All bovines, 4 and 26% of Spanish sheep and goats, respectively, and the deer and wild boar studied, carried IS1311-Cattle type strains. IS1311-Sheep type encompassed 96% and 74% of Spanish sheep and goats, and all three Portuguese sheep. Thirty-seven distinct multiplex PFGE profiles were found, giving 32 novel profiles. Profiles 2-1 and 1-1 accounted for the 85% of cattle isolates. Ten distinct profiles were detected in Spanish sheep, none of them with an incidence higher than 25%. Profile 16-11 (43%) and another three profiles were identified in Spanish caprine cultures. The hierarchical analysis, clustered all profiles found in cattle, "wild" hosts and some small ruminants within the same group. The other group included 11 profiles only found in Spanish sheep and goats, including Spanish pigmented profiles. Differences in growth requirements associated with isolate genotype were observed. CONCLUSION: Cattle in Spain are infected with cattle type strains, while sheep and goats are mainly infected with sheep type strains. Although 7H9 broth based culture media seem to broadly cover the growth requirements of most Map strains, the use of various solid media is recommended to reduce any recovery biases. High genetic homogeneity of isolates from cattle, and heterogeneity of those from sheep and goats have been detected. [Abstract/Link to Full Text]

Martins-Pinheiro M, Marques RC, Menck CF
Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus.
BMC Microbiol. 2007;717.
BACKGROUND: The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. RESULTS: In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA), endonuclease III (nth), O6-methylguanine-DNA methyltransferase (ada gene), photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. CONCLUSION: The absence and the presence of certain genes are discussed and predictions are made considering the particular aspects of the C. crescentus among other known DNA repair pathways. The observed differences enlarge what is known for DNA repair in the Bacterial world, and provide a useful framework for further experimental studies in this organism. [Abstract/Link to Full Text]

Shelobolina ES, Coppi MV, Korenevsky AA, DiDonato LN, Sullivan SA, Konishi H, Xu H, Leang C, Butler JE, Kim BC, Lovley DR
Importance of c-Type cytochromes for U(VI) reduction by Geobacter sulfurreducens.
BMC Microbiol. 2007;716.
BACKGROUND: In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated. RESULTS: The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50-60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24-30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III). CONCLUSION: This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens. [Abstract/Link to Full Text]

Tucker DL, Ott CM, Huff S, Fofanov Y, Pierson DL, Willson RC, Fox GE
Characterization of Escherichia coli MG1655 grown in a low-shear modeled microgravity environment.
BMC Microbiol. 2007;715.
BACKGROUND: Extra-cellular shear force is an important environmental parameter that is significant both medically and in the space environment. Escherichia coli cells grown in a low-shear modeled microgravity (LSMMG) environment produced in a high aspect rotating vessel (HARV) were subjected to transcriptional and physiological analysis. RESULTS: Aerobic LSMMG cultures were grown in rich (LB) and minimal (MOPS + glucose) medium with a normal gravity vector HARV control. Reproducible changes in transcription were seen, but no specific LSMMG responsive genes were identified. Instead, absence of shear and a randomized gravity vector appears to cause local extra-cellular environmental changes, which elicit reproducible cellular responses. In minimal media, the majority of the significantly up- or down-regulated genes of known function were associated with the cell envelope. In rich medium, most LSMMG down-regulated genes were involved in translation. No observable changes in post-culture stress responses and antibiotic sensitivity were seen in cells immediately after exposure to LSMMG. Comparison with earlier studies of Salmonella enterica serovar Typhimurium conducted under similar growth conditions, revealed essentially no similarity in the genes that were significantly up- or down-regulated. CONCLUSION: Comparison of these results to previous studies suggests that different organisms may dramatically differ in their responses to medically significant low-shear and space environments. Depending on their specific response, some organisms, such as Salmonella, may become preadapted in a manner that predisposes them to increased virulence. [Abstract/Link to Full Text]

Johansen TB, Olsen I, Jensen MR, Dahle UR, Holstad G, Djřnne B
New probes used for IS1245 and IS1311 restriction fragment length polymorphism of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates of human and animal origin in Norway.
BMC Microbiol. 2007;714.
BACKGROUND: Mycobacterium avium is an environmental mycobacterium that can be divided into the subspecies avium, hominissuis, paratuberculosis and silvaticum. Some M. avium subspecies are opportunistic pathogens for animals and humans. They are ubiquitous in nature and can be isolated from natural sources of water, soil, plants and bedding material. Isolates of M. avium originating from humans (n = 37), pigs (n = 51) and wild birds (n = 10) in Norway were examined by IS1245 and IS1311 RFLP using new and specific probes and for the presence of IS901 and ISMpa1 by PCR. Analysis and generation of a dendrogram were performed with the software BioNumerics. RESULTS: IS1311 RFLP provided clear results that were easy to interpret, while IS1245 RFLP generated more complex patterns with a higher discriminatory power. The combination of the two methods gave additional discrimination between isolates. All avian isolates except one were M. avium subsp. avium with two copies of IS1311 and one copy of IS1245, while the isolates of human and porcine origin belonged to M. avium subsp.hominissuis. The isolates from human patients were distributed randomly among the clusters of porcine isolates. There were few identical isolates. However, one isolate from a human patient was identical to a porcine isolate. Regional differences were detected among the porcine isolates, while there was no clustering of human isolates according to type of clinical symptoms or geographical location of the patient's home addresses. CONCLUSION: The results demonstrate that a wide range of M. avium subsp.hominissuis are present in pigs and humans in Norway, and that some of these isolates are very similar. It remains to be determined whether humans are infected from pigs or if they are infected from common environmental sources. [Abstract/Link to Full Text]

Mora A, Blanco M, Blanco JE, Dahbi G, López C, Justel P, Alonso MP, Echeita A, Bernárdez MI, González EA, Blanco J
Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003.
BMC Microbiol. 2007;713.
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections. RESULTS: STEC were detected in 95 (12%) of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0%) samples and non-O157 STEC from 90 (11%) samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29%) isolates carried stx1 genes, 49 (51%) possessed stx2 genes, and 19 (20%) both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 43 (45%) and in 25 (26%) of the isolates, respectively. Typing of the eae variants detected four types: gamma1 (nine isolates), beta1 (eight isolates), epsilon1 (three isolates), and theta (two isolates). The majority (68%) of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx1 eae-beta1, O157:H7 stx1stx2 eae-gamma1 and O157:H7 stx2eae-gamma1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore, phage typing of STEC O157:H7 isolates showed that the majority (seven of eight isolates) belonged to the main phage types previously detected in STEC O157:H7 strains associated with severe human illnesses. CONCLUSION: The results of this study do not differ greatly from those reported in other countries with regard to prevalence of O157 and non-O157 STEC in minced beef. As we suspected, serotypes different from O157:H7 also play an important role in food contamination in Spain, including the highly virulent seropathotype O26:H11 stx1 eae-beta1. Thus, our data confirm minced beef in the city of Lugo as vehicles of highly pathogenic STEC. This requires that control measures to be introduced and implemented to increase the safety of minced beef. [Abstract/Link to Full Text]

Weide T, Bockau U, Rave A, Herrmann L, Hartmann MW
A recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila.
BMC Microbiol. 2007;712.
BACKGROUND: Tetrahymena thermophila is one of the best characterized unicellular eukaryotes and its genome is sequenced in its entirety. However, the AT-richness of the genome and an unusual codon usage cause problems in cloning and expression of the ciliate DNA. To overcome these technical hiatuses we developed a Cre-dependent recombinase system. RESULTS: We created novel donor and acceptor vectors that facilitate the transfer of expression cassettes from the donor into novel acceptor plasmid. Expression vectors were used that encode the 19 kDa C-terminus of the MSP1 protein of Plasmodium falciparum and a blasticidin S (bsdR) resistance gene, respectively. The functional expression of these genes was demonstrated by western blot analysis with MSP1 specific antibodies and by a blasticidin growing assay. CONCLUSION: The Cre dependent recombinase system in combination with the modular structure of the donor vectors ease cloning and expression of foreign genes in the ciliate system, providing a powerful tool for protistology research in future. [Abstract/Link to Full Text]

Miller WG, Parker CT, Heath S, Lastovica AJ
Identification of genomic differences between Campylobacter jejuni subsp. jejuni and C. jejuni subsp. doylei at the nap locus leads to the development of a C. jejuni subspeciation multiplex PCR method.
BMC Microbiol. 2007;711.
BACKGROUND: The human bacterial pathogen Campylobacter jejuni contains two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Although Cjd strains are isolated infrequently in many parts of the world, they are obtained primarily from human clinical samples and result in an unusual clinical symptomatology in that, in addition to gastroenteritis, they are associated often with bacteremia. In this study, we describe a novel multiplex PCR method, based on the nitrate reductase (nap) locus, that can be used to unambiguously subspeciate C. jejuni isolates. RESULTS: Internal and flanking napA and napB primer sets were designed, based on existing C. jejuni and Campylobacter coli genome sequences to create two multiplex PCR primer sets, nap mpx1 and nap mpx2. Genomic DNA from 161 C. jejuni subsp. jejuni (Cjj) and 27 C. jejuni subsp. doylei (Cjd) strains were amplified with these multiplex primer sets. The Cjd strains could be distinguished clearly from the Cjj strains using either nap mpx1 or mpx2. In addition, combination of either nap multiplex method with an existing lpxA speciation multiplex method resulted in the unambiguous and simultaneous speciation and subspeciation of the thermophilic Campylobacters. The Cjd nap amplicons were also sequenced: all Cjd strains tested contained identical 2761 bp deletions in napA and several Cjd strains contained deletions in napB. CONCLUSION: The nap multiplex PCR primer sets are robust and give a 100% discrimination of C. jejuni subspecies. The ability to rapidly subspeciate C. jejuni as well as speciate thermophilic Campylobacter species, most of which are pathogenic in humans, in a single amplification will be of value to clinical laboratories in strain identification and the determination of the environmental source of campylobacterioses caused by Cjd. Finally, the sequences of the Cjd napA and napB loci suggest that Cjd strains arose from a common ancestor, providing clues as to the potential evolutionary origin of Cjd. [Abstract/Link to Full Text]

Bohn C, Rigoulay C, Bouloc P
No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus.
BMC Microbiol. 2007;710.
BACKGROUND: The RNA-binding protein Hfq is involved in stress and virulence of several pathogens, probably due to its role as mediator in small RNA (sRNA)-mRNA interactions. In this study, we investigate the function of Hfq in the Gram-positive pathogen Staphylococcus aureus, by constructing hfq null mutant derivatives. RESULTS: We report that unexpectedly, in S. aureus, Hfq does not seem to play a crucial role in stress response, RNAIII or spa mRNA quantity and exoprotein expression, as tested in three virulent genetic backgrounds. Moreover, a global analysis of the RN6390 hfq mutant, which tests approximately 2000 phenotypes, supports our results concerning the non-implication of Hfq in stress response, and shows that Hfq is also not involved in resistance to several chemical agents and antibiotics and does not seem to be implicated in metabolic pathways. CONCLUSION: Our data suggest that although sRNA-mRNA interactions in S. aureus are decisive for gene expression regulation, they do not require the RNA-chaperone protein Hfq. These interactions possibly require an RNA-chaperone protein other than Hfq, which remains to be found. [Abstract/Link to Full Text]

Salazar MI, Richardson JH, Sánchez-Vargas I, Olson KE, Beaty BJ
Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes.
BMC Microbiol. 2007;79.
BACKGROUND: To be transmitted by its mosquito vector, dengue virus (DENV) must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP) is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission. RESULTS: After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi). The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands) increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle. CONCLUSION: Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands) differed in their response to DENV-2 infection. [Abstract/Link to Full Text]

Freestone PP, Haigh RD, Lyte M
Blockade of catecholamine-induced growth by adrenergic and dopaminergic receptor antagonists in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica.
BMC Microbiol. 2007;78.
BACKGROUND: The ability of catecholamines to stimulate bacterial growth was first demonstrated just over a decade ago. Little is still known however, concerning the nature of the putative bacterial adrenergic and/or dopaminergic receptor(s) to which catecholamines (norepinephrine, epinephrine and dopamine) may bind and exert their effects, or even whether the binding properties of such a receptor are similar between different species. RESULTS: Use of specific catecholamine receptor antagonists revealed that only alpha, and not beta, adrenergic antagonists were capable of blocking norepinephrine and epinephrine-induced growth, while antagonism of dopamine-mediated growth was achieved with the use of a dopaminergic antagonist. Both adrenergic and dopaminergic antagonists were highly specific in their mechanism of action, which did not involve blockade of catecholamine-facilitated iron-acquisition. Use of radiolabeled norepinephrine suggested that the adrenergic antagonists could be acting by inhibiting catecholamine uptake. CONCLUSION: The present data demonstrates that the ability of a specific pathogen to respond to a particular hormone is dependent upon the host anatomical region in which the pathogen causes disease as well as the neuroanatomical specificity to which production of the particular hormone is restricted; and that both are anatomically coincidental to each other. As such, the present report suggests that pathogens with a high degree of exclusivity to the gastrointestinal tract have evolved response systems to neuroendocrine hormones such as norepinephrine and dopamine, but not epinephrine, which are found with the enteric nervous system. [Abstract/Link to Full Text]

Patrone JB, Stein DC
Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile.
BMC Microbiol. 2007;77.
BACKGROUND: Neisseria gonorrhoeae is an obligate human pathogen that causes significant worldwide morbidity. N. gonorrhoeae expresses lipooligosaccharide (LOS), a phase variable molecule that plays an important role during pathogenesis of the organism. Alteration in the structure of gonococcal LOS correlates with altered disease presentation. In addition, LOS sialylation occurs readily in vivo, though the role of this sialylation during disease is unknown. RESULTS: Challenge of human monocytes with purified LOS preparations isolated from strains expressing distinct structurally defined LOSs resulted in identical production of the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-12 (IL-12). Similar results were seen when monocytes were challenged with either live or gentamicin-killed whole cell gonococcal variants expressing these LOS structures, although greater cytokine production was observed in comparison with challenge by purified LOS. Challenge of a human primary monocyte model with distinct LOS variants resulted in similar production of TNFalpha, IL-12, interleukin-10 (IL-10), and interleukin-8 (IL-8). A cytokine array was employed to allow measurement of a broad range of cytokines in samples challenge with gonococcal LOS variants as well as variants expressing sialylated LOS. Challenge of primary monocytes with sialylated gonococci was shown to elicit the production of more MCP-2 (monocyte chemoattractant protein-2) in comparison with challenge by unsialylated gonococci. CONCLUSION: We demonstrated that while alterations in the carbohydrate moiety of LOS do not impact the production of most cytokines by human monocytes, whole-cell bacterial challenge is more stimulatory than challenge with purified LOS, implying that other gonococcal cell surface antigens are important for the elicitation of cytokines. Challenge with gonococci expressing sialylated LOS resulted in elicitation of more of the chemokine MCP-2 from challenged cells in comparison with gonococci expressing unsialylated LOS. As MCP-2 is an important chemoattractant, this indicates that in vivo sialylation may play an important role during the pathogenesis of N. gonorrhoeae. [Abstract/Link to Full Text]

Lüdke A, Krämer R, Burkovski A, Schluesener D, Poetsch A
A proteomic study of Corynebacterium glutamicum AAA+ protease FtsH.
BMC Microbiol. 2007;76.
BACKGROUND: The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis. RESULTS: In contrast to the situation in other bacteria, deletion of C. glutamicum ftsH has no significant effect on growth in standard minimal medium or response to heat or osmotic stress. On the proteome level, deletion of the ftsH gene resulted in a strong increase of ten cytoplasmic and membrane proteins, namely biotin carboxylase/biotin carboxyl carrier protein (accBC), glyceraldehyde-3-phosphate dehydrogenase (gap), homocysteine methyltransferase (metE), malate synthase (aceB), isocitrate lyase (aceA), a conserved hypothetical protein (NCgl1985), succinate dehydrogenase A (sdhA), succinate dehydrogenase B (sdhB), succinate dehydrogenase CD (sdhCD), and glutamate binding protein (gluB), while 38 cytoplasmic and membrane-associated proteins showed a decreased abundance. The decreasing amount of succinate dehydrogenase A (sdhA) in the cytoplasmic fraction of the ftsH mutant compared to the wild type and its increasing abundance in the membrane fraction indicates that FtsH might be involved in the cleavage of a membrane anchor of this membrane-associated protein and by this changes its localization. CONCLUSION: The data obtained hint to an involvement of C. glutamicum FtsH protease mainly in regulation of energy and carbon metabolism, while the protease is not involved in stress response, as found in other bacteria. [Abstract/Link to Full Text]

Khoufache K, Puel O, Loiseau N, Delaforge M, Rivollet D, Coste A, Cordonnier C, Escudier E, Botterel F, Bretagne S
Verruculogen associated with Aspergillus fumigatus hyphae and conidia modifies the electrophysiological properties of human nasal epithelial cells.
BMC Microbiol. 2007;75.
BACKGROUND: The role of Aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. In a previous study, we found that filtrates of A. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells (HNEC) compared to those of non pathogenic moulds. RESULTS: We fractionated the organic phase of filtrate from 3-day old A. fumigatus cultures using high-performance liquid chromatography. The different fractions were tested for their ability to modify the electrophysiological properties of HNEC in an in vitro primary culture model.The fraction collected between 20 and 30 min mimicked the effects of the whole filtrate, i.e. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen. Only verruculogen (10(-8) M) had effects similar to the whole filtrate. We verified that verruculogen was produced by a collection of 67 human, animal, plant and environmental A. fumigatus isolates. Using MS-MS analysis, we found that verruculogen was associated with both mycelium and conidia extracts. CONCLUSION: Verruculogen is a secondary metabolite that modifies the electrophysiological properties of HNEC. The role of these modifications in the colonization and invasion of the respiratory epithelium by A. fumigatus on first contact with the epithelium remains to be determined. [Abstract/Link to Full Text]

Subbian S, Mehta PK, Cirillo SL, Cirillo JD
The Mycobacterium marinum mel2 locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species.
BMC Microbiol. 2007;74.
BACKGROUND: Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood. RESULTS: To better understand the role of the M. marinum mel2 locus, we examined these genes for conserved motifs in silico. Striking similarities were observed between the mel2 locus and loci that encode bioluminescence in other bacterial species. Since bioluminescence systems can play a role in resistance to oxidative stress, we postulated that the mel2 locus might be important for mycobacterial resistance to ROS and RNS. We found that an M. marinum mutant in the first gene in this putative operon, melF, confers increased susceptibility to both ROS and RNS. This mutant is more susceptible to ROS and RNS together than either reactive species alone. CONCLUSION: These observations support a role for the M. marinum mel2 locus in resistance to oxidative stress and provide additional evidence that bioluminescence systems may have evolved from oxidative defense mechanisms. [Abstract/Link to Full Text]

Dieye Y, Dyszel JL, Kader R, Ahmer BM
Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium.
BMC Microbiol. 2007;73.
BACKGROUND: The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions. RESULTS: We performed a systematic analysis of the regulation of type III effectors during growth in vitro. We have tested the ability of seven regulatory genes to regulate ten effector genes. Each regulator was expressed in the absence of the other six to avoid cascade effects. Our results confirm and extend the previously reported regulation of TTSS1 and TTSS2 effectors by InvF-SicA and SsrB respectively. CONCLUSION: The set of strains constructed for this study can be used to quickly and systematically study the regulation of newly identified effector genes of Salmonella enterica. The approach we have used can also be applied to study complex regulatory cascades in other bacterial species. [Abstract/Link to Full Text]

Paul L, Mishra PK, Blumenthal RM, Matthews RG
Integration of regulatory signals through involvement of multiple global regulators: control of the Escherichia coli gltBDF operon by Lrp, IHF, Crp, and ArgR.
BMC Microbiol. 2007;72.
BACKGROUND: The glutamate synthase operon (gltBDF) contributes to one of the two main pathways of ammonia assimilation in Escherichia coli. Of the seven most-global regulators, together affecting expression of about half of all E. coli genes, two were previously shown to exert direct, positive control on gltBDF transcription: Lrp and IHF. The involvement of Lrp is unusual in two respects: first, it is insensitive to the usual coregulator leucine, and second, Lrp binds more than 150 bp upstream of the transcription starting point. There was indirect evidence for involvement of a third global regulator, Crp. Given the physiological importance of gltBDF, and the potential opportunity to learn about integration of global regulatory signals, a combination of in vivo and in vitro approaches was used to investigate the involvement of additional regulatory proteins, and to determine their relative binding positions and potential interactions with one another and with RNA polymerase (RNAP). RESULTS: Crp and a more local regulator, ArgR, directly control gltBDF transcription, both acting negatively. Crp-cAMP binds a sequence centered at -65.5 relative to the transcript start. Mutation of conserved nucleotides in the Crp binding site abolishes the Crp-dependent repression. ArgR also binds to the gltBDF promoter region, upstream of the Lrp binding sites, and decreases transcription. RNAP only yields a defined DNAse I footprint under two tested conditions: in the presence of both Lrp and IHF, or in the presence of Crp-cAMP. The DNAse I footprint of RNAP in the presence of Lrp and IHF is altered by ArgR. CONCLUSION: The involvement of nearly half of E. coli's most-global regulatory proteins in the control of gltBDF transcription is striking, but seems consistent with the central metabolic role of this operon. Determining the mechanisms of activation and repression for gltBDF was beyond the scope of this study. However the results are consistent with a model in which IHF bends the DNA to allow stabilizing contacts between Lrp and RNAP, ArgR interferes with such contacts, and Crp introduces an interfering bend in the DNA and/or stabilizes RNAP in a poised but inactive state. [Abstract/Link to Full Text]

de Bruin OM, Ludu JS, Nano FE
The Francisella pathogenicity island protein IglA localizes to the bacterial cytoplasm and is needed for intracellular growth.
BMC Microbiol. 2007;71.
BACKGROUND: Francisella tularensis is a gram negative, facultative intracellular bacterium that is the etiological agent of tularemia. F. novicida is closely related to F. tularensis but has low virulence for humans while being highly virulent in mice. IglA is a 21 kDa protein encoded by a gene that is part of an iglABCD operon located on the Francisella pathogenicity island (FPI). RESULTS: Bioinformatics analysis of the FPI suggests that IglA and IglB are components of a newly described type VI secretion system. In this study, we showed that IglA regulation is controlled by the global regulators MglA and MglB. During intracellular growth IglA production reaches a maximum at about 10 hours post infection. Biochemical fractionation showed that IglA is a soluble cytoplasmic protein and immunoprecipitation experiments demonstrate that it interacts with the downstream-encoded IglB. When the iglB gene was disrupted IglA could not be detected in cell extracts of F. novicida, although IglC could be detected. We further demonstrated that IglA is needed for intracellular growth of F. novicida. A non-polar iglA deletion mutant was defective for growth in mouse macrophage-like cells, and in cis complementation largely restored the wild type macrophage growth phenotype. CONCLUSION: The results of this study demonstrate that IglA and IglB are interacting cytoplasmic proteins that are required for intramacrophage growth. The significance of the interaction may be to secrete effector molecules that affect host cell processes. [Abstract/Link to Full Text]

Chambers CE, Lutter EI, Visser MB, Law PP, Sokol PA
Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome.
BMC Microbiol. 2006;6104.
BACKGROUND: The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs. RESULTS: Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The first-generation consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR. CONCLUSION: Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system. [Abstract/Link to Full Text]

Massonet C, Pintens V, Merckx R, Anné J, Lammertyn E, Van Eldere J
Effect of iron on the expression of sirR and sitABC in biofilm-associated Staphylococcus epidermidis.
BMC Microbiol. 2006;6103.
BACKGROUND: Different gene expression patterns correlate with the altered phenotype in biofilm-associated bacteria. Iron and iron-linked genes are thought to play a key-role in biofilm formation. The expression of Fe-linked genes (sirR, sitABC operon) in Staphylococcus epidermidis, was compared in planktonic versus sessile bacteria in vitro and in vivo in a subcutaneous foreign body rat model. RESULTS: In vitro in a Fe-limited environment, the planktonic form of S. epidermidis produces siderophores and grows slower than in Fe-rich environment. The expression of sirR in planktonic bacteria, in vitro, was not different in medium without Fe or with 1 microM FeCl3. High Fe concentrations (25 microM FeCl3) increased expression of sirR transiently during the early phase of incubation. Expression of sitC in vitro, in planktonic bacteria, was inversely correlated with sirR expression in medium with 25 microM FeCl3: sitC expression decreased for the first 3 hours followed by an up regulation.In sessile bacteria in vitro, sirR expression was high and independent of the Fe concentration. The expression of sitC was not inversely correlated to sirR expression. In vivo, expression levels of sirR and of sitABC were high during the initial phase after implantation and, after a transient decrease, remained stable over a period of two weeks. CONCLUSION: Our data suggest that the expression of sirR and the regulatory effect of sirR on the sitABC operon are different in planktonic and sessile bacteria. [Abstract/Link to Full Text]

Wu Q, Pei J, Turse C, Ficht TA
Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival.
BMC Microbiol. 2006;6102.
BACKGROUND: Random gene inactivation used to identify cellular functions associated with virulence and survival of Brucella spp has relied heavily upon the use of the transposon Tn5 that integrates at G/C base pairs. Transposons of the mariner family do not require species-specific host factors for efficient transposition, integrate nonspecifically at T/A base pairs, and, at a minimum, provide an alternative approach for gene discovery. In this study, plasmid vector pSC189, containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, were used to deliver Himar1 transposable element into the B. melitensis genome. Conjugation was performed efficiently and rapidly in less than one generation in order to minimize the formation of siblings while assuring the highest level of genome coverage. RESULTS: Although previously identified groups or classes of genes required for virulence and survival were represented in the screen, additional novel identifications were revealed and may be attributable to the difference in insertion sequence biases of the two transposons. Mutants identified using a fluorescence-based macrophage screen were further evaluated using gentamicin-based protection assay in macrophages, survival in the mouse splenic clearance model and growth in vitro to identify mutants with reduced growth rates. CONCLUSION: The identification of novel genes within previously described groups was expected, and nearly two-thirds of the 95 genes had not been previously reported as contributing to survival and virulence using random Tn5-based mutagenesis. The results of this work provide added insight with regard to the regulatory elements, nutritional demands and mechanisms required for efficient intracellular growth and survival of the organism. [Abstract/Link to Full Text]

Kariuki S, Revathi G, Kariuki N, Kiiru J, Mwituria J, Hart CA
Characterisation of community acquired non-typhoidal Salmonella from bacteraemia and diarrhoeal infections in children admitted to hospital in Nairobi, Kenya.
BMC Microbiol. 2006;6101.
BACKGROUND: In sub-Saharan Africa community-acquired non-typhoidal Salmonella (NTS) is a major cause of high morbidity and death among children under 5 years of age especially from resource poor settings. The emergence of multidrug resistance is a major challenge in treatment of life threatening invasive NTS infections in these settings. RESULTS: Overall 170 (51.2%) of children presented with bacteraemia alone, 28 (8.4%) with gastroenteritis and bacteraemia and 134 (40.4%) with gastroenteritis alone. NTS serotypes obtained from all the cases included S. Typhimurium (196; 59%), S. Enteritidis (94; 28.3%) and other serotypes in smaller numbers (42; 12.7%); distribution of these serotypes among cases with bacteremia or gastroenteritis was not significantly different. A significantly higher proportion of younger children (< 3 years of age) and those from the slums presented with invasive NTS compared to older children and those from upper socio-economic groups (p < 0.001). One hundred and forty-seven (44.3%) NTS were resistant to 3 or more antibiotics, and out of these 59% were resistant to ampicillin, chloramphenicol and tetracycline. There was no significant difference in antibiotic resistance between the two serotypes, S. Typhimurium and S. Enteritidis. Ceftriaxone and ciprofloxacin were the only antibiotics tested to which all the NTS were fully susceptible. Using Pulsed Field Gel Electrophoresis (PFGE) there were 3 main patterns of S. Typhimurium and 2 main patterns of S. Enteritidis among cases of bacteraemia and gastroenteritis. CONCLUSION: Serotype distribution, antibiotic susceptibility and PFGE patterns of NTS causing bacteraemia and gastroenteritis did not differ significantly. The high prevalence of NTS strains resistant to most of the commonly used antimicrobials is of major public health concern. [Abstract/Link to Full Text]

Derzelle S, Dilasser F
A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae.
BMC Microbiol. 2006;6100.
BACKGROUND: Enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. Rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. In this work, a real time fluorescence resonance energy transfer PCR assay incorporating an internal amplification control (IAC) was developed for the specific detection of E. sakazakii in foods. Performance of the assay, coupled to an automated DNA extraction system and the E. sakazakii ISO-IDF (TS 22964/RM 210) enrichment procedure, was evaluated on infant formulae and samples from production environment. RESULTS: The real-time PCR assay had 100% specificity as assessed using 35 E. sakazakii and 184 non-E. sakazakii strains. According to the E. sakazakii strains tested, the detection limits ranged from 5 to 25 genomic copies. Assays on pure cultures (including real-time PCR and DNA extraction) gave a sensitivity of about 102 to 103 CFU/ml. Out of 41 naturally contaminated infant formulae and environmental samples analysed for the presence of E. sakazakii, 23 were positive by real-time PCR and 22 by the conventional culture method, giving 97.5% concordance with the ISO-IDF reference method. CONCLUSION: This method, combining specific real-time PCR, automated DNA extraction and ISO-IDF standard enrichments, provides a useful tool for rapid screening of E. sakazakii in food and environmental matrices. [Abstract/Link to Full Text]

Halliday JE, Chase-Topping ME, Pearce MC, McKendrick IJ, Allison L, Fenlon D, Low C, Mellor DJ, Gunn GJ, Woolhouse ME
Herd-level risk factors associated with the presence of Phage type 21/28 E. coli O157 on Scottish cattle farms.
BMC Microbiol. 2006;699.
BACKGROUND: E. coli O157 is a bacterial pathogen that is shed by cattle and can cause severe disease in humans. Phage type (PT) 21/28 is a subtype of E. coli O157 that is found across Scotland and is associated with particularly severe human morbidity. METHODS: A cross-sectional survey of Scottish cattle farms was conducted in the period Feb 2002-Feb 2004 to determine the prevalence of E. coli O157 in cattle herds. Data from 88 farms on which E. coli O157 was present were analysed using generalised linear mixed models to identify risk factors for the presence of PT 21/28 specifically. RESULTS: The analysis identified private water supply, and northerly farm location as risk factors for PT 21/28 presence. There was a significant association between the presence of PT 21/28 and an increased number of E. coli O157 positive pat samples from a farm, and PT 21/28 was significantly associated with larger E. coli O157 counts than non-PT 21/28 E. coli O157. CONCLUSION: PT 21/28 has significant risk factors that distinguish it from other phage types of E. coli O157. This finding has implications for the control of E. coli O157 as a whole and suggests that control could be tailored to target the locally dominant PT. [Abstract/Link to Full Text]

Licht TR, Hansen M, Poulsen M, Dragsted LO
Dietary carbohydrate source influences molecular fingerprints of the rat faecal microbiota.
BMC Microbiol. 2006;698.
BACKGROUND: A study was designed to elucidate effects of selected carbohydrates on composition and activity of the intestinal microbiota. Five groups of eight rats were fed a western type diet containing cornstarch (reference group), sucrose, potato starch, inulin (a long- chained fructan) or oligofructose (a short-chained fructan). Fructans are, opposite sucrose and starches, not digestible by mammalian gut enzymes, but are known to be fermentable by specific bacteria in the large intestine. RESULTS: Animals fed with diets containing potato starch, or either of the fructans had a significantly (p < 0.05) higher caecal weight and lower caecal pH when compared to the reference group, indicating increased fermentation. Selective cultivation from faeces revealed a higher amount of lactic acid bacteria cultivable on Rogosa agar in these animals. Additionally, the fructan groups had a lower amount of coliform bacteria in faeces. In the inulin and oligofructose groups, higher levels of butyrate and propionate, respectively, were measured.Principal Component Analysis of profiles of the faecal microbiota obtained by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR amplified bacterial 16S rRNA genes as well as of Reverse Transcriptase-PCR amplified bacterial 16S rRNA resulted in different phylogenetic profiles for each of the five animal groups as revealed by Principal Component Analysis (PCA) of band patterns. CONCLUSION: Even though sucrose and cornstarch are both easily digestible and are not expected to reach the large intestine, the DGGE band patterns obtained indicated that these carbohydrates indeed affected the composition of bacteria in the large gut. Also the two fructans resulted in completely different molecular fingerprints of the faecal microbiota, indicating that even though they are chemically similar, different intestinal bacteria ferment them. Comparison of DNA-based and RNA-based profiles suggested that two species within the phylum Bacteroidetes were not abundant in numbers but had a particularly high ribosome content in the animals fed with inulin. [Abstract/Link to Full Text]

Dryselius R, Nikravesh A, Kulyté A, Goh S, Good L
Variable coordination of cotranscribed genes in Escherichia coli following antisense repression.
BMC Microbiol. 2006;697.
BACKGROUND: A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. RESULTS: To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. CONCLUSION: The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria. [Abstract/Link to Full Text]

---

Recent Articles in Clinical and Diagnostic Laboratory Immunology

---

Dias D, Van Doren J, Schlottmann S, Kelly S, Puchalski D, Ruiz W, Boerckel P, Kessler J, Antonello JM, Green T, Brown M, Smith J, Chirmule N, Barr E, Jansen KU, Esser MT
Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18.
Clin Diagn Lab Immunol. 2005 Aug;12(8):959-69.
A human papillomavirus (HPV) multiplexed competitive Luminex immunoassay first described by Opalka et al. (D. Opalka, C. E. Lachman, S. A. MacMullen, K. U. Jansen, J. F. Smith, N. Chirmule, and M. T. Esser, Clin. Diagn. Lab. Immunol. 10:108--15, 2003) was optimized and validated for use in epidemiology studies and vaccine clinical trials. Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines. [Abstract/Link to Full Text]

Salazar JC, Pope CD, Moore MW, Pope J, Kiely TG, Radolf JD
Lipoprotein-dependent and -independent immune responses to spirochetal infection.
Clin Diagn Lab Immunol. 2005 Aug;12(8):949-58.
In this study, we used the epidermal suction blister technique, in conjunction with multiparameter flow cytometry, to analyze the cellular and cytokine responses elicited by intradermal injection of human volunteers with synthetic analogs for spirochetal lipoproteins and compared the responses to findings previously reported from patients with erythema migrans (EM). Compared with peripheral blood (PB), lipopeptides derived from the N termini of the Borrelia burgdorferi outer surface protein C and the 17-kDa lipoprotein of Treponema pallidum (OspC-L and 17-L, respectively) elicited infiltrates enriched in monocytes/macrophages and dendritic cells (DCs) but also containing substantial percentages of neutrophils and T cells. Monocytoid (CD11c(+)) and plasmacytoid (CD11c(-)) DCs were selectively recruited to the skin in ratios similar to those in PB, but only the former expressed the activation/maturation surface markers CD80, CD83, and DC-SIGN. Monocytes/macrophages and monocytoid DCs, but not plasmacytoid DCs, displayed significant increases in surface expression of Toll-like receptor 1 (TLR1), TLR2, and TLR4. Staining for CD45RO and CD27 revealed that lipopeptides preferentially recruited antigen-experienced T-cell subsets; despite their lack of antigenicity, these agonists induced marked T-cell activation, as evidenced by surface expression of CD69, CD25, and CD71. Lipopeptides also induced significant increases in interleukin 12 (IL-12), IL-10, gamma interferon, and most notably IL-6 without corresponding increases in serum levels of these cytokines. Although lipopeptides and EM lesional infiltrates shared many similarities, differences were noted in a number of immunologic parameters. These studies have provided in situ evidence for a prominent "lipoprotein effect" during human infection while at the same time helping to pinpoint aspects of the cutaneous response that are uniquely driven by spirochetal pathogens. [Abstract/Link to Full Text]

Germenis AE, Yiannaki EE, Zachou K, Roka V, Barbanis S, Liaskos C, Adam K, Kapsoritakis AN, Potamianos S, Dalekos GN
Prevalence and clinical significance of immunoglobulin A antibodies against tissue transglutaminase in patients with diverse chronic liver diseases.
Clin Diagn Lab Immunol. 2005 Aug;12(8):941-8.
The prevalence of celiac disease (CD) and the prevalence and clinical significance of anti-tissue transglutaminase (tTG) antibodies (tTGAbs) in a large series of patients with chronic liver diseases were assessed. We studied 738 patients (462 with chronic viral hepatitis, 117 with autoimmune liver diseases, 113 with alcoholic or nonalcoholic fatty liver disease, and 46 with other liver disorders) and 1,350 healthy controls (HC). Immunoglobulin A (IgA) tTGAbs were measured by enzyme-linked immunosorbent assay and a microsphere-based flow cytometric assay. Positive sera were investigated for IgA antiendomysial antibodies (EmA). IgA tTGAb-positive subjects were invited to undergo a small-intestinal biopsy and HLA-DQ allele typing. Four of 1,350 HC (0.3%) tested tTGAb(+) EmA(+) and underwent a biopsy (CD confirmation in all). Four of 738 liver disease patients tested tTGAbs(+) EmA(+) (0.54%; not statistically significant). Two were HCV infected (1.24%; not statistically significant), and two had transaminasemia of unknown origin. Forty-three patients tested tTGAbs(+) EmA(-) (5.8%; P<0.001 compared to HC). Inhibition experiments verified the existence of specific IgA anti-tTG reactivity. Twenty-six of 43 patients underwent a biopsy (all negative for CD). Binary logistic regression analysis revealed age (P=0.008), cirrhosis (P=0.004), alkaline phosphatase (P=0.026), and antinuclear antibodies (P=0.012) as independent risk factors for tTGAb reactivity among the patients. It was concluded that CD prevalence is the same in HC and patients with chronic liver diseases. The prevalence of tTGAbs is higher in hepatic patients compared to HC, but their specificity for CD diagnosis in this group of patients is low. tTGAbs in patients appear to be associated with the presence of autoimmunity, cirrhosis, and cholestasis, irrespective of the origin of the liver disease. [Abstract/Link to Full Text]

Bryksin AV, Godfrey HP, Carbonaro CA, Wormser GP, Aguero-Rosenfeld ME, Cabello FC
Borrelia burgdorferi BmpA, BmpB, and BmpD proteins are expressed in human infection and contribute to P39 immunoblot reactivity in patients with Lyme disease.
Clin Diagn Lab Immunol. 2005 Aug;12(8):935-40.
The Bmp proteins are a paralogous family of chromosomally encoded Borrelia burgdorferi lipoproteins. They have similar predicted immunogenicities and similar electrophoretic mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P39 reactivity against Borrelia burgdorferi lysate in immunoblots of Lyme disease patients has long been identified with reactivity to BmpA, but responses to other Bmp proteins have not been examined. To determine if patients with Lyme disease developed such responses, immunoglobulin G (IgG) anti-Bmp reactivity in patient and control sera was studied by using soluble recombinant Bmp (rBmp) proteins expressed in Escherichia coli. Although some patient sera contained IgG immunoblot and immunodot reactivities against all four Bmp proteins, analysis of IgG anti-Bmp fine specificity by a competitive enzyme-linked immunosorbent assay with graded doses of soluble homologous and heterologous rBmp proteins showed that only the responses to BmpA, BmpB, and BmpD were specific. This suggests that at least three of the four Bmp proteins are expressed by B. burgdorferi in infected patients and that specific antibodies to them are likely to be present in the P39 band in some patients. [Abstract/Link to Full Text]

Hufnagel M, Kropec A, Theilacker C, Huebner J
Naturally acquired antibodies against four Enterococcus faecalis capsular polysaccharides in healthy human sera.
Clin Diagn Lab Immunol. 2005 Aug;12(8):930-4.
Healthy human sera (HHS) contain naturally acquired enterococcal antibodies which promote neutrophil-mediated killing. The target antigens remain unknown. The present study used a capsular polysaccharide (CPS)-enzyme-linked immunosorbent assay (ELISA) to investigate whether the HHS antibodies of 12 healthy donors bound to the CPS of four E. faecalis serotypes (CPS-A to CPS-D) and then employed an opsonic-killing assay to determine if these antibodies mediated phagocyte-dependent killing. All HHS contained immunoglobulin G (IgG) and IgM antibodies directed against capsular polysaccharides of the four serotypes. Absorption of the sera with homologous and heterologous strains showed a majority of antibodies to be cross-reactive among the prototype strains. The susceptibility of the four prototype strains to opsonic killing varied. Opsonic killing of CPS-A and CPS-B strains was significantly higher than killing of CPS-C and CPS-D strains. Absorption studies revealed that the opsonic killing of HHS was only partially type specific, with cross-reactivity between CPS-A and CPS-B strains and between CPS-C and CPS-D strains. These data indicate that healthy individuals possess opsonic antibodies specific for CPS-A and CPS-B but only low titers of opsonic antibodies against CPS-C and CPS-D. Titers of opsonic antibodies did not correlate with antibody titers measured by ELISA. Whether this lack of correlation is due to the low frequency of opsonic antibodies or to increased resistance to the opsonophagocytic killing of some serotypes remains to be determined. [Abstract/Link to Full Text]

Ko YJ, Choi KS, Nah JJ, Paton DJ, Oem JK, Wilsden G, Kang SY, Jo NI, Lee JH, Kim JH, Lee HW, Park JM
Noninfectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in pigs by enzyme-linked immunosorbent assay.
Clin Diagn Lab Immunol. 2005 Aug;12(8):922-9.
An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n=1,041). When tested using sera (n=186) collected periodically from pigs (n=19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed. [Abstract/Link to Full Text]

Soroka SD, Granade TC, Candal D, Parekh BS
Modification of rapid human immunodeficiency virus (HIV) antibody assay protocols for detecting recent HIV seroconversion.
Clin Diagn Lab Immunol. 2005 Aug;12(8):918-21.
Assay protocols of three rapid human immunodeficiency virus (HIV) assays, OraQuick-1/2, SeroStrip-1/2, and Determine-1/2, were modified to detect recent HIV seroconversion using a higher dilution of serum specimens. Optimal predilution of specimens resulted in negative test results during early periods of seroconversion (about 6 months), when antibody levels were low. A total of 269 seropositive specimens from routine HIV type 1 testing and from commercial sources (low-titer and seroconversion panels) were tested, and results were recorded as negative (score=0) or positive using intensity scores from 0.5 (weak positive) to 4 (strongly positive). The same specimens were previously tested by a less sensitive (LS) enzyme immunoassay (EIA), Abbott 3A 11-LS, and were classified as recent or long-term infections based on the standardized optical density (SOD) cutoff of 0.75. Overall concordance of >94% was observed between 3A 11-LS and modified rapid tests (RT-LSs) for detecting and distinguishing recent HIV seroconversion from long-term HIV infection (kappa statistics=0.894 to 0.901). Moreover, intensity scores on RT-LSs correlated well with median 3A 11-LS SOD values (R(2)>0.98). Our results indicate that rapid HIV tests can be modified to detect recent seroconversion with results comparable to those from less sensitive EIA. [Abstract/Link to Full Text]

Rauter C, Mueller M, Diterich I, Zeller S, Hassler D, Meergans T, Hartung T
Critical evaluation of urine-based PCR assay for diagnosis of Lyme borreliosis.
Clin Diagn Lab Immunol. 2005 Aug;12(8):910-7.
Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at--20 degrees C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 x g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis. [Abstract/Link to Full Text]

Luo L, Sabara MI
Production of a recombinant major inner capsid protein for serological detection of epizootic hemorrhagic disease virus.
Clin Diagn Lab Immunol. 2005 Aug;12(8):904-9.
Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent. [Abstract/Link to Full Text]

Rocha PN, Plumb TJ, Robinson LA, Spurney R, Pisetsky D, Koller BH, Coffman TM
Role of thromboxane A2 in the induction of apoptosis of immature thymocytes by lipopolysaccharide.
Clin Diagn Lab Immunol. 2005 Aug;12(8):896-903.
Lipopolysaccharide (LPS) causes apoptotic deletion of CD4(+) CD8(+) thymocytes, a phenomenon that has been linked to immune dysfunction and poor survival during sepsis. Given the abundance of thromboxane-prostanoid (TP) receptors in CD4(+) CD8(+) thymocytes and in vitro evidence that thromboxane A(2) (TXA(2)) causes apoptosis of these cells, we tested whether enhanced generation of TXA(2) plays a role in LPS-induced thymocyte apoptosis. Mice injected with 50 micro LPS intraperitoneally displayed a marked increase in generation of TXA(2) and prostaglandin E(2) in the thymus as well as apoptotic deletion of CD4(+) CD8(+) thymocytes. Administration of indomethacin or rofecoxib inhibited prostanoid synthesis but did not affect thymocyte death. In contrast, thymocyte apoptosis in response to LPS was significantly attenuated in TP-deficient mice. These studies indicate that TXA(2) mediates a portion of apoptotic thymocyte death caused by LPS. The absence of an effect of global inhibition of prostanoid synthesis suggests a complex role for prostanoids in this model. [Abstract/Link to Full Text]

Pugliese A, Vidotto V, Beltramo T, Torre D
Phagocytic activity in human immunodeficiency virus type 1 infection.
Clin Diagn Lab Immunol. 2005 Aug;12(8):889-95. [Abstract/Link to Full Text]

Liao M, Zhang S, Xuan X, Zhang G, Huang X, Igarashi I, Fujisaki K
Development of rapid immunochromatographic test with recombinant NcSAG1 for detection of antibodies to Neospora caninum in cattle.
Clin Diagn Lab Immunol. 2005 Jul;12(7):885-7.
An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle. [Abstract/Link to Full Text]

Videa E, Coloma MJ, Dos Santos FB, Balmaseda A, Harris E
Immunoglobulin M enzyme-linked immunosorbent assay using recombinant polypeptides for diagnosis of dengue.
Clin Diagn Lab Immunol. 2005 Jul;12(7):882-4.
We demonstrate that a mixture of four recombinant dengue virus E polypeptides corresponding to the N-terminal region of the envelope protein from all serotypes substitutes for standard antigens in two immunoglobulin M enzyme-linked immunosorbent assay formats with 100% concordance, making these polypeptides a useful and accessible reagent for serological diagnosis of dengue in endemic countries. [Abstract/Link to Full Text]

Lin M, Trottier E, Mallory M
Enzyme-linked immunosorbent assay based on a chimeric antigen bearing antigenic regions of structural proteins Erns and E2 for serodiagnosis of classical swine fever virus infection.
Clin Diagn Lab Immunol. 2005 Jul;12(7):877-81.
The antigenic region (residues 109 to 160) of classical swine fever virus (CSFV) protein E(rns) and the N-terminal antigenic region (residues 1 to 136) of protein E2 were constructed in the form of a fused, chimeric protein, C21E(rns)E2, for use as an enzyme-linked immunosorbent assay (ELISA) antigen for the serodiagnosis of CSFV infection. Tested with 238 negative-field (CSFV-free) sera from Canadian sources, the specificity of the ELISA was determined to be 93.7%. All 20 sera from experimentally infected pigs representing a variety of animals, virus strains, and days postinfection (dpi; range, 7 to 210) were detected as positive (100%). In contrast, an ELISA based on an E(rns) fragment (E(rns)(aa 109-160)) or an E2 fragment (E2(aa 1-221)) identified only 18 (90%) of 20 sera from infected pigs as positive, missing two targets collected at 7 dpi. These data suggest that use of the chimeric antigen C21E(rns)E2 would improve serodiagnostic sensitivity and allow for the detection of CSFV infection as early as 7 dpi. [Abstract/Link to Full Text]

Kang SY, Song KJ, Jeong SR, Kim JH, Park S, Kim K, Kwon MH, Shin HJ
Role of the Nfa1 protein in pathogenic Naegleria fowleri cocultured with CHO target cells.
Clin Diagn Lab Immunol. 2005 Jul;12(7):873-6.
Naegleria fowleri, a free-living amoeba, exists as a virulent pathogen which causes fatal primary amoebic meningoencephalitis in experimental animals and humans. Using infected and immune mouse sera, we previously cloned an nfa1 gene from a cDNA library of N. fowleri by immunoscreening. The nfa1 gene (360 bp) produced a recombinant 13.1-kDa protein, and the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. In this study, the role of the Nfa1 protein as a cell contact mechanism of N. fowleri cocultured with target cells was observed by an immunofluorescence assay with an anti-Nfa1 polyclonal antibody. Using confocal microscopic findings, the Nfa1 protein was located on the pseudopodia of N. fowleri trophozoites. The Nfa1 protein in N. fowleri trophozoites cocultured with CHO target cells was also located on pseudopodia, as well as in a food cup formed as a phagocytic structure in close contact with target cells. The amount of nfa1 mRNA of N. fowleri was strongly increased 6 h after coculture. [Abstract/Link to Full Text]

Karem KL, Reynolds M, Braden Z, Lou G, Bernard N, Patton J, Damon IK
characterization of acute-phase humoral immunity to monkeypox: use of immunoglobulin M enzyme-linked immunosorbent assay for detection of monkeypox infection during the 2003 North American outbreak.
Clin Diagn Lab Immunol. 2005 Jul;12(7):867-72.
A monkeypox outbreak occurred in the United States in 2003. Patient's sera were sent to the Centers for Disease Control and Prevention as a part of outbreak response measures. Clinical and epidemiologic information was abstracted from the case investigation forms. Serum samples from patients were tested by using an immunoglobulin M (IgM)-capture and an IgG enzyme-linked immunosorbent assay ELISA against Orthopoxvirus antigen. The detection of antiviral IgG and IgM antibodies and the kinetics of the antiviral IgG and IgM antibody responses were evaluated. Patients were classified as confirmed, probable, or suspect cases or were excluded as cases based on laboratory test results and epidemiologic and clinical criteria. A total of 37 confirmed case patients with monkeypox were identified, and 116 patients were excluded as case patients based on molecular testing or insufficient epidemiology and clinical data to warrant classification as a suspect or probable case. Of 37 confirmed case patients, 36 had a known history (presence or absence) of smallpox vaccination. Of those, 29 of the 36 either had or developed an IgG response, while 34 of the 36 developed an IgM response, regardless of vaccination status. Serum collected > or =5 days for IgM detection or serum collected > or =8 days after rash onset for IgG detection was most efficient for the detection of monkeypox virus infection. IgM ELISA detects recent infection with orthopoxviruses and, in this case, recent infection with monkeypox virus. In addition, analysis of paired sera for IgG and IgM detected seroconversion, another indicator of recent infection. The ELISA results correlated with the virologic PCR and viral culture results, indicating its diagnostic capabilities for monkeypox and potentially other orthopoxvirus infections due to zoonotic transmission or bioterrorism events. [Abstract/Link to Full Text]

Lainka E, Hershfield MS, Santisteban I, Bali P, Seibt A, Neubert J, Friedrich W, Niehues T
polyethylene glycol-conjugated adenosine deaminase (ADA) therapy provides temporary immune reconstitution to a child with delayed-onset ADA deficiency.
Clin Diagn Lab Immunol. 2005 Jul;12(7):861-6.
We describe the effects of polyethylene glycol-conjugated adenosine deaminase (ADA) replacement therapy on lymphocyte counts, activation, apoptosis, proliferation, and cytokine secretion in a 14-month-old girl with "delayed-onset" ADA deficiency and marked immunodysregulation. Pretreatment lymphopenia affected T cells (CD4, 150/microl; CD8, 459/microl), B cells (16/microl), and NK cells (55/microl). T cells were uniformly activated and largely apoptotic (CD4, 59%; CD8, 82%); and T-cell-dependent cytokine levels in plasma were elevated, including the levels of interleukin 2 (IL-2; 26 pg/ml), IL-4 (81 pg/ml), IL-5 (46 pg/ml), gamma interferon (1,430 pg/ml), tumor necrosis factor alpha (210 pg/ml), and IL-10 (168 pg/ml). Mitogen-stimulated peripheral blood mononuclear cells show reduced IL-2 secretion and proliferation. During the first 5 months of therapy there was clinical improvement and partial immune reconstitution, with nearly normal lymphocyte subset numbers, reduced T-cell activation and CD4-cell apoptosis, and decreased plasma cytokine levels. In parallel, IL-2 secretion and the lymphocyte mitogenic response improved. Between 4 and 7 months, immunoglobulin G antibodies to bovine ADA developed and resulted in the complete reversal of immune recovery. [Abstract/Link to Full Text]

Ozdemir O, Niehues
Increasing importance of stem cell gene therapy in adenosine deaminase deficiency?
Clin Vaccine Immunol. 2006 Mar;13(3):433-4; author reply 434-5. [Abstract/Link to Full Text]

TC, Parekh BS, Tih PM, Welty T, Welty E, Bulterys M, Ndikintum G, Nkuoh G, Tancho S
Evaluation of rapid prenatal human immunodeficiency virus testing in rural cameroon.
Clin Diagn Lab Immunol. 2005 Jul;12(7):855-60.
Pregnant women (n = 859) in rural Cameroonian prenatal clinics were screened by two rapid human immunodeficiency virus (HIV) antibody tests (rapid tests [RT]) (Determine and Hema-Strip) using either whole blood or plasma. One additional RT (Capillus, HIV-CHEK, or Sero-Card) was used to resolve discordant results. RT results were compared with HIV-1 enzyme immunoassay (EIA) and Western blot (WB) results of matched dried blood spots (DBS) to assess the accuracy of HIV RTs. DBS EIA/WB identified 83 HIV antibody-reactive, 763 HIV antibody-nonreactive, and 13 indeterminate specimens. RT results were evaluated in serial (two consecutive tests) or parallel (two simultaneous tests) testing algorithms. A serial algorithm using Determine and Hema-Strip yielded sensitivity and specificity results of 97.6% and 99.7%, respectively, whereas a parallel RT algorithm using Determine plus a second RT produced a sensitivity and specificity of 100% and 99.7%, respectively. HIV RTs provide excellent alternatives for identifying HIV infection, and their field performance could be monitored using DBS testing strategies. [Abstract/Link to Full Text]

Yu F, Le MQ, Inoue S, Thai HT, Hasebe F, Del Carmen Parquet M, Morita K
Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay.
Clin Diagn Lab Immunol. 2005 Jul;12(7):848-54.
Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N' protein and (N)Delta(121) protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N' protein-based ELISA showed a highly nonspecific reaction. The (N)Delta(121) protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N' protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV (N)Delta(121) protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the (N)Delta(121) protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist. [Abstract/Link to Full Text]

Toptygina AP, Pukhalsky AL, Alioshkin VA
Immunoglobulin G subclass profile of antimeasles response in vaccinated children and in adults with measles history.
Clin Diagn Lab Immunol. 2005 Jul;12(7):845-7.
The aim of this study was to investigate measles-specific immunoglobulin G (IgG) subclass profile in vaccinated children and in adults with natural infection. Serum samples were collected before and 30 days after vaccination. The sera from 51 late convalescent adults and seven adults with natural measles infection at the 12th day after onset of rash have been also investigated. Measles IgG antibodies and specific IgG subclasses were tested by enzyme-linked immunosorbent techniques. In children younger than 3 years, the predominant subclass was IgG3, which contributed, on average, 63.3% of the total IgG response. The contributions of specific IgG1 and IgG4 to the total IgG antimeasles response were lower (19.9% and 16.8%, respectively), whereas IgG2 was not found. In contrast, in the group of children older than 4 years, just IgG2 was a predominant subclass; it contributed 42.6% of the total IgG response. Other subclasses were also present but the contribution was much lower. In adult volunteers with measles history, IgG2 was a predominant subclass of total IgG. Thus, in early convalescence IgG2 contributed 62% of the total IgG response, whereas in late convalescence the contribution was lower (41.4%). There were no visible differences in IgG subclass composition between subjects with natural infection and vaccinated children except those below 3 years of age. The humoral immune response of such subjects is immature and the IgG2 subclass of virus-specific antibodies has not been revealed in the sera. [Abstract/Link to Full Text]

Buhimschi IA, Buhimschi CS, Weiner CP, Kimura T, Hamar BD, Sfakianaki AK, Norwitz ER, Funai EF, Ratner E
Proteomic but not enzyme-linked immunosorbent assay technology detects amniotic fluid monomeric calgranulins from their complexed calprotectin form.
Clin Diagn Lab Immunol. 2005 Jul;12(7):837-44.
Four proteomic biomarkers (human neutrophil peptide 1 [HNP1], HNP2 [defensins], calgranulin C [Cal-C], and Cal-A) characterize the fingerprint of intra-amniotic inflammation (IAI). We compared proteomic technology using surfaced-enhanced laser desorption-ionization-time of flight (SELDI-TOF) mass spectrometry to enzyme-linked immunosorbent assay (ELISA) for detection of these biomarkers. Amniocentesis was performed on 48 women enrolled in two groups: those with intact membranes (n = 27; gestational age [GA], 26.0 +/- 0.8 weeks) and those with preterm premature rupture of the membranes (PPROM; n = 21; GA, 28.4 +/- 0.9 weeks). Paired abdominal amniotic fluids (aAFs)-vaginal AFs (vAFs) were analyzed in PPROM women. Quantitative aspects of HNP1-3, Cal-C, Cal-A, and calprotectin (a complex of Cal-A with Cal-B) were assessed by ELISA. SELDI-TOF mass spectrometry tracings from 16/48 (33.3%) aAFs and 13/17 (88.2%) vAFs were consistent with IAI (three or four biomarkers present). IAI (by SELDI-TOF mass spectrometry) was associated with increased HNP1-3 and Cal-C measured by ELISA. However, immunoassays detected Cal-A in only 4 of the AFs even though its specific SELDI-TOF mass spectrometry peak was identified in 19/48 AFs. Calprotectin immunoreactivity was decreased in AFs retrieved from women with IAI (P = 0.01). In conclusion, IAI is associated with increased HNP1-3 levels. In the absence of isoform-specific ELISAs, mass spectrometry remains the only way to discriminate the HNP biomarker isoforms. Monomeric Cal-A is not reliably estimated by specific ELISA as it binds to Cal-B to form the calprotectin complex. Cal-C was reliably measured by SELDI-TOF mass spectrometry or specific ELISA. [Abstract/Link to Full Text]

Dale JB, Penfound T, Chiang EY, Long V, Shulman ST, Beall B
Multivalent group A streptococcal vaccine elicits bactericidal antibodies against variant M subtypes.
Clin Diagn Lab Immunol. 2005 Jul;12(7):833-6.
Group A streptococci cause a wide spectrum of clinical illness. One of several strategies for vaccine prevention of these infections is based on the type-specific M protein epitopes. A multivalent M protein-based vaccine containing type-specific determinants from 26 different M serotypes is now in clinical trials. Recent epidemiologic studies have shown that, within some serotypes, the amino-terminal M protein sequence may show natural variation, giving rise to subtypes. This raises the possibility that vaccine-induced antibodies against the parent type may not be as effective in promoting bactericidal killing of variant subtypes. In the present study we used rabbit antisera against the 26-valent M protein-based vaccine in bactericidal tests against M1, M3, and M5 streptococci, which were represented by multiple subtypes. We show that the vaccine antibodies effectively promoted in vitro bactericidal activity despite the fact that the M proteins contained naturally occurring variant sequences in the regions corresponding to the vaccine sequence. Our results show that the variant M proteins generally do not result in significant differences in opsonization promoted by rabbit antisera raised against the 26-valent vaccine, suggesting that a multivalent M protein vaccine may not permit variant subtypes of group A streptococci to escape in a highly immunized population. [Abstract/Link to Full Text]

Aghamohammadi A, Farhoudi A, Moin M, Rezaei N, Kouhi A, Pourpak Z, Yaseri N, Movahedi M, Gharagozlou M, Zandieh F, Yazadni F, Arshi S, Mohammadzadeh I, Ghazi BM, Mahmoudi M, Tahaei S, Isaeian A
Clinical and immunological features of 65 Iranian patients with common variable immunodeficiency.
Clin Diagn Lab Immunol. 2005 Jul;12(7):825-32.
Common variable immunodeficiency (CVID) is a primary immunodeficiency disease characterized by hypogammaglobulinemia and recurrent bacterial infections. The records of 65 patients with CVID (37 males and 28 females) in the age range of 24 to 537 months were reviewed. By the year 2003, 11 patients had died and seven patients could not be located. The total follow-up period was 221 patient-years. The median diagnostic delay (time between onset and diagnosis) in our patient group was 60 months. At the time of diagnosis, the baseline serum immunoglobulin G (IgG), IgM, and IgA levels were below the level normal for the patients' age; the medians for this group were 120, 10, and 0 mg/dl, respectively. All of the patients presented with infectious diseases at the time of onset, the most common of which were otitis media, diarrhea, pneumonia, and sinusitis. Acute and recurrent infections were also found in almost all of the patients, particularly involving respiratory and gastrointestinal systems. The most common infections, before diagnosis and during follow-up, were pneumonia, acute diarrhea, acute sinusitis, and otitis media. CVID should be considered in any patient with a history of recurrent infections and decreased levels of all serum immunoglobulin isotypes. [Abstract/Link to Full Text]

Bonfield TL, John N, Barna BP, Kavuru MS, Thomassen MJ, Yen-Lieberman B
Multiplexed particle-based anti-granulocyte macrophage colony stimulating factor assay used as pulmonary diagnostic test.
Clin Diagn Lab Immunol. 2005 Jul;12(7):821-4.
Pulmonary alveolar proteinosis (PAP) is characterized by the accumulation of lipoproteinaceous material within the lung alveoli. Recent studies indicate that PAP is an autoimmune disease characterized by a neutralizing anti-granulocyte macrophage colony stimulating factor (GM-CSF) antibody. At present the only definitive diagnostic test for PAP is open lung biopsy. We have previously published that anti-GM-CSF is diagnostic for PAP and correlates with disease pathogenesis using a traditional serial anti-GM-CSF antibody titer format (T. L. Bonfield, M. S. Kavuru, and M. J. Thomassen, Clin. Immunol. 105:342-350, 2002). Titer analysis is a semiquantitative method, and often subtle changes in antibody titer are not detectable. In this report we present data to support anti-GM-CSF detection by a quantitative highly sensitive multiplexed particle-based assay which has the potential to be a clinical diagnostic test. [Abstract/Link to Full Text]

Schütz E, Urnovitz HB, Iakoubov L, Schulz-Schaeffer W, Wemheuer W, Brenig B
Bov-tA short interspersed nucleotide element sequences in circulating nucleic acids from sera of cattle with bovine spongiform encephalopathy (BSE) and sera of cattle exposed to BSE.
Clin Diagn Lab Immunol. 2005 Jul;12(7):814-20.
Circulating nucleic acids (CNA) are known to be enriched in repetitive DNA sequences in humans. Here, bovine sera CNA were analyzed to determine if cell stress-related short interspersed nucleotide elements (SINEs) could be detected in sera from cattle associated with bovine spongiform encephalopathy (BSE). Nucleic acids were extracted, amplified, cloned, and sequenced from the sera of protease-resistant prion protein (PrP(res))-positive cattle (n = 2) and sera from BSE-cohort cows (n = 6); 150 out of 163 clones revealed the presence of, on average, an 80-bp sequence from the 3' region of Bov-tA SINE. A PCR protocol was developed that differentially identified SINE-associated CNA in BSE-exposed versus normal cattle. CNA were extracted from a serum vesicular fraction after controlled blood collection and processing procedures. Sera from four confirmed cases of BSE, 137 BSE-exposed cohort animals associated with eight confirmed BSE cases, and 845 healthy, PrP(res)-negative control cows were tested. All four sera from confirmed BSE cases were repeatedly reactive in the assay. BSE-exposed cohorts had a 100-fold higher occurrence of repeatedly reactive individuals per cohort (average = 63%; range = 33% to 91%), compared to healthy controls (average = 0.6%; P < 0.001). This study shows that BSE-confirmed and cohort animals possess a unique profile of SINE-associated serum CNA that can be utilized as a marker that highly correlates to BSE exposure. [Abstract/Link to Full Text]

Fernández RG, Leehan JA, Pastrana RF, Muńiz RO
Effect of malnutrition on K+ current in T lymphocytes.
Clin Diagn Lab Immunol. 2005 Jul;12(7):808-13.
Severe malnutrition in children is frequently associated with infectious diseases. Animal models have been useful for studying the effects of malnutrition. One of the immunosuppressive mechanisms of malnutrition is inhibition of the activation of T lymphocytes. The voltage-dependent K(V) potassium channels are vital for the activation of T lymphocytes. The blockade of K(V) channels inhibits the activation of T lymphocytes. Malnutrition could affect the suitable synthesis of K(V) channels in T lymphocytes, producing changes in the magnitude and/or dependency of the voltage of the K+ current. We reported a significant decrease in the K+ current and activation to a 20 mV more positive membrane potential in T lymphocytes of rats with severe malnutrition. These results indicate that the diminution in the K+ conductance by alteration of K(V) channels in severe malnutrition is one of the mechanisms that inhibit the activation of T lymphocytes. [Abstract/Link to Full Text]

Hamby CV, Llibre M, Utpat S, Wormser GP
Use of Peptide library screening to detect a previously unknown linear diagnostic epitope: proof of principle by use of lyme disease sera.
Clin Diagn Lab Immunol. 2005 Jul;12(7):801-7.
Diagnostic peptides previously isolated from phage-displayed libraries by affinity selection with serum antibodies from patients with Lyme disease were found to give reproducible serum reactivity patterns when tested in two different enzyme-linked immunosorbent assay formats. In addition, the hypothetical possibility that peptides selected by this type of "epitope discovery" technique might identify the original antigens eliciting antibody responses was tested by searching for sequence similarities in bacterial protein databases. In support of this hypothesis, our search uncovered similarities between peptides representing two different sequence motifs and sequences in the VlsE and BBA61 antigens of Borrelia burgdorferi. Utilizing synthetic peptides, we verified that the sequence KAASKETPPALNK, located at the C terminus of the VlsE antigen, had the same reactivity pattern to sera from patients with extracutaneous Lyme disease as the diagnostic peptide SKEKPPSLNWPA, with which it shared a 7-amino-acid-residue match (consensus residues are underlined). A peptide with conservative mutations of five of the consensus residues was nonreactive, strongly suggesting that the VlsE sequence represents the epitope that originally elicited antibody responses in these patients. The diagnostic sensitivity of this new VlsE epitope was relatively low (30%) compared to that (100%) of the well-documented C6 diagnostic peptide of VlsE when tested in our small cohort of 10 patients with Lyme disease. Nonetheless, the identification of this previously unknown epitope serves as a proof of the principle of the hypothetical ability of "epitope discovery" techniques to detect specific microbial antigens with diagnostic relevance in infectious diseases. [Abstract/Link to Full Text]

Herrmann LM, McGuire TC, Hötzel I, Lewis GS, Knowles DP
Surface envelope glycoprotein is B-lymphocyte immunodominant in sheep naturally infected with ovine progressive pneumonia virus.
Clin Diagn Lab Immunol. 2005 Jun;12(6):797-800.
The B-lymphocyte-immunodominant antigen involved in naturally ovine progressive pneumonia virus (OPPV)-infected mature sheep remains unknown. Therefore, the amount of antibody in sera from 10 naturally OPPV-infected sheep was evaluated by immunoprecipitation (IP) of the major viral proteins in [(35)S]methionine/cysteine-labeled OPPV (whole virus) lysate. Using an excess of OPPV proteins in whole-virus lysate, 8 out of 10 sheep had the highest serum antibody IP endpoint titers to the gp135 surface envelope glycoprotein (SU). Also, 2 out of 10 sheep had equivalent serum antibody IP endpoint titers to the transmembrane glycoprotein oligomer (TM90) and SU. Since these data indicate that SU is the immunodominant protein in most mature sheep persistently infected with OPPV, SU-specific diagnostic serological assays can be utilized for OPPV diagnosis. [Abstract/Link to Full Text]

Hostetter J, Kagan R, Steadham E
Opsonization effects on Mycobacterium avium subsp. paratuberculosis--macrophage interactions.
Clin Diagn Lab Immunol. 2005 Jun;12(6):793-6.
High antibody titers in ruminants infected with Mycobacterium avium subsp. paratuberculosis correlates with disease progression. Effects of humoral responses during mycobacterial infection are not completely understood. This study suggests that activation status may be an important factor in determining macrophage ability to limit proliferation of opsonized M. avium subsp. paratuberculosis. [Abstract/Link to Full Text]

Nardelli DT, Cloute JP, Luk KH, Torrealba J, Warner TF, Callister SM, Schell RF
CD4(+) CD25(+) T cells prevent arthritis associated with Borrelia vaccination and infection.
Clin Diagn Lab Immunol. 2005 Jun;12(6):786-92.
CD4(+) CD25(+) T cells are a population of regulatory T cells associated with control of arthritis in anti-interleukin-17 antibody-treated Borrelia-vaccinated and challenged gamma interferon-deficient mice. Here, we present direct evidence that adoptive transfer of enriched CD4(+) CD25(+) T cells from these mice can prevent the development of arthritis in Borrelia-vaccinated and challenged mice. These findings establish a major role for CD4(+) CD25(+) T cells in the prevention of arthritis in Borrelia-vaccinated and challenged animals. [Abstract/Link to Full Text]

Li F, Stevenson RA, Crabb BS, Studdert MJ, Hartley CA
Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.
Clin Diagn Lab Immunol. 2005 Jun;12(6):778-85.
Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA. [Abstract/Link to Full Text]

Livingstone M, Entrican G, Wattegedera S, Buxton D, McKendrick IJ, Longbottom D
Antibody responses to recombinant protein fragments of the major outer membrane protein and polymorphic outer membrane protein POMP90 in Chlamydophila abortus-infected pregnant sheep.
Clin Diagn Lab Immunol. 2005 Jun;12(6):770-7.
Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans. [Abstract/Link to Full Text]

Levine M, Owen WL, Avery KT
Antibody response to actinomyces antigen and dental caries experience: implications for caries susceptibility.
Clin Diagn Lab Immunol. 2005 Jun;12(6):764-9.
Fluoridated dentifrices reduce dental caries in subjects who perform effective oral hygiene. Actinomyces naeslundii increases in teeth-adherent microbial biofilms (plaques) in these subjects, and a well-characterized serum immunoglobulin G (IgG) antibody response (Actinomyces antibody [A-Ab]) is also increased. Other studies suggest that a serum IgG antibody response to streptococcal d-alanyl poly(glycerophosphate) (S-Ab) may indicate caries experience associated strongly with gingival health and exposure to fluoridated water. The aim of this study was to investigate relationships between A-Ab response, oral hygiene, S-Ab response, and caries experience. Measurements were made of A-Ab and S-Ab concentrations, caries experience (number of decayed, missing, and filled teeth [DMFT], number of teeth surfaces [DMFS], and number of decayed teeth needing treated [DT]), exposure to fluoridated water (Flu), mean clinical pocket depth (PD; in millimeters), and extent of plaque (PL) and gingival bleeding on probing (BOP). A-Ab concentration, the dependent variable in a multiple regression analysis, increased with S-Ab concentration and decreased with PL and DMFT adjusted for Flu (R(2) = 0.51, P < 0.002). Residual associations with age, DMFS, DT, and BOP were not significant. In addition, an elevated A-Ab response, defined from immunoprecipitation and immunoassay measurements, indicated a significant, 30% reduction in DMFT after adjustment for significant age and Flu covariance (analysis of variance with covariance F statistic = 10.6, P < 0.003; S-Ab response and interactions not significant). Thus, an elevated A-Ab response indicates less caries in subjects performing effective oral hygiene using fluoridated dentifrices. Conversely, a low A-Ab response is suggestive of decreased A. naeslundii binding to saliva-coated apatite and greater caries experience, as reported by others. [Abstract/Link to Full Text]

---

Recent Articles in Clinical and Molecular Allergy

---

No recent articles are currently available.

---

Recent Articles in Clinical Microbiology Reviews

---

Winstanley P, Ward S, Snow R, Breckenridge A
Therapy of falciparum malaria in sub-saharan Africa: from molecule to policy.
Clin Microbiol Rev. 2004 Jul;17(3):612-37, table of contents.
The burden of falciparum malaria remains as great as ever, and, as has probably always been the case, it is carried mainly by tropical Africa. Of the various means available for the control of malaria, the use of effective drugs remains the most important and is likely to remain so for a considerable time to come. Unfortunately, the extensive development of resistance by the parasite threatens the utility of most of the affordable classes of drug: the development of novel antimalarials has never been more urgently needed. Any attempt to understand the vast complexities of falciparum malaria in Africa requires an ability to think "from molecule to policy." In consequence, the review ambitiously tries to examine the current pharmacopeia, the process by which new drugs are developed and the ways in which drugs are actually used, in both the formal and informal health sectors. The informal sector is particularly important in Africa, where around half of all antimalarial treatments are bought from informal outlets and taken at home without supervision by health care professionals: the potential impact of adherence on clinical outcome is discussed. Given that the full costs are carried by the patient in a large proportion of cases, the importance of drug affordability is explored. The review also discusses the splicing of new drugs into national policy. The various parameters that feed into deliberations on changes in drug policy are discussed. [Abstract/Link to Full Text]

van der Woude MW, Bäumler AJ
Phase and antigenic variation in bacteria.
Clin Microbiol Rev. 2004 Jul;17(3):581-611, table of contents.
Phase and antigenic variation result in a heterogenic phenotype of a clonal bacterial population, in which individual cells either express the phase-variable protein(s) or not, or express one of multiple antigenic forms of the protein, respectively. This form of regulation has been identified mainly, but by no means exclusively, for a wide variety of surface structures in animal pathogens and is implicated as a virulence strategy. This review provides an overview of the many bacterial proteins and structures that are under the control of phase or antigenic variation. The context is mainly within the role of the proteins and variation for pathogenesis, which reflects the main body of literature. The occurrence of phase variation in expression of genes not readily recognizable as virulence factors is highlighted as well, to illustrate that our current knowledge is incomplete. From recent genome sequence analysis, it has become clear that phase variation may be more widespread than is currently recognized, and a brief discussion is included to show how genome sequence analysis can provide novel information, as well as its limitations. The current state of knowledge of the molecular mechanisms leading to phase variation and antigenic variation are reviewed, and the way in which these mechanisms form part of the general regulatory network of the cell is addressed. Arguments both for and against a role of phase and antigenic variation in immune evasion are presented and put into new perspective by distinguishing between a role in bacterial persistence in a host and a role in facilitating evasion of cross-immunity. Finally, examples are presented to illustrate that phase-variable gene expression should be taken into account in the development of diagnostic assays and in the interpretation of experimental results and epidemiological studies. [Abstract/Link to Full Text]

Gerber MA, Shulman ST
Rapid diagnosis of pharyngitis caused by group A streptococci.
Clin Microbiol Rev. 2004 Jul;17(3):571-80, table of contents.
Although commercial rapid antigen detection tests (RADTs) are more expensive than blood agar plate (BAP) cultures, the advantage they offer is the speed with which they provide results. Rapid identification and consequent prompt treatment of patients with pharyngitis due to group A beta-hemolytic streptococci (GABHS) can reduce the risk of spread of GABHS, can allow patients to return to school or work sooner, and may reduce the acute morbidity of this illness. In most studies, RADTs have been compared with BAP cultures as the criterion standard. However, these comparisons are complicated by the fact that there is no universally accepted procedure for performing a BAP culture. The great majority of the RADTs that are currently available have a high specificity (i.e., 95% or greater) and a sensitivity of between 70 and 90% compared with BAP cultures. Few published studies have compared the performance of various RADTs to each other or examined the performance of various RADTs in the office setting. There is also relatively little published information about how physicians in practice actually use RADTs, but the available information suggests that many physicians do not follow recommended guidelines. While the development of easy-to-perform RADTs for the diagnosis of GABHS pharyngitis has altered clinical practice substantially, only limited data about cost-effectiveness are currently available. [Abstract/Link to Full Text]

Johnson EH, Windsor JJ, Clark CG
Emerging from obscurity: biological, clinical, and diagnostic aspects of Dientamoeba fragilis.
Clin Microbiol Rev. 2004 Jul;17(3):553-70, table of contents.
Ever since its first description in 1918, Dientamoeba fragilis has struggled to gain recognition as a significant pathogen. There is little justification for this neglect, however, since there exists a growing body of case reports from numerous countries around the world that have linked this protozoal parasite to clinical manifestations such as diarrhea, abdominal pain, flatulence, and anorexia. A number of studies have even incriminated D. fragilis as a cause of irritable bowel syndrome, allergic colitis, and diarrhea in human immunodeficiency virus patients. Although D. fragilis is most commonly identified using permanently stained fecal smears, recent advances in culturing techniques are simplifying as well as improving the ability of investigators to detect this organism. However, there are limitations in the use of cultures since they cannot be performed on fecal samples that have been fixed. Significant progress has been made in the biological classification of this organism, which originally was described as an ameba. Analyses of small-subunit rRNA gene sequences have clearly demonstrated its close relationship to Histomonas, and it is now known to be a trichomonad. How the organism is transmitted remains a mystery, although there is some evidence that D. fragilis might be transmitted via the ova of the pinworm, Enterobius vermicularis. Also, it remains to be answered whether the two distinct genotypes of D. fragilis recently identified represent organisms with differing virulence. [Abstract/Link to Full Text]

Choi BI, Han JK, Hong ST, Lee KH
Clonorchiasis and cholangiocarcinoma: etiologic relationship and imaging diagnosis.
Clin Microbiol Rev. 2004 Jul;17(3):540-52, table of contents.
Despite a gradual decrease in prevalence, clonorchiasis is still prevalent in East Asia. A large and compelling body of evidence links clonorchiasis and cholangiocarcinoma, although the mechanisms involved are not completely understood. Clonorchiasis induces biliary epithelial hyperplasia and metaplasia, and this could facilitate at least one stage of the carcinogenesis, which is promoting effect. In areas of endemic infection, more clonorchiasis cases are now diagnosed incidentally during radiological examinations such as cholangiography, ultrasonography, and computed tomography. Radiological findings are regarded as pathognomonic for clonorchiasis since they reflect the unique pathological changes of this disorder. These radiological examinations currently play important roles in the diagnosis, staging, and decision-making process involved in the treatment of cholangiocarcinoma. The morphological features and radiological findings of clonorchiasis-associated cholangiocarcinoma are essentially combinations of the findings for the two diseases. The morphological features of clonorchiasis- associated cholangiocarcinoma, observed in radiological examinations, do not differ from those of the usual cholangiocarcinoma. In patients diagnosed with or suspected to have clonorchiasis, radiological findings should be carefully scrutinized for occult cholangiocarcinoma. [Abstract/Link to Full Text]

Clark IA, Alleva LM, Mills AC, Cowden WB
Pathogenesis of malaria and clinically similar conditions.
Clin Microbiol Rev. 2004 Jul;17(3):509-39, table of contents.
There is now wide acceptance of the concept that the similarity between many acute infectious diseases, be they viral, bacterial, or parasitic in origin, is caused by the overproduction of inflammatory cytokines initiated when the organism interacts with the innate immune system. This is also true of certain noninfectious states, such as the tissue injury syndromes. This review discusses the historical origins of these ideas, which began with tumor necrosis factor (TNF) and spread from their origins in malaria research to other fields. As well the more established proinflammatory mediators, such as TNF, interleukin-1, and lymphotoxin, the roles of nitric oxide and carbon monoxide, which are chiefly inhibitory, are discussed. The established and potential roles of two more recently recognized contributors, overactivity of the enzyme poly(ADP-ribose) polymerase 1 (PARP-1) and the escape of high-mobility-group box 1 (HMGB1) protein from its normal location into the circulation, are also put in context. The pathogenesis of the disease caused by falciparum malaria is then considered in the light of what has been learned about the roles of these mediators in these other diseases, as well as in malaria itself. [Abstract/Link to Full Text]

Vilchez RA, Butel JS
Emergent human pathogen simian virus 40 and its role in cancer.
Clin Microbiol Rev. 2004 Jul;17(3):495-508, table of contents.
The polyomavirus simian virus 40 (SV40) is a known oncogenic DNA virus which induces primary brain and bone cancers, malignant mesothelioma, and lymphomas in laboratory animals. Persuasive evidence now indicates that SV40 is causing infections in humans today and represents an emerging pathogen. A meta-analysis of molecular, pathological, and clinical data from 1,793 cancer patients indicates that there is a significant excess risk of SV40 associated with human primary brain cancers, primary bone cancers, malignant mesothelioma, and non-Hodgkin's lymphoma. Experimental data strongly suggest that SV40 may be functionally important in the development of some of those human malignancies. Therefore, the major types of tumors induced by SV40 in laboratory animals are the same as those human malignancies found to contain SV40 markers. The Institute of Medicine recently concluded that "the biological evidence is of moderate strength that SV40 exposure could lead to cancer in humans under natural conditions." This review analyzes the accumulating data that indicate that SV40 is a pathogen which has a possible etiologic role in human malignancies. Future research directions are considered. [Abstract/Link to Full Text]

Grubman MJ, Baxt B
Foot-and-mouth disease.
Clin Microbiol Rev. 2004 Apr;17(2):465-93.
Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. The disease was initially described in the 16th century and was the first animal pathogen identified as a virus. Recent FMD outbreaks in developed countries and their significant economic impact have increased the concern of governments worldwide. This review describes the reemergence of FMD in developed countries that had been disease free for many years and the effect that this has had on disease control strategies. The etiologic agent, FMD virus (FMDV), a member of the Picornaviridae family, is examined in detail at the genetic, structural, and biochemical levels and in terms of its antigenic diversity. The virus replication cycle, including virus-receptor interactions as well as unique aspects of virus translation and shutoff of host macromolecular synthesis, is discussed. This information has been the basis for the development of improved protocols to rapidly identify disease outbreaks, to differentiate vaccinated from infected animals, and to begin to identify and test novel vaccine candidates. Furthermore, this knowledge, coupled with the ability to manipulate FMDV genomes at the molecular level, has provided the framework for examination of disease pathogenesis and the development of a more complete understanding of the virus and host factors involved. [Abstract/Link to Full Text]

Anisimov AP, Lindler LE, Pier GB
Intraspecific diversity of Yersinia pestis.
Clin Microbiol Rev. 2004 Apr;17(2):434-64.
Increased interest in the pathogenic potential of Yersinia pestis has emerged because of the potential threats from bioterrorism. Pathogenic potential is based on genetic factors present in a population of microbes, yet most studies evaluating the role of specific genes in virulence have used a limited number of strains. For Y. pestis this issue is complicated by the fact that most strains available for study in the Americas are clonally derived and thus genetically restricted, emanating from a strain of Y. pestis introduced into the United States in 1902 via marine shipping and subsequent spread of this strain throughout North and South America. In countries from the former Soviet Union (FSU), Mongolia, and China there are large areas of enzootic foci of Y. pestis infection containing genetically diverse strains that have been intensely studied by scientists in these countries. However, the results of these investigations are not generally known outside of these countries. Here we describe the variety of methods used in the FSU to classify Y. pestis strains based on genetic and phenotypic variation and show that there is a high level of diversity in these strains not reflected by ones obtained from sylvatic areas and patients in the Americas. [Abstract/Link to Full Text]

Greub G, Raoult D
Microorganisms resistant to free-living amoebae.
Clin Microbiol Rev. 2004 Apr;17(2):413-33.
Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human "Troy," and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens. [Abstract/Link to Full Text]

Easton AJ, Domachowske JB, Rosenberg HF
Animal pneumoviruses: molecular genetics and pathogenesis.
Clin Microbiol Rev. 2004 Apr;17(2):390-412.
Pneumoviruses are single-stranded, negative-sense, nonsegmented RNA viruses of the family Paramyxoviridae, subfamily Pneumovirinae, and include pathogens that infect humans (respiratory syncytial virus and human metapneumovirus), domestic mammals (bovine, ovine, and caprine respiratory syncytial viruses), rodents (pneumonia virus of mice), and birds (avian metapneumovirus). Among the topics considered in this review are recent studies focused on the roles of the individual virus-encoded components in promoting virus replication as well as in altering and evading innate antiviral host defenses. Advances in the molecular technology of pneumoviruses and the emergence of recombinant pneumoviruses that are leading to improved virus-based vaccine formulations are also discussed. Since pneumovirus infection in natural hosts is associated with a profound inflammatory response that persists despite adequate antiviral therapy, we also review the recent experimental treatment strategies that have focused on combined antiviral, anti-inflammatory, and immunomodulatory approaches. [Abstract/Link to Full Text]

Giri M, Ugen KE, Weiner DB
DNA vaccines against human immunodeficiency virus type 1 in the past decade.
Clin Microbiol Rev. 2004 Apr;17(2):370-89.
This article reviews advances in the field of human immunodeficiency virus type 1 (HIV-1) and AIDS vaccine development over the last decade, with an emphasis on the DNA vaccination approach. Despite the discovery of HIV-1 and AIDS in humans nearly 20 years ago, there is no vaccine yet that can prevent HIV-1 infection. The focus has shifted toward developing vaccines that can control virus replication and disease progression by eliciting broadly cross-reactive T-cell responses. Among several approaches evaluated, the DNA-based modality has shown considerable promise in terms of its ability to elicit cellular immune responses in primate studies. Of great importance are efforts aimed at improvement of the potency of this modality in the clinic. The review discusses principles of DNA vaccine design and the various mechanisms of plasmid-encoded antigen presentation. The review also outlines current DNA-based vaccine strategies and vectors that have successfully been shown to control virus replication and slow disease progression in animal models. Finally, it lists recent strategies that have been developed as well as novel approaches under consideration to enhance the immunogenicity of plasmid-encoded HIV-1 antigen in various animal models. [Abstract/Link to Full Text]

Colmegna I, Cuchacovich R, Espinoza LR
HLA-B27-associated reactive arthritis: pathogenetic and clinical considerations.
Clin Microbiol Rev. 2004 Apr;17(2):348-69.
Current evidence supports the concept that reactive arthritis (ReA) is an immune-mediated synovitis resulting from slow bacterial infections and showing intra-articular persistence of viable, non-culturable bacteria and/or immunogenetic bacterial antigens synthesized by metabolically active bacteria residing in the joint and/or elsewhere in the body. The mechanisms that lead to the development of ReA are complex and basically involve an interaction between an arthritogenic agent and a predisposed host. The way in which a host accommodates to invasive facultative intracellular bacteria is the key to the development of ReA. The details of the molecular pathways that explain the articular and extra-articular manifestations of the disease are still under investigation. Several studies have been done to gain a better understanding of the pathogenesis of ReA; these constitute the basis for a more rational therapeutic approach to this disease. [Abstract/Link to Full Text]

Drevets DA, Leenen PJ, Greenfield RA
Invasion of the central nervous system by intracellular bacteria.
Clin Microbiol Rev. 2004 Apr;17(2):323-47.
Infection of the central nervous system (CNS) is a severe and frequently fatal event during the course of many diseases caused by microbes with predominantly intracellular life cycles. Examples of these include the facultative intracellular bacteria Listeria monocytogenes, Mycobacterium tuberculosis, and Brucella and Salmonella spp. and obligate intracellular microbes of the Rickettsiaceae family and Tropheryma whipplei. Unfortunately, the mechanisms used by intracellular bacterial pathogens to enter the CNS are less well known than those used by bacterial pathogens with an extracellular life cycle. The goal of this review is to elaborate on the means by which intracellular bacterial pathogens establish infection within the CNS. This review encompasses the clinical and pathological findings that pertain to the CNS infection in humans and includes experimental data from animal models that illuminate how these microbes enter the CNS. Recent experimental data showing that L. monocytogenes can invade the CNS by more than one mechanism make it a useful model for discussing the various routes for neuroinvasion used by intracellular bacterial pathogens. [Abstract/Link to Full Text]

Chiu CH, Su LH, Chu C
Salmonella enterica serotype Choleraesuis: epidemiology, pathogenesis, clinical disease, and treatment.
Clin Microbiol Rev. 2004 Apr;17(2):311-22.
Nontyphoid Salmonella strains are important causes of reportable food-borne infection. Among more than 2,000 serotypes, Salmonella enterica serotype Choleraesuis shows the highest predilection to cause systemic infections in humans. The most feared complication of serotype Cholearesuis bacteremia in adults is the development of mycotic aneurysm, which previously was almost uniformally fatal. The advances in diagnostic techniques, surgical care, and antimicrobial therapy have greatly improved the survival of these patients. However, the recent emergence of serotype Choleraesuis that is resistant to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and, notably, fluoroquinolone antibiotics has aroused concern about the use of these agents for the empirical treatment of systemic infection caused by this organism. In view of the serious implications of the situation, the chain of transmission and mechanism of resistance should be carefully studied to reduce the spread of infection and threat to human health. To date, there are no vaccines available to prevent serotype Choleraesuis infections in humans. The availability, in the near future, of the genome sequence of serotype Cholearesuis will facilitate the development of effective vaccines as well as the discovery of new targets for novel antimicrobial agents. [Abstract/Link to Full Text]

Masuoka J
Surface glycans of Candida albicans and other pathogenic fungi: physiological roles, clinical uses, and experimental challenges.
Clin Microbiol Rev. 2004 Apr;17(2):281-310.
Although fungi have always been with us as commensals and pathogens, fungal infections have been increasing in frequency over the past few decades. There is a growing body of literature describing the involvement of carbohydrate groups in various aspects of fungal disease. Carbohydrates comprising the cell wall or capsule, or as a component of glycoproteins, are the fungal cell surface entities most likely to be exposed to the surrounding environment. Thus, the fungus-host interaction is likely to involve carbohydrates before DNA, RNA, or even protein. The interaction between fungal and host cells is also complex, and early studies using whole cells or crude cell fractions often produced seemingly conflicting results. What was needed, and what has been developing, is the ability to identify specific glycan structures and determine how they interact with immune system components. Carbohydrate analysis is complicated by the complexity of glycan structures and by the challenges of separating and detecting carbohydrates experimentally. Advances in carbohydrate chemistry have enabled us to move from the foundation of composition analysis to more rapid characterization of specific structures. This, in turn, will lead to a greater understanding of how fungi coexist with their hosts as commensals or exist in conflict as pathogens. [Abstract/Link to Full Text]

Pfaller MA, Sheehan DJ, Rex JH
Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization.
Clin Microbiol Rev. 2004 Apr;17(2):268-80.
In certain unique clinical settings, the ability of the antimicrobial agent administered to kill the pathogen outright may be quite important. These situations invariably involve infection of a site not easily accessed by host defenses and/or of a structure with essential anatomic or physiologic function such as the heart (endocarditis), central nervous system (meningitis), or bone (osteomyelitis). Likewise, infections in immunosuppressed hosts, especially those who are neutropenic, are often thought to require microbicidal therapy. Proof of the cidal nature of an antimicrobial agent in vitro is tedious, complex, and fraught with error. Although several methods for assessing in vitro bactericidal activity have been standardized (NCCLS M26-A and M21-A), the clinical relevance of these determinations is questionable and the tests are performed infrequently in most laboratories. Most of the clinical data supporting the need for microbicidal therapy and testing have focused on bacterial infections. However, given the fact that most serious fungal infections occur in profoundly immunosuppressed individuals, it is generally assumed that a cidal regimen would be preferable in that setting as well. In view of this clinical concern and the perceived need to assess the fungicidal activity of a variety of agents, we considered that it would be useful to review what is known about the issues and problems in assessing bactericidal activity and the clinical utility of such measurements. Following this review, we discuss the issue of how one defines fungicidal activity in vitro and in vivo and how feasible it might be to determine the fungicidal activity of organism-drug combinations for purposes of both drug development and clinical care. Proposed methods for fungal time-kill determinations and minimal fungicidal concentration determinations are also discussed. [Abstract/Link to Full Text]

Kojic EM, Darouiche RO
Candida infections of medical devices.
Clin Microbiol Rev. 2004 Apr;17(2):255-67.
The number of indwelling medical devices is escalating, and an increasing proportion of device-related infections are being caused by Candida spp. Candida spp. produce biofilms on synthetic materials, which facilitates adhesion of the organisms to devices and renders them relatively refractory to medical therapy. Management of device-related Candida infections can be challenging. Removal of the infected device is generally needed to establish cure of Candida infections of medical devices. However, since the pathogenesis of Candida bloodstream infection is complicated, more studies are necessary to determine the role of catheter exchange in patients with both gastrointestinal tract mucositis and indwelling catheters. The medical and economic impact of these infections is enormous. [Abstract/Link to Full Text]

Talisuna AO, Bloland P, D'Alessandro U
History, dynamics, and public health importance of malaria parasite resistance.
Clin Microbiol Rev. 2004 Jan;17(1):235-54.
Despite considerable efforts, malaria is still one of the most devastating infectious diseases in the tropics. The rapid spread of antimalarial drug resistance currently compounds this grim picture. In this paper, we review the history of antimalarial drug resistance and the methods for monitoring it and assess the current magnitude and burden of parasite resistance to two commonly used drugs: chloroquine and sulfadoxine-pyrimethamine. Furthermore, we review the factors involved in the emergence and spread of drug resistance and highlight its public health importance. Finally, we discuss ways of dealing with such a problem by using combination therapy and suggest some of the research themes needing urgent answers. [Abstract/Link to Full Text]

O'Riordan K, Lee JC
Staphylococcus aureus capsular polysaccharides.
Clin Microbiol Rev. 2004 Jan;17(1):218-34.
Serotype 5 and 8 capsular polysaccharides predominate among clinical isolates of Staphylococcus aureus. The results of experiments in animal models of infection have revealed that staphylococcal capsules are important in the pathogenesis of S. aureus infections. The capsule enhances staphylococcal virulence by impeding phagocytosis, resulting in bacterial persistence in the bloodstream of infected hosts. S. aureus capsules also promote abscess formation in rats. Although the capsule has been shown to modulate S. aureus adherence to endothelial surfaces in vitro, animal studies suggest that it also promotes bacterial colonization and persistence on mucosal surfaces. S. aureus capsular antigens are surface associated, limited in antigenic specificity, and highly conserved among clinical isolates. With the emergence of vancomycin-resistant S. aureus in the United States in 2002, new strategies are needed to combat staphylococcal infections. Purified serotype 5 and 8 capsular polysaccharides offer promise as target antigens for a vaccine to prevent staphylococcal infections, although the inclusion of other antigens is likely to be essential in the development of an effective S. aureus vaccine. The genetics and mechanisms of capsule biosynthesis are complex, and much work remains to enhance our understanding of capsule biosynthesis and its regulation. [Abstract/Link to Full Text]

Keiser PB, Nutman TB
Strongyloides stercoralis in the Immunocompromised Population.
Clin Microbiol Rev. 2004 Jan;17(1):208-17.
Strongyloides stercoralis is an intestinal nematode of humans that infects tens of millions of people worldwide. S. stercoralis is unique among intestinal nematodes in its ability to complete its life cycle within the host through an asexual autoinfective cycle, allowing the infection to persist in the host indefinitely. Under some conditions associated with immunocompromise, this autoinfective cycle can become amplified into a potentially fatal hyperinfection syndrome, characterized by increased numbers of infective filariform larvae in stool and sputum and clinical manifestations of the increased parasite burden and migration, such as gastrointestinal bleeding and respiratory distress. S. stercoralis hyperinfection is often accompanied by sepsis or meningitis with enteric organisms. Glucocorticoid treatment and human T-lymphotropic virus type 1 infection are the two conditions most specifically associated with triggering hyperinfection, but cases have been reported in association with hematologic malignancy, malnutrition, and AIDS. Anthelmintic agents such as ivermectin have been used successfully in treating the hyperinfection syndrome as well as for primary and secondary prevention of hyperinfection in patients whose exposure history and underlying condition put them at increased risk. [Abstract/Link to Full Text]

Oleszak EL, Chang JR, Friedman H, Katsetos CD, Platsoucas CD
Theiler's virus infection: a model for multiple sclerosis.
Clin Microbiol Rev. 2004 Jan;17(1):174-207.
Both genetic background and environmental factors, very probably viruses, appear to play a role in the etiology of multiple sclerosis (MS). Lessons from viral experimental models suggest that many different viruses may trigger inflammatory demyelinating diseases resembling MS. Theiler's virus, a picornavirus, induces in susceptible strains of mice early acute disease resembling encephalomyelitis followed by late chronic demyelinating disease, which is one of the best, if not the best, animal model for MS. During early acute disease the virus replicates in gray matter of the central nervous system but is eliminated to very low titers 2 weeks postinfection. Late chronic demyelinating disease becomes clinically apparent approximately 2 weeks later and is characterized by extensive demyelinating lesions and mononuclear cell infiltrates, progressive spinal cord atrophy, and axonal loss. Myelin damage is immunologically mediated, but it is not clear whether it is due to molecular mimicry or epitope spreading. Cytokines, nitric oxide/reactive nitrogen species, and costimulatory molecules are involved in the pathogenesis of both diseases. Close similarities between Theiler's virus-induced demyelinating disease in mice and MS in humans, include the following: major histocompatibility complex-dependent susceptibility; substantial similarities in neuropathology, including axonal damage and remyelination; and paucity of T-cell apoptosis in demyelinating disease. Both diseases are immunologically mediated. These common features emphasize the close similarities of Theiler's virus-induced demyelinating disease in mice and MS in humans. [Abstract/Link to Full Text]

Sutherst RW
Global change and human vulnerability to vector-borne diseases.
Clin Microbiol Rev. 2004 Jan;17(1):136-73.
Global change includes climate change and climate variability, land use, water storage and irrigation, human population growth and urbanization, trade and travel, and chemical pollution. Impacts on vector-borne diseases, including malaria, dengue fever, infections by other arboviruses, schistosomiasis, trypanosomiasis, onchocerciasis, and leishmaniasis are reviewed. While climate change is global in nature and poses unknown future risks to humans and natural ecosystems, other local changes are occurring more rapidly on a global scale and are having significant effects on vector-borne diseases. History is invaluable as a pointer to future risks, but direct extrapolation is no longer possible because the climate is changing. Researchers are therefore embracing computer simulation models and global change scenarios to explore the risks. Credible ranking of the extent to which different vector-borne diseases will be affected awaits a rigorous analysis. Adaptation to the changes is threatened by the ongoing loss of drugs and pesticides due to the selection of resistant strains of pathogens and vectors. The vulnerability of communities to the changes in impacts depends on their adaptive capacity, which requires both appropriate technology and responsive public health systems. The availability of resources in turn depends on social stability, economic wealth, and priority allocation of resources to public health. [Abstract/Link to Full Text]

Eckert J, Deplazes P
Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern.
Clin Microbiol Rev. 2004 Jan;17(1):107-35.
Echinococcosis in humans is a zoonotic infection caused by larval stages (metacestodes) of cestode species of the genus Echinococcus. Cystic echinococcosis (CE) is caused by Echinococcus granulosus, alveolar echinococcosis (AE) is caused by E. multilocularis, and polycystic forms are caused by either E. vogeli or E. oligarthrus. In untreated cases, AE has a high mortality rate. Although control is essentially feasible, CE remains a considerable health problem in many regions of the northern and southern hemispheres. AE is restricted to the northern hemisphere regions of North America and Eurasia. Recent studies have shown that E. multilocularis, the causative agent of AE, is more widely distributed than previously thought. There are also some hints of an increasing significance of polycystic forms of the disease, which are restricted to Central and South America. Various aspects of human echinococcosis are discussed in this review, including data on the infectivity of genetic variants of E. granulosus to humans, the increasing invasion of cities in Europe and Japan by red foxes, the main definitive hosts of E. multilocularis, and the first demonstration of urban cycles of the parasite. Examples of emergence or reemergence of CE are presented, and the question of potential spreading of E. multilocularis is critically assessed. Furthermore, information is presented on new and improved tools for diagnosing the infection in final hosts (dogs, foxes, and cats) by coproantigen or DNA detection and the application of molecular techniques to epidemiological studies. In the clinical field, the available methods for diagnosing human CE and AE are described and the treatment options are summarized. The development of new chemotherapeutic options for all forms of human echinococcosis remains an urgent requirement. A new option for the control of E. granulosus in the intermediate host population (mainly sheep and cattle) is vaccination. Attempts are made to reduce the prevalence of E. multilocualaris in fox populations by regular baiting with an anthelmintic (praziquantel). Recent data have shown that this control option may be used in restricted areas, for example in cities, with the aim of reducing the infection risk for humans. [Abstract/Link to Full Text]

Primm TP, Lucero CA, Falkinham JO
Health impacts of environmental mycobacteria.
Clin Microbiol Rev. 2004 Jan;17(1):98-106.
Environmental mycobacteria are emerging pathogens causing opportunistic infections in humans and animals. The health impacts of human-mycobacterial interactions are complex and likely much broader than currently recognized. Environmental mycobacteria preferentially survive chlorination in municipal water, using it as a vector to infect humans. Widespread chlorination of water has likely selected more resistant environmental mycobacteria species and potentially explains the shift from M. scrofulaceum to M. avium as a cause of cervical lymphadenitis in children. Thus, human activities have affected mycobacterial ecology. While the slow growth and hydrophobicity of environmental mycobacteria appear to be disadvantages, the unique cell wall architecture also grants high biocide and antibiotic resistance, while hydrophobicity facilitates nutrient acquisition, biofilm formation, and spread by aerosolization. The remarkable stress tolerance of environmental mycobacteria is the major reason they are human pathogens. Environmental mycobacteria invade protozoans, exhibiting parasitic and symbiotic relationships. The molecular mechanisms of mycobacterial intracellular pathogenesis in animals likely evolved from similar mechanisms facilitating survival in protozoans. In addition to outright infection, environmental mycobacteria may also play a role in chronic bowl diseases, allergies, immunity to other pulmonary infections, and the efficacy of bacillus Calmette-Guerin vaccination. [Abstract/Link to Full Text]

Xiao L, Fayer R, Ryan U, Upton SJ
Cryptosporidium taxonomy: recent advances and implications for public health.
Clin Microbiol Rev. 2004 Jan;17(1):72-97.
There has been an explosion of descriptions of new species of Cryptosporidium during the last two decades. This has been accompanied by confusion regarding the criteria for species designation, largely because of the lack of distinct morphologic differences and strict host specificity among Cryptosporidium spp. A review of the biologic species concept, the International Code of Zoological Nomenclature (ICZN), and current practices for Cryptosporidium species designation calls for the establishment of guidelines for naming Cryptosporidium species. All reports of new Cryptosporidium species should include at least four basic components: oocyst morphology, natural host specificity, genetic characterizations, and compliance with the ICZN. Altogether, 13 Cryptosporidium spp. are currently recognized: C. muris, C. andersoni, C. parvum, C. hominis, C. wrairi, C. felis, and C. cannis in mammals; C. baďleyi, C. meleagridis, and C. galli in birds; C. serpentis and C. saurophilum in reptiles; and C. molnari in fish. With the establishment of a framework for naming Cryptosporidium species and the availability of new taxonomic tools, there should be less confusion associated with the taxonomy of the genus Cryptosporidium. The clarification of Cryptosporidium taxonomy is also useful for understanding the biology of Cryptosporidium spp., assessing the public health significance of Cryptosporidium spp. in animals and the environment, characterizing transmission dynamics, and tracking infection and contamination sources. [Abstract/Link to Full Text]

Saiman L, Siegel J
Infection control in cystic fibrosis.
Clin Microbiol Rev. 2004 Jan;17(1):57-71.
Over the past 20 years there has been a greater interest in infection control in cystic fibrosis (CF) as patient-to-patient transmission of pathogens has been increasingly demonstrated in this unique patient population. The CF Foundation sponsored a consensus conference to craft recommendations for infection control practices for CF care providers. This review provides a summary of the literature addressing infection control in CF. Burkholderia cepacia complex, Pseudomonas aeruginosa, and Staphylococcus aureus have all been shown to spread between patients with CF. Standard precautions, transmission-based precautions including contact and droplet precautions, appropriate hand hygiene for health care workers, patients, and their families, and care of respiratory tract equipment to prevent the transmission of infectious agents serve as the foundations of infection control and prevent the acquisition of potential pathogens by patients with CF. The respiratory secretions of all CF patients potentially harbor clinically and epidemiologically important microorganisms, even if they have not yet been detected in cultures from the respiratory tract. CF patients should be educated to contain their secretions and maintain a distance of >3 ft from other CF patients to avoid the transmission of potential pathogens, even if culture results are unavailable or negative. To prevent the acquisition of pathogens from respiratory therapy equipment used in health care settings as well as in the home, such equipment should be cleaned and disinfected. It will be critical to measure the dissemination, implementation, and potential impact of these guidelines to monitor changes in practice and reduction in infections. [Abstract/Link to Full Text]

Schmidt H, Hensel M
Pathogenicity islands in bacterial pathogenesis.
Clin Microbiol Rev. 2004 Jan;17(1):14-56.
In this review, we focus on a group of mobile genetic elements designated pathogenicity islands (PAI). These elements play a pivotal role in the virulence of bacterial pathogens of humans and are also essential for virulence in pathogens of animals and plants. Characteristic molecular features of PAI of important human pathogens and their role in pathogenesis are described. The availability of a large number of genome sequences of pathogenic bacteria and their benign relatives currently offers a unique opportunity for the identification of novel pathogen-specific genomic islands. However, this knowledge has to be complemented by improved model systems for the analysis of virulence functions of bacterial pathogens. PAI apparently have been acquired during the speciation of pathogens from their nonpathogenic or environmental ancestors. The acquisition of PAI not only is an ancient evolutionary event that led to the appearance of bacterial pathogens on a timescale of millions of years but also may represent a mechanism that contributes to the appearance of new pathogens within a human life span. The acquisition of knowledge about PAI, their structure, their mobility, and the pathogenicity factors they encode not only is helpful in gaining a better understanding of bacterial evolution and interactions of pathogens with eukaryotic host cells but also may have important practical implications such as providing delivery systems for vaccination, tools for cell biology, and tools for the development of new strategies for therapy of bacterial infections. [Abstract/Link to Full Text]

Kimberlin DW
Neonatal herpes simplex infection.
Clin Microbiol Rev. 2004 Jan;17(1):1-13.
Tremendous advances have occurred over the past 30 years in the diagnosis and management of neonatal herpes simplex virus (HSV) disease. Mortality in patients with disseminated disease has decreased from 85 to 29%, and that in patients with central nervous system (CNS) disease has decreased from 50 to 4%. Morbidity has been improved more modestly: the proportion of patients with disseminated disease who are developing normally at 1 year has increased from 50 to 83%. While the proportion of patients with neurologic morbidity following CNS disease has remained essentially unchanged over the past three decades, the total number of patients who are developing normally following HSV CNS disease has increased due to the improved survival. Although additional therapeutic advances in the future are possible, more immediate methods for further improvements in outcome for patients with this potentially devastating disease lie in an enhanced awareness of neonatal HSV infection and disease. A thorough understanding of the biology and natural history of HSV in the gravid woman and the neonate provides the basis for such an index of suspicion and is provided in this article. [Abstract/Link to Full Text]

Thomas PA
Current perspectives on ophthalmic mycoses.
Clin Microbiol Rev. 2003 Oct;16(4):730-97.
Fungi may infect the cornea, orbit and other ocular structures. Species of Fusarium, Aspergillus, Candida, dematiaceous fungi, and Scedosporium predominate. Diagnosis is aided by recognition of typical clinical features and by direct microscopic detection of fungi in scrapes, biopsy specimens, and other samples. Culture confirms the diagnosis. Histopathological, immunohistochemical, or DNA-based tests may also be needed. Pathogenesis involves agent (invasiveness, toxigenicity) and host factors. Specific antifungal therapy is instituted as soon as the diagnosis is made. Amphotericin B by various routes is the mainstay of treatment for life-threatening and severe ophthalmic mycoses. Topical natamycin is usually the first choice for filamentous fungal keratitis, and topical amphotericin B is the first choice for yeast keratitis. Increasingly, the triazoles itraconazole and fluconazole are being evaluated as therapeutic options in ophthalmic mycoses. Medical therapy alone does not usually suffice for invasive fungal orbital infections, scleritis, and keratitis due to Fusarium spp., Lasiodiplodia theobromae, and Pythium insidiosum. Surgical debridement is essential in orbital infections, while various surgical procedures may be required for other infections not responding to medical therapy. Corticosteroids are contraindicated in most ophthalmic mycoses; therefore, other methods are being sought to control inflammatory tissue damage. Fungal infections following ophthalmic surgical procedures, in patients with AIDS, and due to use of various ocular biomaterials are unique subsets of ophthalmic mycoses. Future research needs to focus on the development of rapid, species-specific diagnostic aids, broad-spectrum fungicidal compounds that are active by various routes, and therapeutic modalities which curtail the harmful effects of fungus- and host tissue-derived factors. [Abstract/Link to Full Text]

---

Recent Articles in Infection and Immunity

---

Wilkie RP, Vissers MC, Dragunow M, Hampton MB
A functional NADPH oxidase prevents caspase involvement in the clearance of phagocytic neutrophils.
Infect Immun. 2007 Jul;75(7):3256-63.
Neutrophils play a prominent role in host defense. Phagocytosis of bacteria leads to the formation of an active NADPH oxidase complex that generates reactive oxygen species for bactericidal purposes. A critical step in the resolution of inflammation is the uptake of neutrophils by macrophages; however, there are conflicting reports on the mechanisms leading to the apoptosis of phagocytic neutrophils. The aim of this study was to clarify the role of effector caspases in these processes. Caspase activity was measured by DEVDase activity assays or immunofluorescence detection of active caspase-3. With normal human and wild-type murine neutrophils there was no caspase activation following phagocytosis of Staphylococcus aureus. However, caspase activity was observed in phagocytic neutrophils with a defective NADPH oxidase, including neutrophils isolated from X-linked gp91(phox) knockout chronic granulomatous disease mice. These results indicate that a functional NADPH oxidase and the generation of oxidants in the neutrophil phagosome prevent the activation of the cytoplasmic caspase cascade. [Abstract/Link to Full Text]

Deans AM, Nery S, Conway DJ, Kai O, Marsh K, Rowe JA
Invasion pathways and malaria severity in Kenyan Plasmodium falciparum clinical isolates.
Infect Immun. 2007 Jun;75(6):3014-20.
The invasion of erythrocytes by Plasmodium falciparum occurs through multiple pathways that can be studied in vitro by examining the invasion of erythrocytes treated with enzymes such as neuraminidase, trypsin, and chymotrypsin. We have studied the invasion pathways used by 31 Kenyan P. falciparum isolates from children with uncomplicated or severe malaria. Six distinct invasion profiles were detected, out of eight possible profiles. The majority of isolates (23 of 31) showed neuraminidase-resistant, trypsin-sensitive invasion, characteristic of the pathway mediated by an unknown parasite ligand and erythrocyte receptor "X." The neuraminidase-sensitive, trypsin-sensitive phenotype consistent with invasion mediated by the binding of parasite ligand erythrocyte binding antigen 175 to glycophorin A, the most common invasion profile in a previous study of Gambian field isolates, was seen in only 3 of 31 Kenyan isolates. No particular invasion profile was associated with severe P. falciparum malaria, and there was no significant difference in the levels of inhibition by the various enzyme treatments between isolates from children with severe malaria and those from children with uncomplicated malaria (P, >0.1 for all enzymes; Mann-Whitney U test). These results do not support the hypothesis that differences in invasion phenotypes play an important role in malaria virulence and indicate that considerable gaps remain in our knowledge of the molecular basis of invasion pathways in natural P. falciparum infections. [Abstract/Link to Full Text]

Salazar JC, Rathi A, Michael NL, Radolf JD, Jagodzinski LL
Assessment of the kinetics of Treponema pallidum dissemination into blood and tissues in experimental syphilis by real-time quantitative PCR.
Infect Immun. 2007 Jun;75(6):2954-8.
Little is known about the size and kinetics of treponemal burdens in blood and tissues during acquired or experimental syphilitic infection. We used real-time quantitative PCR to measure Treponema pallidum DNA levels in rabbits infected intratesticularly with the prototype Nichols strain. At the outset, we performed a series of in vitro blood spiking experiments to determine the effect of blood processing procedures on the distribution of treponemes in various blood components. T. pallidum DNA levels in plasma and whole blood were approximately 10-fold higher than those in serum and more than 200-fold greater than those in peripheral blood mononuclear cells (PBMCs). Ten rabbits were inoculated intratesticularly with doses of treponemes ranging from 4 x 10(7) to 2 x 10(8) organisms. In five rabbits, T. pallidum DNA levels were measured sequentially in serum, plasma, whole blood, and PBMCs until sacrifice at peak orchitis, at which time brain, kidney, liver, spleen, and testicles were harvested; blood and organs were also harvested at orchitis from the other five rabbits. T. pallidum DNA was detected in plasma within 24 h postinfection. Treponeme levels in whole blood and blood components increased significantly with the development of peak orchitis. Overall, levels in serum and PBMCs were lower than those in plasma and whole blood; this disparity was particularly marked at early time points. Significantly greater numbers of spirochetes were found in the spleen than in liver, kidney, or brain tissue at the time of sacrifice. Our findings highlight the remarkable capacity of T. pallidum to disseminate from the site of infection to blood and tissues, and they identify the spleen as a prime target for treponemal invasion. [Abstract/Link to Full Text]

Nallapareddy SR, Sillanpää J, Ganesh VK, Höök M, Murray BE
Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.
Infect Immun. 2007 Jun;75(6):3192-6.
Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P < 0.0001). Blocking Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents. [Abstract/Link to Full Text]

Miura K, Rikihisa Y
Virulence potential of Ehrlichia chaffeensis strains of distinct genome sequences.
Infect Immun. 2007 Jul;75(7):3604-13.
Human monocytic ehrlichiosis, one of the most frequent life-threatening tick-borne zoonoses, is caused by Ehrlichia chaffeensis that lacks endotoxin and peptidoglycan. While sequence polymorphisms in several genes in E. chaffeensis strains have been reported, global genomic divergence and biological differences among strains are unknown. The objectives of the present study were to compare the genome sequences of strains of E. chaffeensis and to examine the virulence potentials of the strains with defined genome sequences. Genomic DNA was extracted from purified E. chaffeensis strains Wakulla and Liberty, and comparative genome hybridization was performed using a densely tiled microarray of 147,027 chromosome positions of the E. chaffeensis strain Arkansas genome. The results revealed that 4,663 and 5,325 positions in the chromosomes of strains Wakulla and Liberty, respectively, were different from those in the chromosome of strain Arkansas, including three common major polymorphic chromosomal regions. Of various functional categories, the differences were most concentrated in genes predicted to encode cell envelope proteins. Of all the open reading frames (ORFs), 21 omp-1 (p28 gene) paralogs, nine genes encoding hypothetical proteins, two genes encoding ankyrin repeat proteins, and hemE contained the most differences. Several highly polymorphic ORFs were confirmed by sequencing. When the E. chaffeensis strains were inoculated into severe combined immunodeficiency mice, the order of the severity of clinical signs and the bacterial burden detected in mice was Wakulla > Liberty > Arkansas. Severe diffuse inflammation and granulomatous inflammation were evident in the livers of mice infected with strains Wakulla and Arkansas, respectively, but not in the livers of mice infected with strain Liberty. These results revealed distinct virulence phenotypes of E. chaffeensis strains with defined genome sequences. [Abstract/Link to Full Text]

Poly F, Read T, Tribble DR, Baqar S, Lorenzo M, Guerry P
Genome sequence of a clinical isolate of Campylobacter jejuni from Thailand.
Infect Immun. 2007 Jul;75(7):3425-33.
Campylobacter jejuni CG8486, which belongs to the HS4 complex, was isolated from a patient with inflammatory diarrhea in Thailand. This strain caused a diarrheal disease in ferrets comparable to that caused by C. jejuni strain 81-176, but it was much less invasive for epithelial cells in vitro than 81-176. Complete genome sequencing of CG8486 revealed a 1.65-Mb genome that was very similar to the other two published genomes of clinical isolates of C. jejuni, the genomes of 81-176 and NCTC 11168, with a limited number of CG8486-specific genes mapping outside the hypervariable carbohydrate biosynthesis loci. These data suggest that the genes required for induction of inflammatory diarrhea are among the genes shared by CG8486 and 81-176 but that either major changes in the carbohydrate loci and/or more subtle changes in other genes may modulate virulence. [Abstract/Link to Full Text]

Molmeret M, Santic' M, Asare R, Carabeo RA, Abu Kwaik Y
Rapid escape of the dot/icm mutants of Legionella pneumophila into the cytosol of mammalian and protozoan cells.
Infect Immun. 2007 Jul;75(7):3290-304.
The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS, letA/S, and letE regulatory mutants are all localized within intact phagosomes. Importantly, non-dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems. [Abstract/Link to Full Text]

Miajlovic H, Loughman A, Brennan M, Cox D, Foster TJ
Both complement- and fibrinogen-dependent mechanisms contribute to platelet aggregation mediated by Staphylococcus aureus clumping factor B.
Infect Immun. 2007 Jul;75(7):3335-43.
Staphylococcus aureus can stimulate activation and aggregation of platelets, which are thought to be factors in the development of infective endocarditis. Previous studies have identified clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as potent platelet aggregators. These proteins are able to stimulate rapid platelet aggregation by either a fibrinogen- or a fibronectin-dependent process which also requires antibodies specific to each protein. Slower aggregation has been seen in other systems where specific fibrinogen binding ligands are absent and platelet aggregation is mediated by complement and specific antibodies. Bacteria expressing ClfB aggregate platelets with a longer lag time than ClfA or FnBPA and FnBPB. In order to investigate whether ClfB causes platelet aggregation in a complement- or fibrinogen-dependent manner, a non-fibrinogen-binding mutant of ClfB (ClfB Q235A) was constructed. Lactococcus lactis expressing ClfB Q235A was able to stimulate platelet aggregation in platelet-rich plasma without a significant increase in lag time. The requirements for platelet aggregation were investigated using gel-filtered platelets. Fibrinogen and specific anti-ClfB antibodies were found to be sufficient to allow platelet aggregation mediated by the wild-type ClfB protein. It seems that ClfB causes platelet aggregation by a fibrinogen-dependent mechanism. The non-fibrinogen-binding ClfB mutant was unable to stimulate platelet aggregation under these conditions. However, bacteria expressing ClfB Q235A caused platelet aggregation in a complement-dependent manner which required specific anti-ClfB antibodies. [Abstract/Link to Full Text]

Krawczyk CM, Shen H, Pearce EJ
Memory CD4 T cells enhance primary CD8 T-cell responses.
Infect Immun. 2007 Jul;75(7):3556-60.
CD4 T-cell help is required for optimal memory CD8 T-cell responses. We have found that engaging preexisting CD4 Th1, but not Th2, memory cells at the time of CD8 T-cell priming results in increased CD8 effector responses to both bacterial and viral pathogens. The enhanced responses are characterized by increased numbers of cytokine-producing, antigen-specific cells. These findings suggest that engaging endogenous memory Th1 cells may increase cellular responses in an immunotherapy or vaccination setting. [Abstract/Link to Full Text]

Pouliot K, Pan N, Wang S, Lu S, Lien E, Goguen JD
Evaluation of the role of LcrV-Toll-like receptor 2-mediated immunomodulation in the virulence of Yersinia pestis.
Infect Immun. 2007 Jul;75(7):3571-80.
Pathogenic members of the Yersinia genus require the translocator protein LcrV for proper function of the type III secretion apparatus, which is crucial for virulence. LcrV has also been reported to play an independent immunosuppressive role via the induction of interleukin-10 (IL-10) through stimulation of Toll-like receptor 2 (TLR2). To investigate the LcrV-TLR2 interaction in vitro, His-tagged recombinant LcrV (rLcrV) from Yersinia pestis was cloned and expressed in Escherichia coli and purified through Ni-nitrilotriacetic acid column chromatography. High concentrations (5 microg/ml) of rLcrV stimulated TLR2 in vitro. Fractionation of rLcrV preparations via gel filtration revealed that only a minor component consisting of high-molecular-weight multimers or aggregates has TLR2 stimulating activity. Dimer and tetramer forms of rLcrV, which constitute the bulk of the material, do not have this activity. To investigate the potential role of LcrV/TLR2 in plague pathogenesis, we infected wild-type and TLR2(-/-) mice with virulent Y. pestis. No discernible difference between the two mouse strains in severity of disease or kinetics of survival after subcutaneous challenge was observed. IL-6, tumor necrosis factor, and IL-10 levels from spleen homogenates; bacterial load; and the extent of inflammation observed in organs from mice infected intravenously were also indistinguishable in both mouse strains. Taken together, our data indicate that the most abundant molecular species of Y. pestis LcrV do not efficiently activate TLR2-signaling and that TLR2-mediated immunomodulation is unlikely to play a significant role in plague. [Abstract/Link to Full Text]

Andoh M, Zhang G, Russell-Lodrigue KE, Shive HR, Weeks BR, Samuel JE
T cells are essential for bacterial clearance, and gamma interferon, tumor necrosis factor alpha, and B cells are crucial for disease development in Coxiella burnetii infection in mice.
Infect Immun. 2007 Jul;75(7):3245-55.
Coxiella burnetii, the etiological agent of Q fever, has two phase variants. Phase I has a complete lipopolysaccharide (LPS), is highly virulent, and causes Q fever in humans and pathology in experimental animals. Phase II lacks an LPS O side chain, is avirulent, and does not grow well in immunocompetent animals. To understand the pathogenicity of Q fever, we investigated the roles of immune components in animals infected with Nine Mile phase I (NM I) or Nine Mile phase II (NM II) bacteria. Immunodeficient mice, including SCID mice (deficient in T and B cells), SCIDbg mice (deficient in T, B, and NK cells), nude mice (deficient in T cells), muMT mice (deficient in B cells), bg mice (deficient in NK cells), mice deficient in tumor necrosis factor alpha (TNF-alpha(-/-) mice), and mice deficient in gamma interferon (IFN-gamma(-/-) mice), were compared for their responses to infection. SCID, SCIDbg, nude, and IFN-gamma(-/-) mice showed high susceptibility to NM I, and TNF-alpha(-/-) mice showed modest susceptibility. Disease caused by NM I in SCID, SCIDbg, and nude mice progressed slowly, while disease in IFN-gamma(-/-) and TNF-alpha(-/-) mice advanced rapidly. B- and NK-cell deficiencies did not enhance clinical disease development or alter bacterial clearance but did increase the severity of histopathological changes, particularly in the absence of B cells. Mice infected with NM II showed no apparent clinical disease, but T-cell-deficient mice had histopathological changes. These results suggest that T cells are critical for clearance of C. burnetii, either NM I or NM II, that IFN-gamma and TNF-alpha are essential for the early control of infection, and that B cells are important for the prevention of tissue damage. [Abstract/Link to Full Text]

Blumenthal B, Hoffmann C, Aktories K, Backert S, Schmidt G
The cytotoxic necrotizing factors from Yersinia pseudotuberculosis and from Escherichia coli bind to different cellular receptors but take the same route to the cytosol.
Infect Immun. 2007 Jul;75(7):3344-53.
The cytotoxic necrotizing factors CNF1 and CNF2 produced by pathogenic Escherichia coli strains and CNF(Y) of Yersinia pseudotuberculosis constitutively activate small GTPases of the Rho family. They deamidate a glutamine (Gln63 in RhoA), which is crucial for GTP hydrolysis. CNF1 and CNF(Y) exhibit 61% identity on the amino acid level, with equal distribution over the whole molecule. Although the two toxins are homologous in the receptor binding domain, we show that they bind to different cellular receptors. CNF(Y) does not enter Caco-2 and CHO-K1 cells, which are responsive to CNF1. In contrast, HeLa, Hep-2, and HEK 293 cells do respond to both toxins. Competition studies with catalytically inactive mutants of the toxins revealed that binding of CNF1 has no influence on the uptake of CNF(Y) into HeLa cells. In contrast, uptake of CNF1 is retarded after preincubation of HeLa cells with the catalytically inactive mutant of CNF(Y), suggesting that the toxin receptors overlap. Moreover, we compared the pathways of the toxins from receptor binding into the cytosol and showed that both toxins are taken up independent of the presence of clathrin or lipid rafts and are released into the cytosol from acidified endosomes. [Abstract/Link to Full Text]

Atayde VD, Cortez M, Souza R, da Silveira JF, Yoshida N
Expression and cellular localization of molecules of the gp82 family in Trypanosoma cruzi metacyclic trypomastigotes.
Infect Immun. 2007 Jul;75(7):3264-70.
A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and identified by monoclonal antibody (MAb) 3F6, plays a key role in host cell invasion. Apart from the gp82 defined by MAb 3F6, no information is available on members of this protein family. From cDNA clones encoding gp82 proteins sharing 59.1% sequence identity, we produced the recombinant proteins J18 and C03, the former containing and the latter lacking the epitope for MAb 3F6. Polyclonal antibodies to J18 and C03 proteins were generated and used, along with MAb 3F6, to analyze the expression and cellular localization of gp82 family members in metacyclic forms of CL and G strains, which belong to highly divergent genetic groups. By two-dimensional gel electrophoresis and immunoblotting, molecules of 82 to 86 kDa, focusing at pH 4.6 to 5.4, and molecules of 72 to 88 kDa, focusing at pH 4.9 to 5.7, were visualized in CL and G strains, respectively. Flow cytometry and microscopic analysis revealed in both strains similar expression of MAb 3F6-reactive gp82 in live and permeabilized parasites, indicating its surface localization. The reaction of live parasites of both strains with anti-J18 antibodies was weaker than with MAb 3F6 and was increased by permeabilization. Anti-C03 antibodies bound predominantly to flagellar components in permeabilized G strain parasites, but in the CL strain the flagellum was not the preferential target for these antibodies. Host cell invasion of metacyclic forms was inhibited by J18 protein, as well as by MAb 3F6 and anti-J18 antibodies, but not by C03 protein or anti-C03 antibodies. [Abstract/Link to Full Text]

Murphy ER, Payne SM
RyhB, an iron-responsive small RNA molecule, regulates Shigella dysenteriae virulence.
Infect Immun. 2007 Jul;75(7):3470-7.
Regulation of bacterial gene expression by small RNA (sRNA) molecules is an increasingly recognized phenomenon but one that is not yet fully understood. We show that the sRNA RyhB suppresses several virulence-associated phenotypes of Shigella dysenteriae, a causative agent of bacillary dysentery in humans. The virulence genes repressed by S. dysenteriae RyhB include those encoding the type III secretion apparatus, its secreted effectors, and specific chaperones. Suppression of Shigella virulence occurs via RyhB-dependent repression of the transcriptional activator VirB, leading to reduced expression of genes within the VirB regulon. Efficient repression of virB is mediated by a single-stranded region of RyhB that is distinct from the region required for repression of Shigella sodB. Regulation of virB by RyhB implicates iron as an environmental factor contributing to the complex regulation of Shigella virulence determinants. [Abstract/Link to Full Text]

DeRocco AJ, Cornelissen CN
Identification of transferrin-binding domains in TbpB expressed by Neisseria gonorrhoeae.
Infect Immun. 2007 Jul;75(7):3220-32.
The transferrin iron acquisition system of Neisseria gonorrhoeae is necessary for iron uptake from transferrin in the human host and requires the participation of two distinct proteins: TbpA and TbpB. TbpA is a TonB-dependent outer membrane transporter responsible for the transport of iron into the cell. TbpB is a lipid-modified protein, for which a precise role in receptor function has not yet been elucidated. These receptor complex proteins show promise as vaccine candidates; therefore, it is important to identify surface-exposed regions of the proteins required for wild-type functions. In this study we examined TbpB, which has been reported to be surface exposed in its entirety; however, this hypothesis has never been tested experimentally. We placed the hemagglutinin (HA) epitope into TbpB with the dual purpose of examining the surface exposure of particular epitopes as well as their impact on receptor function. Nine insertion mutants were created, placing the epitope downstream of the signal peptidase II cleavage site. We report that the HA epitope is surface accessible in all mutants, indicating that the full-length TbpB is completely surface exposed. By expressing the TbpB-HA fusion proteins in N. gonorrhoeae, we were able to examine the impact of each insertion on the function of TbpB and the transferrin acquisition process. We propose that TbpB is comprised of two transferrin-binding-competent lobes, both of which are critical for efficient iron uptake from human transferrin. [Abstract/Link to Full Text]

Reyes-Leon A, Atherton JC, Argent RH, Puente JL, Torres J
Heterogeneity in the activity of Mexican Helicobacter pylori strains in gastric epithelial cells and its association with diversity in the cagA gene.
Infect Immun. 2007 Jul;75(7):3445-54.
Helicobacter pylori CagA is translocated into gastric epithelial cells by a type IV secretion system and interacts with the Src homology 2 phosphatase, altering cell morphology. Multiple EPIYA motifs in CagA are associated with increased activity in cells and with gastric cancer. The aim of this work was to study the heterogeneity in activity in cells of multiple H. pylori single colonies isolated from a single patient and its association with polymorphism in cagA. The presence of cagA, cagE, cagT, and cag10 was studied with 318 H. pylori isolates from the antra and corpora of 18 patients. AGS gastric epithelial cells were infected with 75 isolates, and interleukin-8 (IL-8) secretion, cytoskeletal changes, CagA translocation, and tyrosine phosphorylation were measured. The cagA 3'-variable region was sequenced for 30 isolates to determine the number and types of EPIYA motifs. Isolates from an individual stomach were usually genetically related and had quantitatively similar phenotypic effects on cells (IL-8 induction and cytoskeletal changes). However, strains from different patients with similar CagA EPIYA motif patterns varied widely in these phenotypes. Among isolates with an EPIYA-ABC pattern, the phenotype was variable: IL-8 induction ranged from 200 to 1,200 pg/ml, and morphological changes occurred in 20 to 70% of cells. In several cases, cagA sequence diversity appeared to explain the lack of CagA activity, as isolates with an EPIYA-ACC pattern or a modified B motif had reduced cell activity. cag pathogenicity island-positive H. pylori isolates displayed a high level of heterogeneity in the capacity to induce IL-8 secretion and morphological changes; an absent or modified B motif was associated with low activity. [Abstract/Link to Full Text]

Gauger EJ, Leatham MP, Mercado-Lubo R, Laux DC, Conway T, Cohen PS
Role of motility and the flhDC Operon in Escherichia coli MG1655 colonization of the mouse intestine.
Infect Immun. 2007 Jul;75(7):3315-24.
Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD(4)C(2). Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD(4)C(2) but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth. [Abstract/Link to Full Text]

Liu M, Alice AF, Naka H, Crosa JH
The HlyU protein is a positive regulator of rtxA1, a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus.
Infect Immun. 2007 Jul;75(7):3282-9.
Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised iron-overloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon. [Abstract/Link to Full Text]

Rolán HG, Tsolis RM
Mice lacking components of adaptive immunity show increased Brucella abortus virB mutant colonization.
Infect Immun. 2007 Jun;75(6):2965-73.
The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng(-/-) or beta(2)m(-/-) mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1(-/-) mice and Igh6(-/-) mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6(-/-) mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus. [Abstract/Link to Full Text]

Woodman ME, Cooley AE, Miller JC, Lazarus JJ, Tucker K, Bykowski T, Botto M, Hellwage J, Wooten RM, Stevenson B
Borrelia burgdorferi binding of host complement regulator factor H is not required for efficient mammalian infection.
Infect Immun. 2007 Jun;75(6):3131-9.
The causative agent of Lyme disease, Borrelia burgdorferi, is naturally resistant to its host's alternative pathway of complement-mediated killing. Several different borrelial outer surface proteins have been identified as being able to bind host factor H, a regulator of the alternative pathway, leading to a hypothesis that such binding is important for borrelial resistance to complement. To test this hypothesis, the development of B. burgdorferi infection was compared between factor H-deficient and wild-type mice. Factor B- and C3-deficient mice were also studied to determine the relative roles of the alternative and classical/lectin pathways in B. burgdorferi survival during mammalian infection. While it was predicted that B. burgdorferi should be impaired in its ability to infect factor H-deficient animals, quantitative analyses of bacterial loads indicated that those mice were infected at levels similar to those of wild-type and factor B- and C3-deficient mice. Ticks fed on infected factor H-deficient or wild-type mice all acquired similar numbers of bacteria. Indirect immunofluorescence analysis of B. burgdorferi acquired by feeding ticks from the blood of infected mice indicated that none of the bacteria had detectable levels of factor H on their outer surfaces, even though such bacteria express high levels of surface proteins capable of binding factor H. These findings demonstrate that the acquisition of host factor H is not essential for mammalian infection by B. burgdorferi and indicate that additional mechanisms are employed by the Lyme disease spirochete to evade complement-mediated killing. [Abstract/Link to Full Text]

Chan KG, Mayer M, Davis EM, Halperin SA, Lin TJ, Lee SF
Role of D-alanylation of Streptococcus gordonii lipoteichoic acid in innate and adaptive immunity.
Infect Immun. 2007 Jun;75(6):3033-42.
In recent years, there has been considerable interest in using the oral commensal gram-positive bacterium Streptococcus gordonii as a live vaccine vector. The present study investigated the role of d-alanylation of lipoteichoic acid (LTA) in the interaction of S. gordonii with the host innate and adaptive immune responses. A mutant strain defective in d-alanylation was generated by inactivation of the dltA gene in a recombinant strain of S. gordonii (PM14) expressing a fragment of the S1 subunit of pertussis toxin. The mutant strain was found to be more susceptible to killing by polymyxin B, nisin, magainin II, and human beta defensins than the parent strain. When it was examined for binding to murine bone marrow-derived dendritic cells (DCs), the dltA mutant exhibited 200- to 400-fold less binding than the parent but similar levels of binding were shown for Toll-like receptor 2 (TLR2) knockout DCs and HEp-2 cells. In a mouse oral colonization study, the mutant showed a colonization ability similar to that of the parent and was not able to induce a significant immune response. The mutant induced significantly less interleukin 12p70 (IL-12p70) and IL-10 than the parent from DCs. LTA purified from the bacteria induced tumor necrosis factor-alpha and IL-6 production from wild-type DCs but not from TLR2 knockout DCs, and the mutant LTA induced a significantly smaller amount of these two cytokines. These results show that d-alanylation of LTA in S. gordonii plays a role in the interaction with the host immune system by contributing to the relative resistance to host defense peptides and by modulating cytokine production by DCs. [Abstract/Link to Full Text]

Su J, Yang J, Zhao D, Kawula TH, Banas JA, Zhang JR
Genome-wide identification of Francisella tularensis virulence determinants.
Infect Immun. 2007 Jun;75(6):3089-101.
Francisella tularensis is a gram-negative pathogen that causes life-threatening infections in humans and has potential for use as a biological weapon. The genetic basis of the F. tularensis virulence is poorly understood. This study screened a total of 3,936 transposon mutants of the live vaccine strain for infection in a mouse model of respiratory tularemia by signature-tagged mutagenesis. We identified 341 mutants attenuated for infection in the lungs. The transposon disruptions were mapped to 95 different genes, virtually all of which are also present in the genomes of other F. tularensis strains, including human pathogenic F. tularensis strain Schu S4. A small subset of these attenuated mutants carried insertions in the genes encoding previously known virulence factors, but the majority of the identified genes have not been previously linked to F. tularensis virulence. Among these are genes encoding putative membrane proteins, proteins associated with stress responses, metabolic proteins, transporter proteins, and proteins with unknown functions. Several attenuated mutants contained disruptions in a putative capsule locus which partially resembles the poly-gamma-glutamate capsule biosynthesis locus of Bacillus anthracis, the anthrax agent. Deletional mutation analysis confirmed that this locus is essential for F. tularensis virulence. [Abstract/Link to Full Text]

Nelson AL, Ratner AJ, Barasch J, Weiser JN
Interleukin-8 secretion in response to aferric enterobactin is potentiated by siderocalin.
Infect Immun. 2007 Jun;75(6):3160-8.
Siderophores are low-molecular-weight iron chelators secreted by microbes to obtain iron under deprivation. We hypothesized that the catecholate siderophore enterobactin, produced by Enterobacteriaceae, serves as a proinflammatory signal for respiratory epithelial cells. Respiratory tract responses were explored, since at this site siderocalin, an enterobactin-binding mammalian gene product, is expressed inducibly at high levels and enterobactin-secreting respiratory flora is rare, suggesting selection against a dependence on enterobactin. Addition of aferric, but not iron-saturated, enterobactin elicits a dose-dependent increase in secretion of the proinflammatory chemokine interleukin-8 by human respiratory epithelial cells in culture. This response to purified enterobactin is potentiated by recombinant siderocalin at physiologically relevant concentrations. Conditioned media from genetically modified Escherichia coli strains expressing various levels of enterobactin induce an enterobactin-mediated proinflammatory response. Siderocalin has been shown to deliver enterobactin to other mammalian cell types, exogenously supplied siderocalin can be detected within epithelial cells, and siderocalin increases delivery of enterobactin to the intracellular compartment. Although many siderophores perturb labile cellular iron pools, only enterobactin elicits interleukin-8 secretion, suggesting that iron chelation is necessary but not sufficient. Thus, aferric enterobactin may be a proinflammatory signal for respiratory epithelial cells, permitting detection of microbial communities that have disturbed local iron homeostasis, and siderocalin expression by the host amplifies this signal. This may be a novel mechanism for the mucosa to respond to metabolic signals of expanding microbial communities. [Abstract/Link to Full Text]

Greene JM, Collins F, Lefkowitz EJ, Roos D, Scheuermann RH, Sobral B, Stevens R, White O, Di Francesco V
National Institute of Allergy and Infectious Diseases bioinformatics resource centers: new assets for pathogen informatics.
Infect Immun. 2007 Jul;75(7):3212-9. [Abstract/Link to Full Text]

Santic M, Asare R, Doric M, Abu Kwaik Y
Host-dependent trigger of caspases and apoptosis by Legionella pneumophila.
Infect Immun. 2007 Jun;75(6):2903-13.
The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice. [Abstract/Link to Full Text]

Plamondon P, Luke NR, Campagnari AA
Identification of a novel two-partner secretion locus in Moraxella catarrhalis.
Infect Immun. 2007 Jun;75(6):2929-36.
Although Moraxella catarrhalis continues to be a significant cause of disease in both children and adults, the steps involved in pathogenesis remain poorly understood. We have identified three open reading frames in the M. catarrhalis genome that encode homologues of the two-partner secretion system (TPS). The sequenced M. catarrhalis hemagglutinin-like locus of strain 7169 has a unique gene organization composed in the order of mchA1, mchB, and mchA2, where mchA1 is divergent. MchA1 and MchA2 are 74% identical at the amino acid level and diverge only in the C-terminal regions. The TPS motif identified in the common N-terminal regions of MchA1 and MchA2 was found to be homologous to the filamentous hemagglutinin of Bordetella pertussis, and MchB has homology to other TpsB transporters. The presence of MchA1 and MchA2 in outer membrane protein preparations and concentrated culture supernatants (CCSs) of strain 7169 was confirmed by immunoblotting using specific antisera. Nanoscale liquid chromatography-tandem mass spectrometry peptide sequencing of the antibody-reactive bands from the CCSs was performed and demonstrated that 13 different peptides mapped to identical regions of MchA1 and MchA2. Quantitative adherence assays revealed a decrease of binding to primary normal human bronchial epithelial cells by the mch mutants 7169mchB and 7169mchA1A2B compared to that by the wild-type strain. These studies show that MchA1, MchA2, and MchB are components of a novel TPS identified in M. catarrhalis and suggest that these proteins may be involved in colonization. [Abstract/Link to Full Text]

Ulett GC, Valle J, Beloin C, Sherlock O, Ghigo JM, Schembri MA
Functional analysis of antigen 43 in uropathogenic Escherichia coli reveals a role in long-term persistence in the urinary tract.
Infect Immun. 2007 Jul;75(7):3233-44.
Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with the virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter subgroup of proteins. The best characterized of these proteins, antigen 43 (Ag43), is a self-recognizing adhesin that is associated with cell aggregation and biofilm formation in E. coli K-12. The sequenced genome of prototype UPEC strain CFT073 contains two variant Ag43-encoding genes located on pathogenicity islands. The biological significance of both of these genes and their role in UPEC pathogenesis have not been investigated previously. Here we performed a detailed molecular characterization analysis of Ag43a (c3655) and Ag43b (c1273) from UPEC CFT073. Expression of Ag43a and Ag43b in a K-12 background revealed that they possess different functional properties. Ag43a produced a strong aggregation phenotype and promoted significant biofilm growth. Deletion mutants and strains constitutively expressing Ag43a and Ag43b were also constructed using CFT073. When these mutants were analyzed in a mouse model of UTI, Ag43a (but not Ag43b) promoted long-term persistence in the urinary bladder. Our findings demonstrate that Ag43a contributes to UPEC disease pathogenesis and reveal that there are pathogenicity-adapted variants of Ag43 with distinct virulence-related functions. [Abstract/Link to Full Text]

Worthington ZE, Carbonetti NH
Evading the proteasome: absence of lysine residues contributes to pertussis toxin activity by evasion of proteasome degradation.
Infect Immun. 2007 Jun;75(6):2946-53.
Pertussis toxin (PT) is an important virulence factor produced by Bordetella pertussis. PT holotoxin comprises one enzymatically active A subunit (S1), associated with a pentamer of B subunits. PT is an ADP-ribosyltransferase that modifies several mammalian heterotrimeric G proteins. Some bacterial toxins are believed to undergo retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway involves the removal of misfolded proteins from the ER and degradation upon their return to the cytosol; this pathway may be exploited by PT and other toxins. In the cytosol, ERAD substrates are ubiquitinated at lysine residues, targeting them to the proteasome for degradation. We hypothesize that S1 avoids ubiquitination and proteasome degradation due to its lack of lysine residues. We predicted that the addition of lysine residues would reduce PT toxicity by allowing ubiquitination and degradation to occur. Variant forms of PT were engineered, replacing one, two, or three arginines with lysines in a variety of locations on S1. Several variants were identified with wild-type in vitro enzymatic activity but reduced cellular activity, consistent with our hypothesis. Significant recovery of the cellular activity of these variants was observed when CHO cells were pretreated with a proteasome inhibitor. We concluded that the replacement of arginine residues with lysine in the S1 subunit of PT renders the toxin subject to proteasomal degradation, suggesting that wild-type PT avoids proteasome degradation due to an absence of lysine residues. [Abstract/Link to Full Text]

Auerbuch V, Isberg RR
Growth of Yersinia pseudotuberculosis in mice occurs independently of Toll-like receptor 2 expression and induction of interleukin-10.
Infect Immun. 2007 Jul;75(7):3561-70.
Pathogenic Yersinia translocates effector proteins into target cells via a type III secretion system (TTSS), modulating the host immune response. A component of the TTSS translocon, LcrV, has been implicated in preventing inflammation through Toll-like receptor 2 (TLR2) by inducing expression of the anti-inflammatory cytokine interleukin-10 (IL-10). TLR2(-/-) mice were reported to be less susceptible to the enteropathogen Yersinia enterocolitica. To determine whether TLR2 also plays a role in recognition of the enteropathogen Yersinia pseudotuberculosis and whether this results in an immune response that is detrimental to the host, we evaluated the macrophage cytokine response to live Y. pseudotuberculosis and analyzed the susceptibility of TLR2(-/-) mice to enteropathogenic Yersinia. We find that Yersinia induction of macrophage IL-10 occurs independently of TLR2 and LcrV and is blocked by the TTSS. In particular, the TTSS effector protein YopJ, which inhibits production of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), also inhibits IL-10 expression. Consistent with these results, IL-10 is undetectable in Y. pseudotuberculosis-infected mouse tissues until advanced stages of infection. In addition, we find that TLR2(-/-) mice (derived independently from those used in previous studies) do not display altered susceptibility to enteropathogenic Yersinia compared to wild-type mice. Tissue levels of IL-10, as well as the inflammatory cytokines TNF-alpha, IL-6, and gamma interferon and the chemokine macrophage chemotactic protein 1, are similar in TLR2(+/+) and TLR2(-/-) mice during enteropathogenic Yersinia infection. Therefore, the absence of TLR2 alone does not affect the cytokine response of macrophages to, or the in vivo growth and survival of, enteropathogenic Yersinia. [Abstract/Link to Full Text]

Ueti MW, Reagan JO, Knowles DP, Scoles GA, Shkap V, Palmer GH
Identification of midgut and salivary glands as specific and distinct barriers to efficient tick-borne transmission of Anaplasma marginale.
Infect Immun. 2007 Jun;75(6):2959-64.
Understanding the determinants of efficient tick-borne microbial transmission is needed to better predict the emergence of highly transmissible pathogen strains and disease outbreaks. Although the basic developmental cycle of Anaplasma and Ehrlichia spp. within the tick has been delineated, there are marked differences in the ability of specific strains to be efficiently tick transmitted. Using the highly transmissible St. Maries strain of Anaplasma marginale in Dermacentor andersoni as a positive control and two unrelated nontransmissible strains, we identified distinct barriers to efficient transmission within the tick. The Mississippi strain was unable to establish infection at the level of the midgut epithelium despite successful ingestion of infected blood following acquisition feeding on a bacteremic animal host. This inability to colonize the midgut epithelium prevented subsequent development within the salivary glands and transmission. In contrast, A. marginale subsp. centrale colonized the midgut and then the salivary glands, replicating to a titer indistinguishable from that of the highly transmissible St. Maries strain and at least 100 times greater than that previously associated with successful transmission. Nonetheless, A. marginale subsp. centrale was not transmitted, even when a large number of infected ticks was used for transmission feeding. These results establish that there are at least two specific barriers to efficient tick-borne transmission, the midgut and salivary glands, and highlight the complexity of the pathogen-tick interaction. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Bacteriology

---

Russell RM, Sharp FC, Rasko DA, Sperandio V
QseA and GrlR/GrlA regulation of the locus of enterocyte effacement genes in enterohemorrhagic Escherichia coli.
J Bacteriol. 2007 Jul;189(14):5387-92.
Transcription of the locus of enterocyte effacement (LEE) genes in enterohemorrhagic Escherichia coli (EHEC) is regulated by the LEE-encoded Ler and GrlR/GrlA proteins as well as the non-LEE-encoded regulator QseA. This work demonstrates that GrlR/GrlA activate LEE2 transcription in a Ler-independent fashion, whereas transcription of grlRA is activated by QseA in both Ler-dependent and -independent manners. [Abstract/Link to Full Text]

Stephanou NC, Gao F, Bongiorno P, Ehrt S, Schnappinger D, Shuman S, Glickman MS
Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks.
J Bacteriol. 2007 Jul;189(14):5237-46.
Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis Delta recA Delta ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs. [Abstract/Link to Full Text]

Kaufman GE, Yother J
CcpA-dependent and -independent control of beta-galactosidase expression in Streptococcus pneumoniae occurs via regulation of an upstream phosphotransferase system-encoding operon.
J Bacteriol. 2007 Jul;189(14):5183-92.
A spontaneous mutant of Streptococcus pneumoniae strain D39 exhibiting elevated beta-galactosidase activity was identified. We determined that the beta-galactosidase activity was due to BgaA, a surface protein in S. pneumoniae, and that the expression of bgaA was regulated. Transcription analyses demonstrated expression of bgaA in the constitutive beta-galactosidase (BgaA(C)) mutant, but not in the parent. beta-Galactosidase expression was induced in the parent under specific growth conditions; however, the levels did not reach those of the BgaA(C) mutant. We localized the mutation resulting in the BgaA(C) phenotype to a region upstream of bgaA and in the promoter of a phosphoenolpyruvate-dependent phosphotransferase system (PTS) operon. The mutation was in a catabolite-responsive element (cre) and affected the binding of CcpA (catabolite control protein A), a key regulator of many carbon metabolism genes. The pts operon and bgaA were cotranscribed, and their transcription was regulated by CcpA. Deletion of ccpA altered beta-galactosidase activity, leading to a sevenfold increase in the parent but a fivefold decrease in the BgaA(C) mutant. The resulting beta-galactosidase activities were the same in the two strains, suggesting the presence of a second repressor. The presence of glucose in the growth medium resulted in pts-bgaA repression by both CcpA and the second repressor, with the latter being important in responding to the glucose concentration. Expression of beta-galactosidase is important for S. pneumoniae adherence during colonization of the nasopharynx, a site normally devoid of glucose. CcpA and environmental glucose concentrations thus appear to play important roles in the regulation of a niche-specific virulence factor. [Abstract/Link to Full Text]

Kristoffersen SM, Ravnum S, Tourasse NJ, Řkstad OA, Kolstř AB, Davies W
Low Concentrations of bile salts induce stress responses and reduce motility in Bacillus cereus ATCC 14570.
J Bacteriol. 2007 Jul;189(14):5302-13.
Tolerance to bile salts was investigated in forty Bacillus cereus strains, including 17 environmental isolates, 11 dairy isolates, 3 isolates from food poisoning outbreaks, and 9 other clinical isolates. Growth of all strains was observed at low bile salt concentrations, but no growth was observed on LB agar plates containing more than 0.005% bile salts. Preincubation of the B. cereus type strain, ATCC 14579, in low levels of bile salts did not increase tolerance levels. B. cereus ATCC 14579 was grown to mid-exponential growth phase and shifted to medium containing bile salts (0.005%). Global expression patterns were determined by hybridization of total cDNA to a 70-mer oligonucleotide microarray. A general stress response and a specific response to bile salts were observed. The general response was similar to that observed in cultures grown in the absence of bile salts but at a higher (twofold) cell density. Up-regulation of several putative multidrug exporters and transcriptional regulators and down-regulation of most motility genes were observed as part of the specific response. Motility experiments in soft agar showed that motility decreased following bile salts exposure, in accordance with the transcriptional data. Genes encoding putative virulence factors were either unaffected or down-regulated. [Abstract/Link to Full Text]

Pilonieta MC, Bodero MD, Munson GP
CfaD-dependent expression of a novel extracytoplasmic protein from enterotoxigenic Escherichia coli.
J Bacteriol. 2007 Jul;189(14):5060-7.
H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions -23 to -56, and the other extends from positions -73 to -103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC. [Abstract/Link to Full Text]

Yamaichi Y, Fogel MA, McLeod SM, Hui MP, Waldor MK
Distinct centromere-like parS sites on the two chromosomes of Vibrio spp.
J Bacteriol. 2007 Jul;189(14):5314-24.
Vibrio cholerae, the cause of cholera, has two circular chromosomes. The parAB genes on each V. cholerae chromosome act to control chromosome segregation in a replicon-specific fashion. The chromosome I (ChrI) parAB genes (parAB1) govern the localization of the origin region of ChrI, while the chromosome II (ChrII) parAB genes (parAB2) control the segregation of ChrII. In addition to ParA and ParB proteins, Par systems require ParB binding sites (parS). Here we identified the parS sites on both V. cholerae chromosomes. We found three clustered origin-proximal ParB1 binding parS1 sites on ChrI. Deletion of these three parS1 sites abrogated yellow fluorescent protein (YFP)-ParB1 focus formation in vivo and resulted in mislocalization of the ChrI origin region. However, as observed in a parA1 mutant, mislocalization of the ChrI origin region in the parS1 mutant did not compromise V. cholerae growth, suggesting that additional (non-Par-related) mechanisms may mediate the partitioning of ChrI. We also identified 10 ParB2 binding parS2 sites, which differed in sequence from parS1. Fluorescent derivatives of ParB1 and ParB2 formed foci only with the cognate parS sequence. parABS2 appears to form a functional partitioning system, as we found that parABS2 was sufficient to stabilize an ordinarily unstable plasmid in Escherichia coli. Most parS2 sites were located within 70 kb of the ChrII origin of replication, but one parS2 site was found in the terminus region of ChrI. In contrast, in other sequenced vibrio species, the distribution of parS1 and parS2 sites was entirely chromosome specific. [Abstract/Link to Full Text]

Gibbons HS, Wolschendorf F, Abshire M, Niederweis M, Braunstein M
Identification of two Mycobacterium smegmatis lipoproteins exported by a SecA2-dependent pathway.
J Bacteriol. 2007 Jul;189(14):5090-100.
The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and DeltasecA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a DeltasecA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis. [Abstract/Link to Full Text]

Servinsky MD, Julin DA
Effect of a recD mutation on DNA damage resistance and transformation in Deinococcus radiodurans.
J Bacteriol. 2007 Jul;189(14):5101-7.
The bacterium Deinococcus radiodurans is resistant to extremely high levels of DNA-damaging agents such as UV light, ionizing radiation, and chemicals such as hydrogen peroxide and mitomycin C. The organism is able to repair large numbers of double-strand breaks caused by ionizing radiation, in spite of the lack of the RecBCD enzyme, which is essential for double-strand DNA break repair in Escherichia coli and many other bacteria. The D. radiodurans genome sequence indicates that the organism lacks recB and recC genes, but there is a gene encoding a protein with significant similarity to the RecD protein of E. coli and other bacteria. We have generated D. radiodurans strains with a disruption or deletion of the recD gene. The recD mutants are more sensitive than wild-type cells to irradiation with gamma rays and UV light and to treatment with hydrogen peroxide, but they are not sensitive to treatment with mitomycin C and methyl methanesulfonate. The recD mutants also show greater efficiency of transformation by exogenous homologous DNA. These results are the first indication that the D. radiodurans RecD protein has a role in DNA damage repair and/or homologous recombination in the organism. [Abstract/Link to Full Text]

Dodd D, Reese JG, Louer CR, Ballard JD, Spies MA, Blanke SR
Functional comparison of the two Bacillus anthracis glutamate racemases.
J Bacteriol. 2007 Jul;189(14):5265-75.
Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because L-glutamate stereoisomerization to D-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of L-glutamate and D-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of L-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features. [Abstract/Link to Full Text]

Duerkop BA, Ulrich RL, Greenberg EP
Octanoyl-homoserine lactone is the cognate signal for Burkholderia mallei BmaR1-BmaI1 quorum sensing.
J Bacteriol. 2007 Jul;189(14):5034-40.
Acyl-homoserine lactones (HSLs) serve as quorum-sensing signals for many Proteobacteria. Members of the LuxI family of signal generators catalyze the production of acyl-HSLs, which bind to a cognate receptor in the LuxR family of transcription factors. The obligate animal pathogen Burkholderia mallei produces several acyl-HSLs, and the B. mallei genome has four luxR and two luxI homologs, each of which has been established as a virulence factor. To begin to delineate the relevant acyl-HSL signals for B. mallei LuxR homologs, we analyzed the BmaR1-BmaI1 system. A comparison of acyl-HSL profiles from B. mallei ATCC 23344 and a B. mallei bmaI1 mutant indicates that octanoyl-HSL synthesis is BmaI1 dependent. Furthermore, octanoyl-HSL is the predominant acyl-HSL produced by BmaI1 in recombinant Escherichia coli. The synthesis of soluble BmaR1 in recombinant E. coli requires octanoyl-HSL or decanoyl-HSL. Insoluble aggregates of BmaR1 are produced in the presence of other acyl-HSLs and in the absence of acyl-HSLs. The bmaI1 promoter is activated by BmaR1 and octanoyl-HSL, and a 20-bp inverted repeat in the bmaI1 promoter is required for bmaI1 activation. Purified BmaR1 binds to this promoter region. These findings implicate octanoyl-HSL as the signal for BmaR1-BmaI1 quorum sensing and show that octanoyl-HSL and BmaR1 activate bmaI1 transcription. [Abstract/Link to Full Text]

Lee YM, Dodson KW, Hultgren SJ
Adaptor function of PapF depends on donor strand exchange in P-pilus biogenesis of Escherichia coli.
J Bacteriol. 2007 Jul;189(14):5276-83.
P-pilus biogenesis occurs via the highly conserved chaperone-usher pathway and involves the strict coordination of multiple subunit proteins. All nonadhesin structural P-pilus subunits possess the same topology, consisting of two domains: an incomplete immunoglobulin-like fold (pilin body) and an N-terminal extension. Pilus subunits form interactions with one another through donor strand exchange, occurring at the usher, in which the N-terminal extension of an incoming subunit completes the pilin body of the preceding subunit, allowing the incorporation of the subunit into the pilus fiber. In this study, pilus subunits in which the N-terminal extension was either deleted or swapped with that of another subunit were used to examine the role of each domain of PapF in functions involving donor strand exchange and hierarchical assembly. We found that the N-terminal extension of PapF is required to adapt the PapG adhesin to the tip of the fiber. The pilin body of PapF is required to efficiently initiate assembly of the remainder of the pilus, with the assistance of the N-terminal extension. Thus, distinct functions were assigned to each region of the PapF subunit. In conclusion, all pilin subunits possess the same overall architectural topology; however, each N-terminal extension and pilin body has specific functions in pilus biogenesis. [Abstract/Link to Full Text]

Mashimo T, Hashimoto M, Yamaguchi S, Aizawa S
Temperature-hypersensitive sites of the flagellar switch component FliG in Salmonella enterica serovar typhimurium.
J Bacteriol. 2007 Jul;189(14):5153-60.
Three flagellar proteins, FliG, FliM, and FliN (FliGMN), are the components of the C ring of the flagellar motor. The genes encoding these proteins are multifunctional; they show three different phenotypes (Fla(-), Mot(-), and Che(-)), depending on the sites and types of mutations. Some of the Mot(-) mutants previously characterized are found to be motile. Reexamination of all Mot(-) mutants in fliGMN genes so far studied revealed that many of them are actually temperature sensitive (TS); that is, they are motile at 20 degrees C but nonmotile at 37 degrees C. There were two types of TS mutants: one caused a loss of function that was not reversed by a return to the permissive temperature (rigid TS), and the other caused a loss that was reversed (hyper-TS). The rigid TS mutants showed an all-or-none phenotype; that is, once a structure was formed, the structure and function were stable against temperature shifts. All of fliM and fliN and most of the fliG TS mutants belong to this group. On the other hand, the hyper-TS mutants (three of the fliG mutants) showed a temporal swimming/stop phenotype, responding to temporal temperature shifts when the structure was formed at a permissive temperature. Those hyper-TS mutation sites are localized in the C-terminal domain of the FliG molecules at sites that are different from the previously proposed functional sites. We discuss a role for this new region of FliG in the torque generation of the flagellar motor. [Abstract/Link to Full Text]

Mueller RS, McDougald D, Cusumano D, Sodhi N, Kjelleberg S, Azam F, Bartlett DH
Vibrio cholerae strains possess multiple strategies for abiotic and biotic surface colonization.
J Bacteriol. 2007 Jul;189(14):5348-60.
Despite its notoriety as a human pathogen, Vibrio cholerae is an aquatic microbe suited to live in freshwater, estuarine, and marine environments where biofilm formation may provide a selective advantage. Here we report characterization of biofilms formed on abiotic and biotic surfaces by two non-O1/O139 V. cholerae strains, TP and SIO, and by the O1 V. cholerae strain N16961 in addition to the isolation of 44 transposon mutants of SIO and TP impaired in biofilm formation. During the course of characterizing the mutants, 30 loci which have not previously been associated with V. cholerae biofilms were identified. These loci code for proteins which perform a wide variety of functions, including amino acid metabolism, ion transport, and gene regulation. Also, when the plankton colonization abilities of strains N16961, SIO, and TP were examined, each strain showed increased colonization of dead plankton compared with colonization of live plankton (the dinoflagellate Lingulodinium polyedrum and the copepod Tigriopus californicus). Surprisingly, most of the biofilm mutants were not impaired in plankton colonization. Only mutants impaired in motility or chemotaxis showed reduced colonization. These results indicate the presence of both conserved and variable genes which influence the surface colonization properties of different V. cholerae subspecies. [Abstract/Link to Full Text]

Sakuragi Y, Kolter R
Quorum-sensing regulation of the biofilm matrix genes (pel) of Pseudomonas aeruginosa.
J Bacteriol. 2007 Jul;189(14):5383-6.
Quorum sensing (QS) has been previously shown to play an important role in the development of Pseudomonas aeruginosa biofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of the pel operon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced in lasI and rhlI mutants. Chemical complementation of the lasI mutant with 3-oxo-dodecanoyl homoserine lactone restores pel transcription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis. [Abstract/Link to Full Text]

Magnusson LU, Gummesson B, Joksimovi? P, Farewell A, Nyström T
Identical, independent, and opposing roles of ppGpp and DksA in Escherichia coli.
J Bacteriol. 2007 Jul;189(14):5193-202.
The recent discovery that the protein DksA acts as a coregulator of genes controlled by ppGpp led us to investigate the similarities and differences between the relaxed phenotype of a ppGpp-deficient mutant and the phenotype of a strain lacking DksA. We demonstrate that the absence of DksA and ppGpp has similar effects on many of the observed phenotypes but that DksA and ppGpp also have independent and sometimes opposing roles in the cell. Specifically, we show that overexpression of DksA can compensate for the loss of ppGpp with respect to transcription of the promoters P(uspA), P(livJ), and P(rrnBP1) as well as amino acid auxotrophy, cell-cell aggregation, motility, filamentation, and stationary phase morphology, suggesting that DksA can function without ppGpp in regulating gene expression. In addition, ppGpp and DksA have opposing effects on adhesion. In the course of our analysis, we also discovered new features of the relaxed mutant, namely, defects in cell-cell aggregation and motility. [Abstract/Link to Full Text]

Ajdi? D, Pham VT
Global transcriptional analysis of Streptococcus mutans sugar transporters using microarrays.
J Bacteriol. 2007 Jul;189(14):5049-59.
The transport of carbohydrates by Streptococcus mutans is accomplished by the phosphoenolpyruvate-phosphotransferase system (PTS) and ATP-binding cassette (ABC) transporters. To undertake a global transcriptional analysis of all S. mutans sugar transporters simultaneously, we used a whole-genome expression microarray. Global transcription profiles of S. mutans UA159 were determined for several monosaccharides (glucose, fructose, galactose, and mannose), disaccharides (sucrose, lactose, maltose, and trehalose), a beta-glucoside (cellobiose), oligosaccharides (raffinose, stachyose, and maltotriose), and a sugar alcohol (mannitol). The results revealed that PTSs were responsible for transport of monosaccharides, disaccharides, beta-glucosides, and sugar alcohol. Six PTSs were transcribed only if a specific sugar was present in the growth medium; thus, they were regulated at the transcriptional level. These included transporters for fructose, lactose, cellobiose, and trehalose and two transporters for mannitol. Three PTSs were repressed under all conditions tested. Interestingly, five PTSs were always highly expressed regardless of the sugar source used, presumably suggesting their availability for immediate uptake of most common dietary sugars (glucose, fructose, maltose, and sucrose). The ABC transporters were found to be specific for oligosaccharides, raffinose, stachyose, and isomaltosaccharides. Compared to the PTSs, the ABC transporters showed higher transcription under several tested conditions, suggesting that they might be transporting multiple substrates. [Abstract/Link to Full Text]

Byrne GA, Russell DA, Chen X, Meijer WG
Transcriptional regulation of the virR operon of the intracellular pathogen Rhodococcus equi.
J Bacteriol. 2007 Jul;189(14):5082-9.
The virR operon, located on the virulence plasmid of the intracellular pathogen Rhodococcus equi, contains five genes, two of which (virR and orf8) encode transcriptional regulators. The first gene of the operon (virR), encoding a LysR-type transcriptional regulator, is transcribed at a constitutive low level, whereas the four downstream genes are induced by low pH and high growth temperature. Differential regulation of the virR operon genes could not be explained by differential mRNA stability, as there were no major differences in mRNA half-lives of the transcripts representing each of the five genes within the virR operon. Transcription of virR is driven by the P(virR) promoter, with a transcription start site 53 bp upstream of the virR initiation codon. The four genes downstream of virR are transcribed from P(virR) and from a second promoter, P(orf5), located 585 bp downstream of the virR initiation codon. VirR binds to a site overlapping the initiation codon of virR, resulting in negative autoregulation of the virR gene, explaining its low constitutive transcription level. The P(orf5) promoter is induced by high temperature and low pH, thus explaining the observed differential gene expression of the virR operon. VirR has a positive effect on P(orf5) activity, whereas the response regulator encoded by orf8 is not involved in regulating transcription of the virR operon. The P(virR) promoter is strikingly similar to those recognized by the principal sigma factors of Streptomyces and Mycobacterium, whereas the P(orf5) promoter does not share sequence similarity with P(virR). This suggests that P(orf5) is recognized by an alternative sigma factor. [Abstract/Link to Full Text]

Friedrich A, Arvidson CG, Shafer WM, Lee EH, So M
Two ABC transporter operons and the antimicrobial resistance gene mtrF are pilT responsive in Neisseria gonorrhoeae.
J Bacteriol. 2007 Jul;189(14):5399-402.
Retraction of type IV pili is mediated by PilT. We show that loss of pilT function leads to upregulation of mtrF (multiple transferable resistance) and two operons encoding putative ABC transporters in Neisseria gonorrhoeae MS11. This effect occurs indirectly through the transcriptional regulator FarR, which until now has been shown to regulate only farAB. L-Glutamine can reverse pilT downregulation of the ABC transporter operons and mtrF. [Abstract/Link to Full Text]

Juliao PC, Marrs CF, Xie J, Gilsdorf JR
Histidine auxotrophy in commensal and disease-causing nontypeable Haemophilus influenzae.
J Bacteriol. 2007 Jul;189(14):4994-5001.
Histidine biosynthesis is one of the best studied metabolic pathways in bacteria. Although this pathway is thought to be highly conserved within and between bacterial species, a previous study identified a genetic region within the histidine operon (his) of nontypeable strains of Haemophilus influenzae (NTHI) that was more prevalent among otitis media strains than among throat commensal NTHI strains. In the present study, we further characterized this region and showed that genes in the complete his operon (hisG, -D, -C, -NB, -H, -A, -F, and -IE) are >99% conserved among four fully sequenced NTHI strains, are present in the same location in these four genomes, and are situated in the same gene order. Using PCR and dot blot hybridization, we determined that the his operon was significantly more prevalent in otitis media NTHI strains (106/121; 87.7%) than in throat strains (74/137; 54%) (prevalence ratio, 1.62; P<0.0001), suggesting a possible role in middle ear survival and/or acute otitis media. NTHI strains lacking the his operon showed attenuated growth in histidine-restricted media, confirming them as his-negative auxotrophs. Our results suggest that the ability to make histidine is an important factor in bacterial growth and survival in the middle ear, where nutrients such as histidine may be found in limited amounts. Those isolates lacking the histidine pathway were still able to survive well in the throat, which suggests that histidine is readily available in the throat environment. [Abstract/Link to Full Text]

Daines DA, Wu MH, Yuan SY
VapC-1 of nontypeable Haemophilus influenzae is a ribonuclease.
J Bacteriol. 2007 Jul;189(14):5041-8.
Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage. [Abstract/Link to Full Text]

Wang X, Kikuchi T, Rikihisa Y
Proteomic identification of a novel Anaplasma phagocytophilum DNA binding protein that regulates a putative transcription factor.
J Bacteriol. 2007 Jul;189(13):4880-6.
Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium. Little is known about the gene regulatory mechanisms for this bacterium. A gene encoding a putative transcription factor, tr1, upstream of three tandem genes encoding outer membrane proteins, including the major outer membrane protein P44, is driven by a strong promoter. In the present study, gel mobility shift assays revealed the presence of A. phagocytophilum proteins that interact with the promoter region of tr1. These proteins interacting with the tr1 promoter region were purified by biotin-labeled DNA affinity chromatography from a large amount of host cell-free bacteria. Mass spectrometry identified the major protein as an A. phagocytophilum 12.5-kDa hypothetical protein, which was named ApxR. In a DNase I protection assay, recombinant ApxR (rApxR) bound cooperatively to four 24- or 25-bp sites within 235 bp upstream of tr1: regions III and IV proximal to tr1 had higher affinity than regions I and II did. Deletion assays showed that regions III and IV were essential for rApxR binding, whereas regions I and II upstream of regions III and IV were not. The primary cis-acting region was region IV, since region IV alone was sufficient for rApxR to strongly transactivate the downstream gene in a lacZ reporter assay. Addition of regions I, II, and III did not enhance transactivation. These results show that ApxR is a novel transcriptional regulator that directly regulates tr1. [Abstract/Link to Full Text]

Bentchikou E, Servant P, Coste G, Sommer S
Additive effects of SbcCD and PolX deficiencies in the in vivo repair of DNA double-strand breaks in Deinococcus radiodurans.
J Bacteriol. 2007 Jul;189(13):4784-90.
Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated the sbcC and/or sbcD genes of Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following gamma-irradiation. It has been recently reported that D. radiodurans DNA polymerase X (PolX) possesses a structure-modulated 3'-to-5' exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex from Escherichia coli. We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant DeltasbcCD DeltapolX(Dr) (where Dr indicates D. radiodurans) bacteria are much more sensitive to gamma-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair. [Abstract/Link to Full Text]

Vakharia-Rao H, Kastead KA, Savenkova MI, Bulathsinghala CM, Postle K
Deletion and substitution analysis of the Escherichia coli TonB Q160 region.
J Bacteriol. 2007 Jul;189(13):4662-70.
The active transport of iron siderophores and vitamin B(12) across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonBDelta7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonBDelta7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter. [Abstract/Link to Full Text]

Ikeda R, Saito F, Matsuo M, Kurokawa K, Sekimizu K, Yamaguchi M, Kawamoto S
Contribution of the mannan backbone of cryptococcal glucuronoxylomannan and a glycolytic enzyme of Staphylococcus aureus to contact-mediated killing of Cryptococcus neoformans.
J Bacteriol. 2007 Jul;189(13):4815-26.
The fungal pathogen Cryptococcus neoformans is killed by the bacterium Staphylococcus aureus, and the killing is inhibited by soluble capsular polysaccharides. To investigate the mechanism of killing, cells in coculture were examined by scanning and transmission electron microscopy. S. aureus attached to the capsule of C. neoformans, and the ultrastructure of the attached C. neoformans cells was characteristic of dead cells. To identify the molecules that contributed to the fungal-bacterial interaction, we treated each with NaIO(4) or protease. Treatment of C. neoformans with NaIO(4) promoted adherence. It was inferred that cleavage of xylose and glucuronic acid side chains of glucuronoxylomannan (GXM) allowed S. aureus to recognize mannose residues in the backbone, which resisted periodate oxidation. On the other hand, treatment of S. aureus with protease decreased adherence, suggesting that protein contributed to attachment in S. aureus. In confirmation, side chain-cleaved polysaccharide or defined alpha-(1-->3)-mannan inhibited the killing at lower concentrations than native GXM did. Also, these polysaccharides reduced the adherence of the two species and induced clumping of pure S. aureus cells. alpha-(1-->3)-Mannooligosaccharides with a degree of polymerization (DP) of >/=3 induced cluster formation of S. aureus in a dose-dependent manner. Surface plasmon resonance analyses showed interaction of GXM and surface protein from S. aureus; the interaction was inhibited by oligosaccharides with a DP of > or =3. Conformations of alpha-(1-->3) oligosaccharides were predicted. The three-dimensional structures of mannooligosaccharides larger than triose appeared curved and could be imagined to be recognized by a hypothetical staphylococcal lectin. Native polyacrylamide gel electrophoresis of staphylococcal protein followed by electroblotting, enzyme-linked immunolectin assay, protein staining, and N-terminal amino acid sequencing suggested that the candidate protein was triosephosphate isomerase (TPI). The enzymatic activities were confirmed by using whole cells of S. aureus. TPI point mutants of S. aureus decreased the ability to interact with C. neoformans. Thus, TPI on S. aureus adheres to the capsule of C. neoformans by recognizing the structure of mannotriose units in the backbone of GXM; we suggest that this contact is required for killing of C. neoformans. [Abstract/Link to Full Text]

Nakunst D, Larisch C, Hüser AT, Tauch A, Pühler A, Kalinowski J
The extracytoplasmic function-type sigma factor SigM of Corynebacterium glutamicum ATCC 13032 is involved in transcription of disulfide stress-related genes.
J Bacteriol. 2007 Jul;189(13):4696-707.
The gene for the extracytoplasmic function (ECF) sigma factor SigM was deleted from the chromosome of the gram-positive soil bacterium Corynebacterium glutamicum to elucidate the role of the SigM protein in the regulation of gene expression. Comparative DNA microarray hybridizations of the C. glutamicum wild type and sigM-deficient mutant C. glutamicum DN1 revealed 23 genes with enhanced expression in the sigM-proficient strain, encoding functions in the assembly of iron-sulfur clusters (suf operon), thioredoxin reductase (trxB), thioredoxins (trxC, trxB1), chaperones (groES, groEL, clpB), and proteins involved in the heat shock response (hspR, dnaJ, grpE). Deletion of the sigM gene rendered the C. glutamicum cells more sensitive to heat, cold, and the presence of the thiol oxidant diamide. Transcription of the sigM gene increased under different stress conditions, including heat shock, cold shock, and disulfide stress caused by diamide treatment, suggesting a regulatory role for SigM under thiol-oxidative stress conditions. Stress-responsive promoters were determined upstream of the suf operon and of the trxB, trxC, and trxB1 genes. The deduced SigM consensus promoter is characterized by the -35 hexamer gGGAAT and the -10 hexamer YGTTGR. Transcription of the sigM gene is apparently controlled by the ECF sigma factor SigH, since a sigH mutant was unable to enhance the expression of sigM and the SigM regulon under thiol-oxidative stress conditions. A typical SigH-responsive promoter was mapped upstream of the sigM gene. The ECF sigma factor SigM is apparently part of a regulatory cascade, and its transcription is controlled by SigH under conditions of thiol-oxidative stress. [Abstract/Link to Full Text]

Folster JP, Dhulipala V, Nicholas RA, Shafer WM
Differential regulation of ponA and pilMNOPQ expression by the MtrR transcriptional regulatory protein in Neisseria gonorrhoeae.
J Bacteriol. 2007 Jul;189(13):4569-77.
Neisseria gonorrhoeae utilizes the mtrCDE-encoded efflux pump system to resist not only host-derived, hydrophobic antimicrobials that bathe mucosal surfaces, which likely aids in its ability to colonize and infect numerous sites within the human host, but also antibiotics that have been used clinically to treat infections. Recently, overexpression of the MtrC-MtrD-MtrE efflux pump was shown to be critically involved in the capacity of gonococci to develop chromosomally mediated resistance to penicillin G, which for over 40 years was used to treat gonococcal infections. Mutations in either the promoter or the coding sequence of the mtrR gene, which encodes a repressor of the efflux pump operon, decrease gonococcal susceptibility to penicillin. We now describe the capacity of MtrR to directly or indirectly influence the expression of two other loci that are involved in gonococcal susceptibility to penicillin: ponA, which encodes penicillin-binding protein 1 (PBP 1), and the pilMNOPQ operon, which encodes components of the type IV pilus secretion system, with PilQ acting as a channel for entry for penicillin. We determined that MtrR increases the expression of ponA directly or indirectly, resulting in increased levels of PBP 1, while repressing the expression of the divergently transcribed pilM gene, the first gene in the pilMNOPQ operon. Taken together with other studies, the results presented herein indicate that transcriptional regulation of gonococcal genes by MtrR is centrally involved in determining levels of gonococcal susceptibility to penicillin and provides a framework for understanding how resistance developed over the years. [Abstract/Link to Full Text]

Barjon C, Wecker K, Izadi-Pruneyre N, Delepelaire P
Mutagenesis and molecular modeling reveal three key extracellular loops of the membrane receptor HasR that are involved in hemophore HasA binding.
J Bacteriol. 2007 Jul;189(14):5379-82.
On the basis of the three-dimensional model of the heme/hemophore TonB-dependent outer membrane receptor HasR, mutants with six-residue deletions in the 11 putative extracellular loops were generated. Although all mutants continued to be active TonB-dependent heme transporters, mutations in three loops abolished hemophore HasA binding both in vivo and in vitro. [Abstract/Link to Full Text]

Thijs IM, De Keersmaecker SC, Fadda A, Engelen K, Zhao H, McClelland M, Marchal K, Vanderleyden J
Delineation of the Salmonella enterica serovar Typhimurium HilA regulon through genome-wide location and transcript analysis.
J Bacteriol. 2007 Jul;189(13):4587-96.
The Salmonella enterica serovar Typhimurium HilA protein is the key regulator for the invasion of epithelial cells. By a combination of genome-wide location and transcript analysis, the HilA-dependent regulon has been delineated. Under invasion-inducing conditions, HilA binds to most of the known target genes and a number of new target genes. The sopB, sopE, and sopA genes, encoding effector proteins secreted by the type III secretion system on Salmonella pathogenicity island 1 (SPI-1), were identified as being both bound by HilA and differentially regulated in an HilA mutant. This suggests a cooperative role for HilA and InvF in the regulation of SPI-1-secreted effectors. Also, siiA, the first gene of SPI-4, is both bound by HilA and differentially regulated in an HilA mutant, thus linking this pathogenicity island to the invasion key regulator. Finally, the interactions of HilA with the SPI-2 secretion system gene ssaH and the flagellar gene flhD imply a repressor function for HilA under invasion-inducing conditions. [Abstract/Link to Full Text]

Kawasaki K, China K, Nishijima M
Release of the lipopolysaccharide deacylase PagL from latency compensates for a lack of lipopolysaccharide aminoarabinose modification-dependent resistance to the antimicrobial peptide polymyxin B in Salmonella enterica.
J Bacteriol. 2007 Jul;189(13):4911-9.
Salmonella enterica modifies its lipopolysaccharide (LPS), including the lipid A portion, to adapt to its environments. The lipid A 3-O-deacylase PagL exhibits latency; deacylation of lipid A is not usually observed in vivo despite the expression of PagL, which is under the control of a two-component regulatory system, PhoP-PhoQ. In contrast, PagL is released from latency in pmrA and pmrE mutants, both of which are deficient in aminoarabinose-modified lipid A, although the biological significance of this is not clear. The attachment of aminoarabinose to lipid A decreases the net anionic charge at the membrane's surface and reduces electrostatic repulsion between neighboring LPS molecules, leading to increases in bacterial resistance to cationic antimicrobial peptides, including polymyxin B. Here we examined the effects of the release of PagL from latency on resistance to polymyxin B. The pmrA pagL and pmrE pagL double mutants were more susceptible to polymyxin B than were the parental pmrA and pmrE mutants, respectively. Furthermore, introduction of the PagL expression plasmid into the pmrA pagL double mutant increased the resistance to polymyxin B. In addition, PagL-dependent deacylation of lipid A was observed in a mutant in which lipid A could not be modified with phosphoethanolamine, which partly contributes to the PmrA-dependent resistance to polymyxin B. These results, taken together, suggest that the release of PagL from latency compensates for the loss of resistance to polymyxin B that is due to a lack of other modifications to LPS. [Abstract/Link to Full Text]

Williams KP, Sobral BW, Dickerman AW
A robust species tree for the alphaproteobacteria.
J Bacteriol. 2007 Jul;189(13):4578-86.
The branching order and coherence of the alphaproteobacterial orders have not been well established, and not all studies have agreed that mitochondria arose from within the Rickettsiales. A species tree for 72 alphaproteobacteria was produced from a concatenation of alignments for 104 well-behaved protein families. Coherence was upheld for four of the five orders with current standing that were represented here by more than one species. However, the family Hyphomonadaceae was split from the other Rhodobacterales, forming an expanded group with Caulobacterales that also included Parvularcula. The three earliest-branching alphaproteobacterial orders were the Rickettsiales, followed by the Rhodospirillales and then the Sphingomonadales. The principal uncertainty is whether the expanded Caulobacterales group is more closely associated with the Rhodobacterales or the Rhizobiales. The mitochondrial branch was placed within the Rickettsiales as a sister to the combined Anaplasmataceae and Rickettsiaceae, all subtended by the Pelagibacter branch. Pelagibacter genes will serve as useful additions to the bacterial outgroup in future evolutionary studies of mitochondrial genes, including those that have transferred to the eukaryotic nucleus. [Abstract/Link to Full Text]

Sánchez-Sutil MC, Gómez-Santos N, Moraleda-Muńoz A, Martins LO, Pérez J, Muńoz-Dorado J
Differential expression of the three multicopper oxidases from Myxococcus xanthus.
J Bacteriol. 2007 Jul;189(13):4887-98.
Myxococcus xanthus is a soil bacterium that undergoes a unique life cycle among the prokaryotes upon starvation, which includes the formation of macroscopic structures, the fruiting bodies, and the differentiation of vegetative rods into coccoid myxospores. This peculiarity offers the opportunity to study the copper response in this bacterium in two different stages. In fact, M. xanthus vegetative rods exhibit 15-fold-greater resistance against copper than developing cells. However, cells pre-adapted to this metal reach the same levels of resistance during both stages. Analysis of the M. xanthus genome reveals that many of the genes involved in copper resistance are redundant, three of which encode proteins of the multicopper oxidase family (MCO). Each MCO gene exhibits a different expression profile in response to external copper addition. Promoters of cuoA and cuoB respond to Cu(II) ions during growth and development; however, they show a 10-fold-increased copper sensitivity during development. The promoter of cuoC shows copper-independent induction upon starvation, but it is copper up-regulated during growth. Phenotypic analyses of deletion mutants reveal that CuoB is involved in the primary copper-adaptive response; CuoA and CuoC are necessary for the maintenance of copper tolerance; and CuoC is required for normal development. These roles seem to be carried out through cuprous oxidase activity. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Clinical Microbiology

---

Thilesen CM, Nicolaidis M, Lökebö JE, Falsen E, Jorde AT, Müller F
Leptotrichia amnionii, an emerging pathogen of the female urogenital tract.
J Clin Microbiol. 2007 Jul;45(7):2344-7.
Leptotrichia amnionii, a recently described, very fastidious, gram-negative anaerobic bacterium, is an opportunistic pathogen of the female urogenital tract. We report a case of second-trimester abortion in a patient with chorioamnionitis and L. amnionii bacteremia and a case of renal abscess in a female 5 weeks postpartum. [Abstract/Link to Full Text]

Louisirirotchanakul S, Lerdsamran H, Wiriyarat W, Sangsiriwut K, Chaichoune K, Pooruk P, Songserm T, Kitphati R, Sawanpanyalert P, Komoltri C, Auewarakul P, Puthavathana P
Erythrocyte binding preference of avian influenza H5N1 viruses.
J Clin Microbiol. 2007 Jul;45(7):2284-6.
Five erythrocyte species (horse, goose, chicken, guinea pig, and human) were used to agglutinate avian influenza H5N1 viruses by hemagglutination assay and to detect specific antibody by hemagglutination inhibition test. We found that goose erythrocytes confer a greater advantage over other erythrocyte species in both assays. [Abstract/Link to Full Text]

Cooke FJ, Wain J, Fookes M, Ivens A, Thomson N, Brown DJ, Threlfall EJ, Gunn G, Foster G, Dougan G
Prophage sequences defining hot spots of genome variation in Salmonella enterica serovar Typhimurium can be used to discriminate between field isolates.
J Clin Microbiol. 2007 Aug;45(8):2590-8.
Sixty-one Salmonella enterica serovar Typhimurium isolates of animal and human origin, matched by phage type, antimicrobial resistance pattern, and place of isolation, were analyzed by microbiological and molecular techniques, including pulsed-field gel electrophoresis (PFGE) and plasmid profiling. PFGE identified 10 profiles that clustered by phage type and antibiotic resistance pattern with human and animal isolates distributed among different PFGE profiles. Genomic DNA was purified from 23 representative strains and hybridized to the composite Salmonella DNA microarray, and specific genomic regions that exhibited significant variation between isolates were identified. Bioinformatic analysis showed that variable regions of DNA were associated with prophage-like elements. Subsequently, simple multiplex PCR assays were designed on the basis of these variable regions that could be used to discriminate between S. enterica serovar Typhimurium isolates from the same geographical region. These multiplex PCR assays, based on prophage-like elements and Salmonella genomic island 1, provide a simple method for identifying new variants of S. enterica serovar Typhimurium in the field. [Abstract/Link to Full Text]

Al-Benwan K, Abbott S, Janda JM, Huys G, Albert MJ
Cystitis caused by Aeromonas caviae.
J Clin Microbiol. 2007 Jul;45(7):2348-50.
Aeromonas sp. organisms rarely cause urinary tract infection. We report for the first time a case of urinary tract infection caused by A. caviae in an adult patient with a history of increased frequency of urination, dysuria, hematuria, and weight loss. [Abstract/Link to Full Text]

Klungthong C, Gibbons RV, Thaisomboonsuk B, Nisalak A, Kalayanarooj S, Thirawuth V, Nutkumhang N, Mammen MP, Jarman RG
Dengue virus detection using whole blood for reverse transcriptase PCR and virus isolation.
J Clin Microbiol. 2007 Aug;45(8):2480-5.
Dengue is one of the most important diseases in the tropical and subtropical regions of the world, with an estimated 2.5 billion people being at risk. Detection of dengue virus infections has great importance for the clinical management of patients, surveillance, and clinical trial assessments. Traditionally, blood samples are collected in serum separator tubes, processed for serum, and then taken to the laboratory for analysis. The use of whole blood has the potential advantages of requiring less blood, providing quicker results, and perhaps providing better sensitivity during the acute phase of the disease. We compared the results obtained by reverse transcriptase PCR (RT-PCR) with blood drawn into tubes containing EDTA and those obtained by RT-PCR with blood samples in serum separator tubes from 108 individuals clinically suspected of being infected with dengue virus. We determined that the extraction of RNA from whole blood followed by RT-PCR resulted in a higher detection rate than the use of serum or plasma. Using a selection of these samples, we also found that our ability to detect virus by direct C6/36 cell culture and mosquito inoculation was enhanced by using whole blood but not to the same extent as that seen by the use of RT-PCR. [Abstract/Link to Full Text]

Pham NT, Khamrin P, Nguyen TA, Kanti DS, Phan TG, Okitsu S, Ushijima H
Isolation and molecular characterization of Aichi viruses from fecal specimens collected in Japan, Bangladesh, Thailand, and Vietnam.
J Clin Microbiol. 2007 Jul;45(7):2287-8.
Aichi virus is a new member of the family Picornaviridae, genus Kobuvirus, and is associated with human gastroenteritis. This study detected Aichi virus in 28 of 912 fecal specimens which were negative for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus and were collected in Japan, Bangladesh, Thailand, and Vietnam during 2002 to 2005. [Abstract/Link to Full Text]

Maaroufi Y, De Bruyne JM, Heymans C, Crokaert F
Real-time PCR for determining capsular serotypes of Haemophilus influenzae.
J Clin Microbiol. 2007 Jul;45(7):2305-8.
A two-step real-time PCR assay targeting all six capsulation loci of Haemophilus influenzae (i.e., serotypes a to f) was developed and compared with a previously published qualitative PCR assay by using 131 H. influenzae clinical isolates. There was a 98.5% concordance between the two tests. The sensitivity of detection of capsular type-specific reference strains of H. influenzae a to c (10(1) CFU/PCR) was higher than that for type e (10(3) CFU/PCR) and types d and f (10(4) CFU/PCR), and a broader dynamic range was obtained (5 to 8 log(10) units). No cross-reaction was observed with bacteria commonly isolated from the respiratory tract. We showed that both PCR assays are more reliable than slide agglutination serotyping. The real-time PCR-based assay seems to be an alternative of choice for the epidemiological follow-up of H. influenzae invasive infections. [Abstract/Link to Full Text]

Taylor NS, Xu S, Nambiar P, Dewhirst FE, Fox JG
Enterohepatic Helicobacter species are prevalent in mice from commercial and academic institutions in Asia, Europe, and North America.
J Clin Microbiol. 2007 Jul;45(7):2166-72.
The discovery of Helicobacter hepaticus and its role in hepatitis, hepatocellular carcinoma, typhlocolitis, and lower-bowel carcinoma in murine colonies was followed by the isolation and characterization of other Helicobacter spp. involved in enterohepatic disease. Colonization of mouse colonies with members of the family Helicobacteriaceae has become an increasing concern for the research community. From 2001 to 2005, shipments of selected gift mice from other institutions and mice received from specified commercial vendors were screened for Helicobacter spp. by culture of cecal tissue. The identities of the isolates were confirmed by genus-specific PCR, followed by species-specific PCR and restriction fragment length polymorphism analysis. Sequencing of the 16S rRNA gene was performed if the species identity was not apparent. The survey included 79 mice from 34 sources: 2 commercial sources and 16 research sources from the United States and 1 commercial source and 15 research sources from Canada, Europe, or Asia. Helicobacter spp. were cultured from the ceca of 62 of 79 mice. No Helicobacter spp. were found in mice from advertised Helicobacter-free production areas from two U.S. vendors. Multiple Helicobacter spp. were found in mice from one vendor's acknowledged Helicobacter-infected production area. The European commercial vendor had mice infected with novel Helicobacter sp. strain MIT 96-1001. Of the U.S. academic institutions, 6 of 16 (37%) had mice infected with Helicobacter hepaticus; but monoinfection with H. bilis, H. mastomyrinus, H. rodentium, and MIT 96-1001 was also encountered, as were mice infected simultaneously with two Helicobacter spp. Non-U.S. academic institutions had mice that were either monoinfected with H. hepaticus, monoinfected with seven other Helicobacter spp., or infected with a combination of Helicobacter spp. This survey indicates that 30 of 34 (88%) commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States have mouse colonies infected with Helicobacter spp. Mice from 20 of the 34 institutions (59%) were most commonly colonized with H. hepaticus alone or in combination with other Helicobacter spp. These results indicate that a broad range of Helicobacter spp. infect mouse research colonies. The potential impact of these organisms on in vivo experiments continues to be an important issue for mice being used for biomedical research. [Abstract/Link to Full Text]

O'Donnell K, Sarver BA, Brandt M, Chang DC, Noble-Wang J, Park BJ, Sutton DA, Benjamin L, Lindsley M, Padhye A, Geiser DM, Ward TJ
Phylogenetic diversity and microsphere array-based genotyping of human pathogenic Fusaria, including isolates from the multistate contact lens-associated U.S. keratitis outbreaks of 2005 and 2006.
J Clin Microbiol. 2007 Jul;45(7):2235-48.
In 2005 and 2006, outbreaks of Fusarium keratitis associated with soft contact lens use occurred in multiple U.S. states and Puerto Rico. A case-control study conducted by the Centers for Disease Control and Prevention (CDC) showed a significant association between infections and the use of one particular brand of lens solution. To characterize the full spectrum of the causal agents involved and their potential sources, partial DNA sequences from three loci (RPB2, EF-1alpha, and nuclear ribosomal rRNA) totaling 3.48 kb were obtained from 91 corneal and 100 isolates from the patient's environment (e.g., contact lens and lens cases). We also sequenced a 1.8-kb region encoding the RNA polymerase II second largest subunit (RPB2) from 126 additional pathogenic isolates to better understand how the keratitis outbreak isolates fit within the full phylogenetic spectrum of clinically important fusaria. These analyses resulted in the most robust phylogenetic framework for Fusarium to date. In addition, RPB2 nucleotide variation within a 72-isolate panel was used to design 34 allele-specific probes to identify representatives of all medically important species complexes and 10 of the most important human pathogenic Fusarium in a single-well diagnostic assay, using flow cytometry and fluorescent microsphere technology. The multilocus data revealed that one haplotype from each of the three most common species comprised 55% of CDC's corneal and environmental isolates and that the corneal isolates comprised 29 haplotypes distributed among 16 species. The high degree of phylogenetic diversity represented among the corneal isolates is consistent with multiple sources of contamination. [Abstract/Link to Full Text]

Leaw SN, Chang HC, Barton R, Bouchara JP, Chang TC
Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array.
J Clin Microbiol. 2007 Jul;45(7):2220-9.
The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies. [Abstract/Link to Full Text]

Tcheremenskaia O, Marucci G, De Petris S, Ruggeri FM, Dovecar D, Sternak SL, Matyasova I, Dhimolea MK, Mladenova Z, Fiore L
Molecular epidemiology of rotavirus in Central and Southeastern Europe.
J Clin Microbiol. 2007 Jul;45(7):2197-204.
A surveillance network was implemented by the Istituto Superiore di Sanitŕ of Rome in collaboration with laboratories of virology in Czech Republic, Slovenia, Croatia, Albania, and Bulgaria. About 1,500 rotavirus-positive stool samples were collected from children with severe gastroenteritis admitted to hospitals or outpatient wards between 2004 and 2006. The G and P genotypes were determined by reverse transcription-nested PCR. Significant differences were found in the geographical distributions of rotavirus genotypes between countries participating in the study. The prevalence of "common" G/P combinations, G1P[8], G3P[8], G4P[8], and G2P[4], ranged between 50 and 85%. The G9 genotype, which is emerging worldwide, was identified in 2 to 35% of all samples depending on the country. Unusual combinations, such as G1 or G4 associated with P[4] or G2 with P[8], which may have arisen by reassortment between human strains, were found in samples from 3 to 20% of patients. The uncommon genotypes G8P[8] and G10P[6], which may have an animal origin, were also identified. Double infections with two rotavirus strains were observed in between 1.7 and 14% of cases studied. Our findings might implicate challenges for rotavirus vaccine implementation in a wide geographic area of the Balkans and Central-Eastern Europe and underscore the importance of extensive strain surveillance for success in vaccine development. [Abstract/Link to Full Text]

Carbonnelle E, Beretti JL, Cottyn S, Quesne G, Berche P, Nassif X, Ferroni A
Rapid identification of Staphylococci isolated in clinical microbiology laboratories by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
J Clin Microbiol. 2007 Jul;45(7):2156-61.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) of intact bacteria yields a reproducible spectrum depending upon growth conditions, strain, or species. Using whole viable bacteria we describe here the application of MALDI-TOF-MS to the identification of coagulase-negative staphylococci (CoNS). Our aim was, once a bacterium has been recognized as Micrococcaceae, to identify peaks in the spectrum that can be used to identify the species or subspecies. MALDI-TOF-MS was performed using bacteria obtained from one isolated colony. One reference strain for each of the 23 clinically relevant species or subspecies of Micrococcaceae was selected. For each reference strain, the MALDI-TOF-MS profile of 10 colonies obtained from 10 different passages was analyzed. For each strain, only peaks that were conserved in the spectra of all 10 isolated colonies and with a relative intensity above 0.1 were retained, thus leading to a set of 3 to 14 selected peaks per strain. The MALDI-TOF-MS profile of 196 tested strains was then compared with that of the set of selected peaks of each of the 23 reference strains. In all cases the best hit was with the set of peaks of the reference strain belonging to the same species as that of the tested strain, thus demonstrating that the 23 sets of selected peaks can be used as a database for the rapid species identification of CoNS. Similar results were obtained using four different growth conditions. Extending this strategy to other groups of relevant pathogenic bacteria will allow rapid bacterial identification. [Abstract/Link to Full Text]

Decousser JW, Prouzet-Mauléon V, Bartizel C, Gin T, Colin JP, Fadel N, Holler C, Pollet J, Megraud F
Fatal relapse of a purulent pleurisy caused by Campylobacter fetus subsp. fetus.
J Clin Microbiol. 2007 Jul;45(7):2334-6.
Campylobacter fetus is associated with invasive disease, while other Campylobacter species, such as C. coli and C. jejuni, are a common cause of bacterial diarrhea. Bacteremia has been well described, but pleurisy remains very uncommon. We report the recurrent isolation of a C. fetus subsp. fetus strain during two episodes of pleural effusion with a fatal outcome. [Abstract/Link to Full Text]

Jinno S, Jirakulaporn T, Bankowski MJ, Kim W, Wong R
Rare case of Nocardia asteroides pericarditis in a human immunodeficiency virus-infected patient.
J Clin Microbiol. 2007 Jul;45(7):2330-3.
Nocardia asteroides was isolated after prolonged culture from the pericardial fluid of a human immunodeficiency virus-infected patient. The lengthy duration required for culture growth and identification of this N. asteroides isolate affected both initial therapeutic decisions and patient management. A proposed algorithm for the microbiological workup of pericardial fluid for possible Nocardia spp. is described in an effort to improve the timeliness of results. [Abstract/Link to Full Text]

Nascimento MC, Ferreira S, Sabino E, Hamilton I, Parry J, Pannuti CS, Mayaud P
Performance of the HerpeSelect (Focus) and Kalon enzyme-linked immunosorbent assays for detection of antibodies against herpes simplex virus type 2 by use of monoclonal antibody-blocking enzyme immunoassay and clinicovirological reference standards in Brazil.
J Clin Microbiol. 2007 Jul;45(7):2309-11.
A total of 586 serum samples were used to evaluate the performance of type-specific herpes simplex virus type 2 (HSV-2) commercial enzyme-linked immunosorbent assays (ELISAs) by using the monoclonal antibody-blocking enzyme immunoassay (MAb-EIA) and a clinicovirological panel as reference standards. The Kalon and HerpeSelect ELISAs had similar sensitivities (93.5% and 93.8% compared with the results obtained by MAb-EIA, respectively, and 100% for both ELISAs compared with the results obtained with a clinicovirological panel). The Kalon ELISA had a higher specificity (96.5% and 96.8% compared with the results obtained by MAb-EIA and with a clinicovirological panel, respectively) than the HerpeSelect ELISA (86.9% and 94% compared with the results obtained by MAb-EIA and with a clinicovirological panel, respectively). A higher cutoff significantly improved the specificity of the HerpeSelect ELISA. [Abstract/Link to Full Text]

Al-Hajoj SA, Zozio T, Al-Rabiah F, Mohammad V, Al-Nasser M, Sola C, Rastogi N
First insight into the population structure of Mycobacterium tuberculosis in Saudi Arabia.
J Clin Microbiol. 2007 Aug;45(8):2467-73.
This study constitutes a first attempt to describe the genetic population structure and drug resistance of the tubercle bacilli circulating in Saudi Arabia. A total of 1,505 clinical isolates of M. tuberculosis, isolated between 2002 and 2005 from seven regions of Saudi Arabia, were studied. The sample studied showed a male-to-female sex ratio of 1.27, with half of the cases among foreign-born individuals and 47% within the 21- to 40-year-old age group; a total resistance rate of 19.7%; and multiple drug resistance of 4.5%. Upon spoligotyping, a total of 387 individual patterns were obtained (clustering rate, 86.4%; 182 clusters containing between 2 and 130 isolates per cluster). A total of 94% of the strains matched the spoligotype patterns in an international database. Nearly 81% of the isolates in this study belonged to established phylogeographic clades: Central Asian (CAS), 22.5%; ill-defined T clade, 19.5%; East African-Indian (EAI), 13.5%; Haarlem, 7.5%; Latin American-Mediterranean, 7.2%; Beijing, 4.4%; Manu, 2.7%; X, 0.9%; and Bovis, 0.9%. Two clonal complexes with unique spoligotyping signatures (octal codes 703777707770371 and 467777377413771) specific to Saudi Arabia were identified. These belonged to the CAS and EAI clades, respectively, as confirmed upon secondary typing using mycobacterial interspersed repetitive units (MIRUs). The results obtained underline the predominance of historic clones of principal genetic group 1, which are responsible for roughly 45% of all tuberculosis cases in Saudi Arabia. The high rate of clustering observed might be an indication of rapid ongoing transmission within certain communities and/or subpopulations in Saudi Arabia; nonetheless, spoligotyping is known to overestimate clustering, and only a systematic second-line typing, such as MIRUs, coupled with a better tuberculosis registry and epidemiological investigations would allow us to know the exact rate of ongoing transmission and associated risk factors in Saudi Arabia. [Abstract/Link to Full Text]

Godschalk PC, van Belkum A, van den Braak N, van Netten D, Ang CW, Jacobs BC, Gilbert M, Endtz HP
PCR-restriction fragment length polymorphism analysis of Campylobacter jejuni genes involved in lipooligosaccharide biosynthesis identifies putative molecular markers for Guillain-Barré syndrome.
J Clin Microbiol. 2007 Jul;45(7):2316-20.
Molecular mimicry of Campylobacter jejuni lipooligosaccharides (LOS) by gangliosides in peripheral nerve tissue probably triggers the Guillain-Barré syndrome due to the induction of cross-reactive antibodies. PCR-restriction fragment length polymorphism analysis of C. jejuni genes involved in the biosynthesis of LOS demonstrated that specific genes were associated with the expression of ganglioside mimics and the development of neuropathy. [Abstract/Link to Full Text]

van de Pol AC, van Loon AM, Wolfs TF, Jansen NJ, Nijhuis M, Breteler EK, Schuurman R, Rossen JW
Increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time PCR in samples from patients with respiratory symptoms.
J Clin Microbiol. 2007 Jul;45(7):2260-2.
Respiratory samples (n = 267) from hospitalized patients with respiratory symptoms were tested by real-time PCR, viral culture, and direct immunofluorescence for respiratory syncytial virus, influenza virus, parainfluenza viruses, and adenoviruses. Compared with conventional diagnostic tests, real-time PCR increased the diagnostic yields for these viruses from 24% to 43% and from 3.5% to 36% for children and adults, respectively. [Abstract/Link to Full Text]

Anil M, Ozkalay N, Helvaci M, Agus N, Guler O, Dikerler A, Kanar B
Meningitis due to Gemella haemolysans in a pediatric case.
J Clin Microbiol. 2007 Jul;45(7):2337-9.
Gemella haemolysans is a rare pathogen in cases of bacterial meningitis. We present a case of meningitis due to G. haemolysans in a 17-month-old boy. This is the first reported case of Gemella meningitis in a child. The patient completely recovered following intravenous therapy with linezolid and chloramphenicol. [Abstract/Link to Full Text]

Shukla SK, Aswani V, Stockwell PJ, Reed KD
Contribution of polymorphisms in ankA, gltA, and groESL in defining genetic variants of Anaplasma phagocytophilum.
J Clin Microbiol. 2007 Jul;45(7):2312-5.
Analysis of several nucleotide polymorphisms in polymorphic genes (ankA, gltA, and groESL) from 16S rRNA gene-based genetic variants of Anaplasma phagocytophilum from dogs in the western United States defined at least two sets of multigene polymorphisms to further characterize these variants. The multigene polymorphism approach holds promise for development of a genotyping scheme for this important pathogen. [Abstract/Link to Full Text]

Li H, McCormac MA, Estes RW, Sefers SE, Dare RK, Chappell JD, Erdman DD, Wright PF, Tang YW
Simultaneous detection and high-throughput identification of a panel of RNA viruses causing respiratory tract infections.
J Clin Microbiol. 2007 Jul;45(7):2105-9.
Clinical presentations for viral respiratory tract infections are often nonspecific, and a rapid, high-throughput laboratory technique that can detect a panel of common viral pathogens is clinically desirable. We evaluated two multiplex reverse transcription-PCR (RT-PCR) products coupled with microarray-based systems for simultaneous detection of common respiratory tract viral pathogens. The NGEN respiratory virus analyte-specific assay (Nanogen, San Diego, CA) detects influenza A virus (Flu-A) and Flu-B, parainfluenza virus 1 (PIV-1), PIV-2, and PIV-3, and respiratory syncytial virus (RSV), while the ResPlex II assay (Genaco Biomedical Products, Inc., Huntsville, AL) detects Flu-A, Flu-B, PIV-1, PIV-2, PIV-3, PIV-4, RSV, human metapneumovirus (hMPV), rhinoviruses (RhVs), enteroviruses (EnVs), and severe acute respiratory syndrome (SARS) coronavirus (CoV). A total of 360 frozen respiratory specimens collected for a full year were tested, and results were compared to those obtained with a combined reference standard of cell culture and monoplex real-time TaqMan RT-PCR assays. NGEN and ResPlex II gave comparable sensitivities for Flu-A (82.8 to 86.2%), Flu-B (90.0 to 100.0%), PIV-1 (87.5 to 93.8%), PIV-3 (66.7 to 72.2%), and RSV (63.3 to 73.3%); both assays achieved excellent specificities (99.1 to 100.0%) for these five common viruses. The ResPlex II assay detected hMPV in 13 (3.6%) specimens, with a sensitivity of 80.0% and specificity of 99.7%. The ResPlex II assay also differentiated RSV-A and RSV-B and gave positive results for RhV and EnV in 31 (8.6%) and 19 (5.3%) specimens, respectively. PIV-2, PIV-4, and SARS CoV were not detected in the specimens tested. The two systems can process 80 (NGEN) and 96 (ResPlex II) tests per run, with a hands-on time of approximately 60 min and test turnaround times of 6 h (ResPlex II) and 9 h (NGEN). Multiple-panel testing detected an additional unsuspected 9 (3.4%) PIV-1 and 10 (3.7%) PIV-3 infections. While test sensitivities for RSV and PIV-3 need improvement, both the NGEN and ResPlex II assays provide user-friendly and high-throughput tools for simultaneous detection and identification of a panel of common respiratory viral pathogens in a single test format. The multiplex approach enhances diagnosis through detection of respiratory viral etiologic agents in cases in which the presence of the agent was not suspected and a test was not ordered by the clinicians. [Abstract/Link to Full Text]

Zbinden A, Böttger EC, Bosshard PP, Zbinden R
Evaluation of the colorimetric VITEK 2 card for identification of gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing.
J Clin Microbiol. 2007 Jul;45(7):2270-3.
Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern. [Abstract/Link to Full Text]

Creti R, Imperi M, Baldassarri L, Pataracchia M, Recchia S, Alfarone G, Orefici G
emm Types, virulence factors, and antibiotic resistance of invasive Streptococcus pyogenes isolates from Italy: What has changed in 11 years?
J Clin Microbiol. 2007 Jul;45(7):2249-56.
To investigate the epidemiology and characteristics of invasive group A streptococcal (GAS) disease over 11 years in Italy, this study compared the emm types and the superantigen toxin genes speA and speC as well as the erythromycin, clindamycin, and tetracycline susceptibilities of 207 invasive GAS strains collected during two national enhanced surveillance periods (1994 to 1996 and 2003 to 2005) and the time between each set of surveillance periods. The present study demonstrated that emm1 strains were consistently responsible for about 20% of invasive GAS infections, while variations in the frequencies of the other types were noted, although the causes of most cases of invasive infections were restricted to emm1, emm3, emm4, emm6, emm12, and emm18. During the 1994 to 1996 surveillance period, an emm89 epidemic clone spread across the northern part of Italy. A restricted macrolide resistance phenotype-type distribution of the bacteriophage-encoded speA toxin as well as of macrolide resistance genes was noted over time. Indeed, the recent acquisition of macrolide resistance in previously susceptible emm types was observed. [Abstract/Link to Full Text]

Chiu CY, Alizadeh AA, Rouskin S, Merker JD, Yeh E, Yagi S, Schnurr D, Patterson BK, Ganem D, DeRisi JL
Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.
J Clin Microbiol. 2007 Jul;45(7):2340-3.
A pan-virus DNA microarray (Virochip) was used to detect a human metapneumovirus (hMPV) strain associated with a critical respiratory tract infection in an elderly adult with chronic lymphocytic leukemia. This infection had previously eluded diagnosis despite extensive microbiological testing for possible etiologic agents. The patient's hMPV strain did not grow in viral culture, and only one of five specific reverse transcription-PCR assays for hMPV was positive. [Abstract/Link to Full Text]

Stevens MP, Garland SM, Rudland E, Tan J, Quinn MA, Tabrizi SN
Comparison of the Digene Hybrid Capture 2 assay and Roche AMPLICOR and LINEAR ARRAY human papillomavirus (HPV) tests in detecting high-risk HPV genotypes in specimens from women with previous abnormal Pap smear results.
J Clin Microbiol. 2007 Jul;45(7):2130-7.
The development of cervical cancer is strongly associated with the presence of persistent high-risk (HR) human papillomavirus (HPV) infection. Recently, the commercially manufactured PCR-based Roche AMPLICOR (AMP) and LINEAR ARRAY (LA) HPV tests have become available for HPV detection. However, knowledge of their clinical performance compared to the U.S. Food and Drug Administration-approved Hybrid Capture 2 (HC2) assay is limited. This study evaluated the concordance between the HC2, AMP, and LA tests in detecting HR-HPV among a cohort of 1,679 women with previous abnormal Pap smear results. Overall, 1,393 specimens (81.3%) generated concordant results for HR-HPV presence or absence by the three assays. The concordance levels were substantial between the HC2 and AMP tests (84.4%, kappa = 0.6419) and between the HC2 and LA tests (84.0%, kappa = 0.6341) and nearly perfect between the AMP and LA tests (97.8%, kappa = 0.9441). HR-HPV prevalence, as detected by the AMP or LA tests, was significantly higher among women with cytological or histological high-grade disease (CIN2 or greater) than that detected by HC2 (P < 0.0001). The AMP and LA tests exhibited greater sensitivity, but lower specificity, than HC2 for detecting HR-HPV among this cohort of women with underlying cervical abnormalities, particularly among subjects with histologically proven high-grade disease. Both PCR-based HPV tests may be valuable in the management of care for women with underlying cervical abnormalities, in predicting treatment success, and in studying the clearance or acquisition of new infections. [Abstract/Link to Full Text]

Hakim H, Gibson C, Pan J, Srivastava K, Gu Z, Bankowski MJ, Hayden RT
Comparison of various blood compartments and reporting units for the detection and quantification of Epstein-Barr virus in peripheral blood.
J Clin Microbiol. 2007 Jul;45(7):2151-5.
Epstein-Barr virus (EBV) infection is associated with a broad spectrum of disease. While quantification of EBV nucleic acid in the peripheral blood has been demonstrated to be useful for diagnosis and patient care, the optimal sample type and reporting format for such testing remain uncertain. Using quantitative real-time PCR (QRT-PCR), we evaluated EBV in whole blood (WB), peripheral blood mononuclear cells (PBMC), and plasma in 249 samples from 122 patients. In WB and PBMC, results were reported both in viral copies/ml and in copies/microg of total DNA. Trendings of quantitative values over time among the different sample types were compared. The sensitivities of QRT-PCR using WB and that using PBMC did not differ significantly (P = 0.33), and both were more sensitive than plasma alone (P < 0.0001). EBV viral load results from WB and PBMC paired sample types also showed a significant correlation (P < 0.05), as did results reported in copies/ml and copies/microg DNA for both WB and PBMC (R2 > 0.93). EBV viral loads detected using WB and PBMC trended very closely for the few patients who had multiple positive samples available for analysis. WB and PBMC show comparable sensitivities and a close quantitative correlation when assayed for EBV by QRT-PCR. The close correlation between copies/ml and copies/microg DNA also suggests that normalization to cell number or genomic DNA in cellular specimens may not be necessary. [Abstract/Link to Full Text]

Vinh DC, Garceau R, Martinez G, Wiebe D, Burdz T, Reimer A, Bernard K
Legionella jordanis lower respiratory tract infection: case report and review.
J Clin Microbiol. 2007 Jul;45(7):2321-3.
Legionella jordanis was first described in 1982 after isolation from environmental sources and is otherwise a very rare human pathogen. Here, we report the recovery of L. jordanis from a bronchoalveolar lavage specimen from a patient who presented with an indolent lower respiratory tract infection associated with constitutional symptoms. This case is the first culture-positive case of infection involving this species in Canada. [Abstract/Link to Full Text]

Garnier F, Janapatla RP, Charpentier E, Masson G, Grélaud C, Stach JF, Denis F, Ploy MC
Insertion sequence 1515 in the ply gene of a type 1 clinical isolate of Streptococcus pneumoniae abolishes pneumolysin expression.
J Clin Microbiol. 2007 Jul;45(7):2296-7.
A serotype 1 Streptococcus pneumoniae strain isolated by blood culture from a woman with pneumonia was found to harbor insertion sequence (IS) 1515 in the pneumolysin gene, abolishing pneumolysin expression. To our knowledge, this is the first report of an IS in the pneumolysin gene of S. pneumoniae. [Abstract/Link to Full Text]

Brown SD, Traczewski MM
Comparative in vitro antimicrobial activity of tigecycline, a new glycylcycline compound, in freshly prepared medium and quality control.
J Clin Microbiol. 2007 Jul;45(7):2173-9.
The in vitro spectra of activity of tigecycline and tetracycline were determined for 2,490 bacterial isolates representing 50 different species or phenotypic groups. All isolates were tested simultaneously by broth microdilution using freshly prepared Mueller-Hinton broth and by disk diffusion. Portions of these data were submitted to the Food and Drug Administration (FDA) in support of the sponsor's application for new drug approval. In a separate study, MIC and disk diffusion quality control ranges were determined. The tigecycline MICs at which 50%/90% of bacteria were inhibited were (in microg/ml) as follows: for Streptococcus spp., 0.06/0.12; for Moraxella catarrhalis, 0.06/0.12; for Staphylococcus spp., 0.12/0.25; for Enterococcus spp., 0.12/0.25; for Listeria monocytogenes, 0.12/0.12; for Neisseria meningitidis, 0.12/0.25; for Haemophilus spp., 0.25/0.5; for Enterobacteriaceae, 0.05/2.0; for non-Enterobacteriaceae, 0.5/8.0. Tigecycline was consistently more potent than tetracycline against all species studied. The data from this study confirm the FDA-approved MIC and disk diffusion breakpoints for tigecycline for Streptococcus spp. other than Streptococcus pneumoniae, enterococci, and Enterobacteriaceae. Provisional breakpoints for Haemophilus spp. and S. pneumoniae are proposed based on the data from this study. The following MIC and/or disk diffusion quality control ranges are proposed: Staphylococcus aureus ATCC 29213, 0.03 to 0.25 microg/ml; S. aureus ATCC 25923, 20 to 25 mm; Escherichia coli ATCC 25922, 0.03 to 0.25 microg/ml and 20 to 27 mm; Pseudomonas aeruginosa ATCC 27853, 9 to 13 mm, Enterococcus faecalis ATCC 29212, 0.03 to 0.12 microg/ml; S. pneumoniae ATCC 49619, 0.015 to 0.12 microg/ml and 23 to 29 mm; Haemophilus influenzae ATCC 49247, 0.06 to 0.5 microg/ml and 23 to 31 mm; and Neisseria gonorrhoeae ATCC 49226, 30 to 40 mm. [Abstract/Link to Full Text]

Cameron ML, Tsang RS
Analysis of phenotypic variants of the serogroup C ET-15 clone of Neisseria meningitidis by pulsed-field gel electrophoresis.
J Clin Microbiol. 2007 Jul;45(7):2351-2. [Abstract/Link to Full Text]

Popovich K, Hota B, Rice T, Aroutcheva A, Weinstein RA
Phenotypic prediction rule for community-associated methicillin-resistant Staphylococcus aureus.
J Clin Microbiol. 2007 Jul;45(7):2293-5.
Recent studies have suggested that community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are encroaching upon nosocomial settings. We assessed the performance characteristics of a rule using the antimicrobial phenotype to predict genotype. This rule could be applied for epidemiologic purposes to describe the trend in CA-MRSA infections over time. [Abstract/Link to Full Text]

Tenover FC, Vaughn RR, McDougal LK, Fosheim GE, McGowan JE
Multiple-locus variable-number tandem-repeat assay analysis of methicillin-resistant Staphylococcus aureus strains.
J Clin Microbiol. 2007 Jul;45(7):2215-9.
Our objective was to determine if a multiple-locus variable-number tandem-repeat assay (MLVA) for Staphylococcus aureus could predict pulsed-field gel electrophoresis (PFGE) types (i.e., USA types), thus allowing us to replace PFGE with a simpler and more rapid typing method. One hundred three well-characterized isolates representing 13 major lineages of S. aureus were tested by both PFGE and MLVA. MLVA was performed using a rapid DNA extraction technique and PCR primers for sdrCDE, clfA, clfB, sspA, and spa. PFGE was performed with genomic DNA fragments generated using SmaI, as per CDC protocols. Banding patterns were analyzed both visually and with BioNumerics software. All isolates were typeable with MLVA and PFGE. MLVA patterns were highly reproducible. PFGE separated the isolates into 13 types with 42 subtypes. Using any band difference to designate a novel MLVA type, MLVA produced 45 types, including 9 clusters containing multiple isolates. Using BioNumerics and a cutoff of >75% relatedness, MLVA produced 28 types, 11 of which contained >1 isolate. Epidemiologically related outbreak isolates of USA300-0114 from five states clustered in one MLVA pattern. USA100 isolates were present in several unrelated (<40%) MLVA types. A cutoff of >80% separated outbreak strains of USA300-0114 into three distinct MLVA types. MLVA did not differentiate community methicillin-resistant S. aureus (MRSA) lineages (USA300, USA400, USA1000, and USA1100) from health care MRSA lineages (USA100, USA200, or USA500). The ability of MLVA to differentiate among strains that are indistinguishable by PFGE may be of epidemiologic value and warrants further study. [Abstract/Link to Full Text]

Bourbeau PP, Riley JA, Shoemaker BC, Jones KS
Use of CultureSwab Plus swabs with Amies gel agar for testing of naris specimens with the GeneOhm MRSA assay.
J Clin Microbiol. 2007 Jul;45(7):2281-3.
The GeneOhm MRSA assay detects nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA). We compared the use of seeded swabs with liquid Stuart's medium and that of seeded swabs with Amies gel for the assay. Overall, the swabs with liquid Stuart's medium detected significantly greater numbers of MRSA than the swabs with Amies gel (P = 0.0003). [Abstract/Link to Full Text]

Ho EC, Cheng PK, Lau AW, Wong AH, Lim WW
Atypical norovirus epidemic in Hong Kong during summer of 2006 caused by a new genogroup II/4 variant.
J Clin Microbiol. 2007 Jul;45(7):2205-11.
An atypically high level of norovirus activity was noticed in Hong Kong beginning in early May 2006. A study was carried out to investigate whether this was caused by a new norovirus variant. Epidemiological data including monthly positivity rates and the numbers of outbreaks per month from January to July 2006 were analyzed and compared to those from 2002 to 2005. In a comparison with the epidemiological data from 2001 to 2005, an atypical peak of norovirus-associated gastroenteritis outbreak was observed beginning in May 2006, concurring with a striking increase in norovirus activity. Most of the outbreaks (>60%) were located in homes for the elderly. Phylogenetic analysis for both RdRp and 5' capsid regions showed that this epidemic was caused by a new genogroup II/4 variant. This variant was genetically distinct from the predominant variants of 2002 and 2004 but was closely related to one of the 95/96-subset variants which caused an epidemic in Hong Kong in 2001, suggesting that the 95/96 subset may be starting to recirculate. [Abstract/Link to Full Text]

Fiscus SA, Wiener J, Abrams EJ, Bulterys M, Cachafeiro A, Respess RA
Ultrasensitive p24 antigen assay for diagnosis of perinatal human immunodeficiency virus type 1 infection.
J Clin Microbiol. 2007 Jul;45(7):2274-7.
We evaluated an ultrasensitive p24 antigen enzyme immunosorbent assay on 802 plasma specimens from 582 infants and children of 0 to 180 days of age. Overall sensitivity and specificity were 91.7% and 98.5%, respectively. After exclusion of infants of less than 7 days of age, the sensitivity and specificity were 93.7% and 98.3%, respectively. [Abstract/Link to Full Text]

Neske F, Blessing K, Tollmann F, Schubert J, Rethwilm A, Kreth HW, Weissbrich B
Real-time PCR for diagnosis of human bocavirus infections and phylogenetic analysis.
J Clin Microbiol. 2007 Jul;45(7):2116-22.
The human bocavirus (hBoV) was first described in 2005 in respiratory tract samples. The clinical relevance of hBoV is still unclear. The aim of our study was to establish a real-time PCR assay for the detection and quantification of hBoV DNA, to apply the real-time assay for the analysis of stool and serum samples for the presence of hBoV DNA, and to perform a phylogenetic analysis of the hBoV positive samples. A total of 834 nasopharyngeal aspirates (NPA), 10 serum samples, and 31 stool samples of children with acute respiratory diseases were retrospectively tested. For phylogenetic analysis, 968 bp of the VP2 gene were sequenced from 69 hBoV-positive NPA samples. The qualitative results of the real-time hBoV PCR were in good agreement with a conventional hBoV PCR. We found that 12% of the NPA were positive for hBoV DNA. The median viral load in the NPA was 4.9 x 10(3) copies/ml (range, 2.7 x 10 degrees to 1.5 x 10(11) copies/ml). There was no difference of the hBoV load in NPA between children with or without known coinfection, but the load was significantly higher in children with bronchitis than in children with the diagnosis of febrile seizures. hBoV DNA was found in 1 of 10 serum samples and in 14 of 31 stool samples. hBoV sequence identity was >99% in the VP2 region. In conclusion, hBoV DNA can be found in NPA samples at very high titers. In addition to being found in the respiratory tract, hBoV was found in stool samples. The clinical relevance of these findings remains to be determined. [Abstract/Link to Full Text]

Lam MM, Clarridge JE, Young EJ, Mizuki S
The other group G Streptococcus: increased detection of Streptococcus canis ulcer infections in dog owners.
J Clin Microbiol. 2007 Jul;45(7):2327-9.
beta-Hemolytic Lancefield group G Streptococcus dysgalactiae and Streptococcus canis cannot be distinguished when only Lancefield typing is performed. Phenotypic testing and 16S rRNA gene sequencing identified S. canis associated with ulcer infections in dog owners. Because S. canis may be incorrectly identified (published biochemical descriptions are inconsistent), there may be an underestimation of the true number of infections. Identification of group G streptococci to the species level could have epidemiological and clinical implications. [Abstract/Link to Full Text]

Espinel-Ingroff A, Fothergill A, Ghannoum M, Manavathu E, Ostrosky-Zeichner L, Pfaller MA, Rinaldi MG, Schell W, Walsh TJ
Quality control and reference guidelines for CLSI broth microdilution method (M38-A document) for susceptibility testing of anidulafungin against molds.
J Clin Microbiol. 2007 Jul;45(7):2180-2.
The CLSI (formerly NCCLS) M38-A document for antifungal susceptibility testing of filamentous fungi does not describe guidelines for echinocandins. A multicenter study (eight centers) evaluated inter- and intralaboratory reproducibilities of two reading times (24 and 48 h or 48 and 72 h) and two end points (MICs and minimum effective concentrations [MECs]) for evaluating anidulafungin against molds. Anidulafungin MICs (>or=50% inhibition) and MECs (morphological hyphal changes) were determined for seven Aspergillus isolates (four species) and one isolate each of Fusarium moniliforme, Fusarium solani, and Paecilomyces variotii and for two Scedosporium apiospermum isolates. The inter- and intralaboratory reproducibilities of 10 replicate tests performed in each laboratory on 10 different days for each isolate was 100% at 24 h (MECs, <or=0.015 microg/ml) for six Aspergillus and P. variotii isolates. The reproducibility was 94 to 96.7% at 72 h (MECs, 1 to 8 microg/ml) for S. apiospermum and 96.7 to 97.5% at 48 h (MICs, >or=32 microg/ml) for both Fusarium isolates. Introduction of these identified optimum testing conditions for anidulafungin into future versions of the M38 document is warranted. [Abstract/Link to Full Text]

Affolabi D, Odoun M, Martin A, Palomino JC, Anagonou S, Portaels F
Evaluation of direct detection of Mycobacterium tuberculosis rifampin resistance by a nitrate reductase assay applied to sputum samples in Cotonou, Benin.
J Clin Microbiol. 2007 Jul;45(7):2123-5.
The aim of this study was to evaluate a nitrate reductase assay (NRA) performed on smear-positive sputa for the direct detection of rifampin resistance in Mycobacterium tuberculosis. A total of 213 smear-positive sputa with a positivity score of 1+ or more (>1 acid-fast bacillus per field by fluorescence microscopy) were used in the study. The samples were decontaminated using the modified Petroff method, and portions of the resulting suspension were used to perform the NRA. The NRA results were compared with the reference indirect proportion method for 177 specimens for which comparable results were available. NRA results were obtained at day 10 for 15 specimens (9%), results for 88 specimens (50%) were obtained at day 14, results for 66 specimens (37%) were obtained at day 18, and results for the remaining 8 specimens (4%) were obtained at day 28. Thus, 96% of NRA results were obtained in 18 days. Of the 177 specimens, there was only one discrepancy (susceptible according to the NRA and resistant according to the indirect proportion method). NRA is simple to perform and provides a rapid, accurate, and cost-effective means for the detection of rifampin resistance in M. tuberculosis isolates. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Immune Based Therapies and Vaccines

---

No recent articles are currently available.

---

Recent Articles in Journal of Virology

---

Ikegami T, Won S, Peters CJ, Makino S
Characterization of Rift Valley fever virus transcriptional terminations.
J Virol. 2007 Aug;81(16):8421-38.
Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome and causes a mosquito-borne disease among humans and livestock in sub-Saharan African and Arabian Peninsula countries. Phlebovirus L, M, and N mRNAs are synthesized from the virus-sense RNA segments, while NSs mRNA is transcribed from the anti-virus-sense S segment. The present study determined the 3' termini of all RVFV mRNAs. The 3' termini of N and NSs mRNAs were mapped within the S-segment intergenic region and were complementary to each other by 30 to 60 nucleotides. The termini of M and L mRNAs were mapped within 122 to 107 nucleotides and 16 to 41 nucleotides, respectively, from the 5' ends of their templates. Viral RNA elements that control phlebovirus transcriptional terminations are largely unknown. Our studies suggested the importance of a pentanucleotide sequence, CGUCG, for N, NSs, and M mRNA transcription terminations. Homopolymeric tracts of C sequences, which were located upstream of the pentanucleotide sequence, promoted N and M mRNA terminations. Likewise, the homopolymeric tracts of G sequences that are found upstream of the pentanucleotide sequence promoted NSs mRNA termination. The L-segment 5'-untranslated region (L-5' UTR) had neither the pentanucleotide sequence nor homopolymeric sequences, yet replacement of the S-segment intergenic region with the L-5' UTR exerted N mRNA termination in an infectious virus. The L-5' UTR contained two 13-nucleotide-long complete complementary sequences, and their sequence complementarities were important for L mRNA termination. A computer-mediated RNA secondary structure analysis further suggested that RNA secondary structures formed by the sections of the two 13-nucleotide-long sequences and by the sequence between them may have a role in L mRNA termination. Our data demonstrated that viral RNA elements that govern L mRNA termination differed from those that regulate mRNA terminations in the M and S segments. [Abstract/Link to Full Text]

Lavillette D, Pécheur EI, Donot P, Fresquet J, Molle J, Corbau R, Dreux M, Penin F, Cosset FL
Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus.
J Virol. 2007 Aug;81(16):8752-65.
Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH. [Abstract/Link to Full Text]

Rolland M, Jensen MA, Nickle DC, Yan J, Learn GH, Heath L, Weiner D, Mullins JI
Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins.
J Virol. 2007 Aug;81(16):8507-14.
The extensive diversity of human immunodeficiency virus type 1 (HIV-1) and its capacity to mutate and escape host immune responses are major challenges for AIDS vaccine development. Ancestral sequences, which minimize the genetic distance to circulating strains, provide an opportunity to design immunogens with the potential to elicit broad recognition of HIV epitopes. We developed a phylogenetics-informed algorithm to reconstruct ancestral HIV sequences, called Center of Tree (COT). COT sequences have potentially significant benefits over isolate-based strategies, as they minimize the evolutionary distances to circulating strains. COT sequences are designed to surmount the potential pitfalls stemming from sampling bias with the consensus method and outlier bias with the most-recent-common-ancestor approach. We computationally derived COT sequences from circulating HIV-1 subtype B sequences for the genes encoding the major viral structural protein (Gag) and two regulatory proteins, Tat and Nef. COT genes were synthesized de novo and expressed in mammalian cells, and the proteins were characterized. COT Gag was shown to generate virus-like particles, while COT Tat transactivated gene expression from the HIV-1 long terminal repeat and COT Nef mediated downregulation of cell surface major histocompatibility complex class I. Thus, retrodicted ancestral COT proteins can retain the biological functions of extant HIV-1 proteins. Additionally, COT proteins were immunogenic, as they elicited antigen-specific cytotoxic T-lymphocyte responses in mice. These data support the utility of the COT approach to create novel and biologically active ancestral proteins as a starting point for studies of the structure, function, and biological fitness of highly variable genes, as well as for the rational design of globally relevant vaccine candidates. [Abstract/Link to Full Text]

Cameron MJ, Ran L, Xu L, Danesh A, Bermejo-Martin JF, Cameron CM, Muller MP, Gold WL, Richardson SE, Poutanen SM, Willey BM, DeVries ME, Fang Y, Seneviratne C, Bosinger SE, Persad D, Wilkinson P, Greller LD, Somogyi R, Humar A, Keshavjee S, Louie M, Loeb MB, Brunton J, McGeer AJ, Kelvin DJ
Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome.
J Virol. 2007 Aug;81(16):8692-706.
It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-alpha, IFN-gamma, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination. [Abstract/Link to Full Text]

Ramanathan HN, Chung DH, Plane SJ, Sztul E, Chu YK, Guttieri MC, McDowell M, Ali G, Jonsson CB
Dynein-dependent transport of the hantaan virus nucleocapsid protein to the endoplasmic reticulum-Golgi intermediate compartment.
J Virol. 2007 Aug;81(16):8634-47.
In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly, deriving the virion envelope from the endoplasmic reticulum (ER) or Golgi compartment. While it is generally accepted that Old World hantaviruses assemble and bud into the Golgi compartment, some studies with New World hantaviruses have raised the possibility of maturation at the plasma membrane as well. Overall, the steps leading to virion assembly remain largely undetermined for hantaviruses. Because hantaviruses do not have matrix proteins, the nucleocapsid protein (N) has been proposed to play a key role in assembly. Herein, we examine the intracellular trafficking and morphogenesis of the prototype Old World hantavirus, Hantaan virus (HTNV). Using confocal microscopy, we show that N colocalized with the ER-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells, not with the ER, Golgi compartment, or early endosomes. Brefeldin A, which effectively disperses the ER, the ERGIC, and Golgi membranes, redistributed N with the ERGIC, implicating membrane association; however, subcellular fractionation experiments showed the majority of N in particulate fractions. Confocal microscopy revealed that N was juxtaposed to and distributed along microtubules and, over time, became surrounded by vimentin cages. To probe cytoskeletal association further, we probed trafficking of N in cells treated with nocodazole and cytochalasin D, which depolymerize microtubules and actin, respectively. We show that nocodazole, but not cytochalasin D, affected the distribution of N and reduced levels of intracellular viral RNA. These results suggested the involvement of microtubules in trafficking of N, whose movement could occur via molecular motors such as dynein. Overexpression of dynamitin, which is associated with dynein-mediated transport, creates a dominant-negative phenotype blocking transport on microtubules. Overexpression of dynamitin reduced N accumulation in the perinuclear region, which further supports microtubule components in N trafficking. The combined results of these experiments support targeting of N to the ERGIC prior to its movement to the Golgi compartment and the requirement of an intact ERGIC for viral replication and, thus, the possibility of virus factories in this region. [Abstract/Link to Full Text]

Guerra S, Nájera JL, González JM, López-Fernández LA, Climent N, Gatell JM, Gallart T, Esteban M
Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.
J Virol. 2007 Aug;81(16):8707-21.
Although recombinants based on the attenuated poxvirus vectors MVA and NYVAC are currently in clinical trials, the nature of the genes triggered by these vectors in antigen-presenting cells is poorly characterized. Using microarray technology and various analysis conditions, we compared specific changes in gene expression profiling following MVA and NYVAC infection of immature human monocyte-derived dendritic cells (MDDC). Microarray analysis was performed at 6 h postinfection, since these viruses induced extensive cytopathic effects, rRNA breakdown, and apoptosis at late times postinfection. MVA- and NYVAC-infected MDDC shared upregulation of 195 genes compared to uninfected cells: MVA specifically upregulated 359 genes, and NYVAC upregulated 165 genes. Microarray comparison of NYVAC and MVA infection revealed 544 genes with distinct expression patterns after poxvirus infection and 283 genes specifically upregulated after MVA infection. Both vectors upregulated genes for cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex genes. mRNA levels for interleukin 12beta (IL-12beta), beta interferon, and tumor necrosis factor alpha were higher after MVA infection than after NYVAC infection. The expression profiles of transcription factors such as NF-kappaB/Rel and STAT were regulated similarly by both viruses; in contrast, OASL, MDA5, and IRIG-I expression increased only during MVA infection. Type I interferon, IL-6, and Toll-like receptor pathways were specifically induced after MVA infection. Following MVA or NYVAC infection in MDDC, we found similarities as well as differences between these virus strains in the expression of cellular genes with immunological function, which should have an impact when these vectors are used as recombinant vaccines. [Abstract/Link to Full Text]

Beerens N, Selisko B, Ricagno S, Imbert I, van der Zanden L, Snijder EJ, Canard B
De novo initiation of RNA synthesis by the arterivirus RNA-dependent RNA polymerase.
J Virol. 2007 Aug;81(16):8384-95.
All plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3' end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3'-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis. [Abstract/Link to Full Text]

Choudhary SK, Vrisekoop N, Jansen CA, Otto SA, Schuitemaker H, Miedema F, Camerini D
Low immune activation despite high levels of pathogenic human immunodeficiency virus type 1 results in long-term asymptomatic disease.
J Virol. 2007 Aug;81(16):8838-42.
Long-term asymptomatic human immunodeficiency virus (HIV)-infected individuals (LTA) usually have low viral load and low immune activation. To discern whether viral load or immune activation is dominant in determining progression to AIDS, we studied three exceptional LTA with high viral loads. HIV type 1 isolates from these LTA were as pathogenic as viruses from progressors in organ culture. Despite high viral loads, these LTA had low levels of proliferating and activated T cells compared to progressors, like other LTA. In contrast to those in progressors, HIV-specific CD4(+) T-cell responses in these LTA were maintained. Thus, low immune activation despite a high viral load preserved HIV-specific T-cell responses and resulted in a long-term asymptomatic phenotype. [Abstract/Link to Full Text]

Loffredo JT, Maxwell J, Qi Y, Glidden CE, Borchardt GJ, Soma T, Bean AT, Beal DR, Wilson NA, Rehrauer WM, Lifson JD, Carrington M, Watkins DI
Mamu-B*08-positive macaques control simian immunodeficiency virus replication.
J Virol. 2007 Aug;81(16):8827-32.
Certain major histocompatibility complex (MHC) class I alleles are associated with the control of human immunodeficiency virus and simian immunodeficiency virus (SIV) replication. We have designed sequence-specific primers for detection of the rhesus macaque MHC class I allele Mamu-B*08 by PCR and screened a cohort of SIV-infected macaques for this allele. Analysis of 196 SIV(mac)239-infected Indian rhesus macaques revealed that Mamu-B*08 was significantly overrepresented in elite controllers; 38% of elite controllers were Mamu-B*08 positive compared to 3% of progressors (P = 0.00001). Mamu-B*08 was also associated with a 7.34-fold decrease in chronic phase viremia (P = 0.002). Mamu-B*08-positive macaques may, therefore, provide a good model to understand the correlates of MHC class I allele-associated immune protection and viral containment in human elite controllers. [Abstract/Link to Full Text]

Yang X, Lipchina I, Lifton M, Wang L, Sodroski J
Antibody binding in proximity to the receptor/glycoprotein complex leads to a basal level of virus neutralization.
J Virol. 2007 Aug;81(16):8809-13.
Hypothetically, antibodies may neutralize enveloped viruses by diverse mechanisms, such as disruption of receptor binding, interference with conformational changes required for virus entry, steric hindrance, or virus aggregation. Here, we demonstrate that retroviral infection mediated by the avian sarcoma-leukosis virus (ASLV-A) envelope glycoproteins can be neutralized by an antibody directed against a functionally unimportant component of a chimeric receptor protein. Thus, the binding of an antibody in proximity to the retroviral envelope glycoprotein-receptor complex, without binding to the entry machinery itself, results in neutralization. This finding provides additional support for the hypothesis that steric hindrance is sufficient for antibody-mediated neutralization of retroviruses. [Abstract/Link to Full Text]

Kueng HJ, Leb VM, Haiderer D, Raposo G, Thery C, Derdak SV, Schmetterer KG, Neunkirchner A, Sillaber C, Seed B, Pickl WF
General strategy for decoration of enveloped viruses with functionally active lipid-modified cytokines.
J Virol. 2007 Aug;81(16):8666-76.
Viral particles preferentially incorporate extra- and intracellular constituents of host cell lipid rafts, a phenomenon central to pseudotyping. Based on this mechanism, we have developed a system for the predictable decoration of enveloped viruses with functionally active cytokines that circumvents the need to modify viral proteins themselves. Human interleukin-2 (hIL-2), hIL-4, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), and murine IL-2 (mIL-2) were used as model cytokines and fused at their C terminus to the glycosylphosphatidylinositol (GPI) acceptor sequence of human Fcgamma receptor III (CD16b). We show here that genetically modified cytokines are all well expressed on 293 producer cells. However, only molecules equipped with GPI anchors but not those linked to transmembrane/intracellular regions of type I membrane proteins are efficiently targeted to lipid rafts and consequently to virus-like particles (VLP) induced by Moloney murine leukemia virus Gag-Pol. hIL-4::GPI and hGM-CSF::GPI coexpressed on VLP were found to differentiate monocytes towards dendritic cells. Apart from myeloid-committed cell types, VLP-bound cytokines also act efficiently on lymphocytes. hIL-2::GPI strongly costimulated T-cell receptor (TCR)/CD3 dependent T-cell activation in vitro and mIL-2::GPI-coactivated antigen-specific T cells in vivo. On a molar basis, the functional activity of VLP-bound hIL-2::GPI was found to be comparable to that of soluble hIL-2. VLP decorated with hIL-2::GPI and coexpressing a TCR/CD3 ligand have an IL-2-specific activity of 5 x 10(4) units/mg protein. Virus particles decorated with lipid-modified cytokines might help to improve viral strains for vaccination purposes, the propagation of factor-dependent cell types, as well as gene transfer by viral systems in the future. [Abstract/Link to Full Text]

Jones CT, Murray CL, Eastman DK, Tassello J, Rice CM
Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus.
J Virol. 2007 Aug;81(16):8374-83.
Hepatitis C virus (HCV) infection is a global health concern affecting an estimated 3% of the world's population. Recently, cell culture systems have been established, allowing recapitulation of the complete virus life cycle for the first time. Since the HCV proteins p7 and NS2 are not predicted to be major components of the virion, nor are they required for RNA replication, we investigated whether they might have other roles in the viral life cycle. Here we utilize the recently described infectious J6/JFH chimera to establish that the p7 and NS2 proteins are essential for HCV infectivity. Furthermore, unprocessed forms of p7 and NS2 were not required for this activity. Mutation of two conserved basic residues, previously shown to be important for the ion channel activity of p7 in vitro, drastically impaired infectious virus production. The protease domain of NS2 was required for infectivity, whereas its catalytic active site was dispensable. We conclude that p7 and NS2 function at an early stage of virion morphogenesis, prior to the assembly of infectious virus. [Abstract/Link to Full Text]

Patkar CG, Jones CT, Chang YH, Warrier R, Kuhn RJ
Functional requirements of the yellow fever virus capsid protein.
J Virol. 2007 Jun;81(12):6471-81.
Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning. [Abstract/Link to Full Text]

Takeuchi H, Buckler-White A, Goila-Gaur R, Miyagi E, Khan MA, Opi S, Kao S, Sokolskaja E, Pertel T, Luban J, Strebel K
Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells.
J Virol. 2007 Aug;81(15):8080-90.
Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1. [Abstract/Link to Full Text]

Inoue Y, Tanaka N, Tanaka Y, Inoue S, Morita K, Zhuang M, Hattori T, Sugamura K
Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted.
J Virol. 2007 Aug;81(16):8722-9.
The penetration of various viruses into host cells is accomplished by hijacking the host endocytosis machinery. In the case of severe acute respiratory syndrome coronavirus (SARS-CoV) infection, viral entry is reported to require a low pH in intracytoplasmic vesicles; however, little is known about how SARS-CoV invades such compartments. Here we demonstrate that SARS-CoV mainly utilizes the clathrin-mediated endocytosis pathway for its entry to target cells by using infectious SARS-CoV, as well as a SARS-CoV pseudovirus packaged in the SARS-CoV envelope. The SARS-CoV entered caveolin-1-negative HepG2 cells, and the entry was significantly inhibited by treatment with chlorpromazine, an inhibitor for clathrin-dependent endocytosis, and by small interfering RNA-mediated gene silencing for the clathrin heavy chain. Furthermore, the SARS-CoV entered COS7 cells transfected with the mutant of ACE2 with the cytoplasmic tail deleted, SARS-CoV receptor, as well as the wild-type ACE2, and their entries were significantly inhibited by treatment with chlorpromazine. In addition, ACE2 translocated into EEA1-positive early endosomes immediately after the virus attachment to ACE2. These results suggest that when SARS-CoV binds ACE2 it is internalized and penetrates early endosomes in a clathrin-dependent manner and that the cytoplasmic tail of ACE2 is not required for the penetration of SARS-CoV. [Abstract/Link to Full Text]

Núńez JI, Molina N, Baranowski E, Domingo E, Clark S, Burman A, Berryman S, Jackson T, Sobrino F
Guinea pig-adapted foot-and-mouth disease virus with altered receptor recognition can productively infect a natural host.
J Virol. 2007 Aug;81(16):8497-506.
We report that adaptation to infect the guinea pig did not modify the capacity of foot-and-mouth disease virus (FMDV) to kill suckling mice and to cause an acute and transmissible disease in the pig, an important natural host for this pathogen. Adaptive amino acid replacements (I(248)-->T in 2C, Q(44)-->R in 3A, and L(147)-->P in VP1), selected upon serial passages of a type C FMDV isolated from swine (biological clone C-S8c1) in the guinea pig, were maintained after virus multiplication in swine and suckling mice. However, the adaptive replacement L(147)-->P, next to the integrin-binding RGD motif at the GH loop in VP1, abolished growth of the virus in different established cell lines and modified its antigenicity. In contrast, primary bovine thyroid cell cultures could be productively infected by viruses with replacement L(147)-->P, and this infection was inhibited by antibodies to alphavbeta6 and by an FMDV-derived RGD-containing peptide, suggesting that integrin alphavbeta6 may be used as a receptor for these mutants in the animal (porcine, guinea pig, and suckling mice) host. Substitution T(248)-->N in 2C was not detectable in C-S8c1 but was present in a low proportion of the guinea pig-adapted virus. This substitution became rapidly dominant in the viral population after the reintroduction of the guinea pig-adapted virus into pigs. These observations illustrate how the appearance of minority variant viruses in an unnatural host can result in the dominance of these viruses on reinfection of the original host species. [Abstract/Link to Full Text]

Murayama A, Date T, Morikawa K, Akazawa D, Miyamoto M, Kaga M, Ishii K, Suzuki T, Kato T, Mizokami M, Wakita T
The NS3 helicase and NS5B-to-3'X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells.
J Virol. 2007 Aug;81(15):8030-40.
The JFH-1 strain of hepatitis C virus (HCV) is a genotype 2a strain that can replicate autonomously in Huh7 cells. The J6 strain is also a genotype 2a strain, but its full genomic RNA does not replicate in Huh7 cells. However, chimeric J6/JFH-1 RNA that has J6 structural-protein-coding regions and JFH-1 nonstructural-protein-coding regions can replicate autonomously and produce infectious HCV particles. In order to determine the mechanisms underlying JFH-1 RNA replication, we constructed various J6/JFH-1 chimeras and tested their RNA replication and virus particle production abilities in Huh7 cells. Via subgenomic-RNA-replication assays, we found that both the JFH-1 NS5B-to-3'X (N5BX) and the NS3 helicase (N3H) regions are important for the replication of the J6CF replicon. We applied these results to full-length genomic RNA replication and analyzed replication using Northern blotting. We found that a chimeric J6 clone with JFH-1 N3H and N5BX could replicate autonomously but that a chimeric J6 clone with only JFH-1 N5BX had no replication ability. Finally, we tested the virus production abilities of these clones and found that a chimeric J6 clone with JFH-1 N3H and N5BX could produce infectious HCV particles. In conclusion, the JFH-1 NS3 helicase and NS5B-to-3'X regions are important for efficient replication and virus particle formation of HCV genotype 2a strains. [Abstract/Link to Full Text]

Quakkelaar ED, van Alphen FP, Boeser-Nunnink BD, van Nuenen AC, Pantophlet R, Schuitemaker H
Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies.
J Virol. 2007 Aug;81(16):8533-42.
The ability of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) specific human monoclonal antibodies (MAbs) b12, 2G12, 2F5, and 4E10 to neutralize recently transmitted viruses has not yet been explored in detail. We investigated the neutralization sensitivity of subtype B HIV-1 variants obtained from four primary HIV infection cases and six transmission couples (four homosexual and two parenteral) to these MAbs. Sexually transmitted HIV-1 variants isolated within the first 2 months after seroconversion were generally sensitive to 2F5, moderately resistant to 4E10 and b12, and initially resistant but later more sensitive to 2G12 neutralization. In the four homosexual transmission couples, MAb neutralization sensitivity of HIV in recipients did not correlate with the MAb neutralization sensitivity of HIV from their source partners, whereas the neutralization sensitivity of donor and recipient viruses involved in parenteral transmission was more similar. For a fraction (11%) of the HIV-1 variants analyzed here, neutralization by 2G12 could not be predicted by the presence of N-linked glycosylation sites previously described to be involved in 2G12 binding. Resistance to 2F5 and 4E10 neutralization did also not correlate with mutations in the respective core epitopes. Overall, we observed that the neutralization resistance of recently transmitted subtype B HIV-1 variants was relatively high. Although 8 of 10 patients had viruses that were sensitive to neutralization by at least one of the four broadly neutralizing antibodies studied, 4 of 10 patients harbored at least one virus variant that seemed resistant to all four antibodies. Our results suggest that vaccine antigens that only elicit antibodies equivalent to b12, 2G12, 2F5, and 4E10 may not be sufficient to protect against all contemporary HIV-1 variants and that additional cross-neutralizing specificities need to be sought. [Abstract/Link to Full Text]

Zhang P, Samuel CE
Protein kinase PKR plays a stimulus- and virus-dependent role in apoptotic death and virus multiplication in human cells.
J Virol. 2007 Aug;81(15):8192-200.
The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNA-induced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-alpha)-induced apoptosis was comparable between the HeLa lines. NF-kappaB was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-alpha. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus, was unaffected by the PKR knockdown. Surprisingly, the yield of mutant adenovirus that fails to encode VAI RNA was not enhanced in PKR-deficient cells, indicating the importance of host factors in addition to PKR in conferring the VAI RNA phenotype. [Abstract/Link to Full Text]

Amonsen M, Smith DF, Cummings RD, Air GM
Human parainfluenza viruses hPIV1 and hPIV3 bind oligosaccharides with alpha2-3-linked sialic acids that are distinct from those bound by H5 avian influenza virus hemagglutinin.
J Virol. 2007 Aug;81(15):8341-5.
We investigated the binding of human parainfluenza virus types 1 and 3 (hPIV1 and hPIV3, respectively) to the glycan array of the Consortium for Functional Glycomics and binding and their release from erythrocytes under conditions where neuraminidase is inactive or active. hPIV1 and hPIV3 bind modifications of Neu5Acalpha2-3Galbeta1-4GlcNAc, including the sialyl-Lewis(x) motif and structures containing 6-sulfogalactose. hPIV1 and hPIV3 thus bind typical N-linked glycans, in contrast to avian influenza virus H5 hemagglutinin (J. Stevens, O. Blixt, T. M. Tumpey, J. K. Taubenberger, J. C. Paulson, and I. A. Wilson, Science 312:404-410, 2006), which binds less-common motifs. While the receptor is not the sole determinant of tropism, hPIV or H5 influenza virus infection of specific cells that express receptors may contribute to their different pathologies. [Abstract/Link to Full Text]

Besecker MI, Furness CL, Coen DM, Griffiths A
Expression of extremely low levels of thymidine kinase from an acyclovir-resistant herpes simplex virus mutant supports reactivation from latently infected mouse trigeminal ganglia.
J Virol. 2007 Aug;81(15):8356-60.
A single-cytosine-deletion in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. A laboratory strain engineered to carry this mutation did not generate sufficient TK activity for detection by plaque autoradiography, which detected 0.25% wild-type activity. However, a drug sensitivity assay suggested that extremely low levels of TK are generated by this virus. The virus was estimated to express 0.09% of wild-type TK activity via a ribosomal frameshift 24 nucleotides upstream of the mutation. Remarkably, this appeared to be sufficient active TK to support a low level of reactivation from latently infected mouse trigeminal ganglia. [Abstract/Link to Full Text]

Lobritz MA, Marozsan AJ, Troyer RM, Arts EJ
Natural variation in the V3 crown of human immunodeficiency virus type 1 affects replicative fitness and entry inhibitor sensitivity.
J Virol. 2007 Aug;81(15):8258-69.
Natural polymorphisms in the heterogeneous human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein may have an impact on both sensitivity to entry inhibitors and viral replicative fitness. Of significant interest is variation in the V3 crown due to its involvement in direct engagement with the coreceptor. Two positions in the crown (318 and 319) appear to be important in determining intrinsic susceptibility to multiple entry inhibitors. Thus, we evaluated a series of natural polymorphisms at positions 318 and 319 in three distinct CCR5-tropic envelope genetic backgrounds to address their role in replicative fitness and sensitivity to entry inhibitors. Change at position 319 to each of the three major consensus amino acids (A, T, and R) resulted in variation in sensitivity to entry inhibitors and altered replicative fitness, but the effects of any one amino acid depended on the envelope context. Change of the nearly invariant tyrosine at position 318 to a rare arginine resulted in increased sensitivity to entry inhibitors and decreased replicative fitness independent of envelope context. Polymorphisms in the V3 crown that showed increased susceptibility to entry inhibitors also exhibited decreased entry efficiency, replicative fitness in primary peripheral blood mononuclear cells, and ability to replicate in primary macrophages. These findings suggest that differences in coreceptor affinity and/or avidity may underlie these phenotypic characteristics. [Abstract/Link to Full Text]

Hong PW, Nguyen S, Young S, Su SV, Lee B
Identification of the optimal DC-SIGN binding site on human immunodeficiency virus type 1 gp120.
J Virol. 2007 Aug;81(15):8325-36.
Human immunodeficiency virus type 1 (HIV-1) envelope (gp120) binding to DC-SIGN, a C-type lectin that can facilitate HIV infection in cis and in trans, is largely dependent on high-mannose-content moieties. Here, we delineate the N-linked glycosylation (N-glycan) sites in gp120 that contribute to optimal DC-SIGN binding. Soluble DC-SIGN was able to block 2G12 binding to gp120, but not vice versa, suggesting that DC-SIGN binds to a more flexible combination of N-glycans than 2G12. Consistent with this observation, HIV strain JRCSF gp120 prebound to 2G12 was 10-fold more sensitive to mannan competition than gp120 that was not prebound in a DC-SIGN cell surface binding assay. The analysis of multiple mutant forms of the 2G12 epitope revealed one triple glycosylation mutant form, termed 134mut (carrying N293Q, N382Q, and N388Q mutations), that exhibited a significant increase in sensitivity to both mannan competition and endoglycosidase H digestion compared to that of the 124mut form (carrying N293Q, N328Q, and N388Q mutations) and wild-type gp120 in a DC-SIGN binding assay. Importantly, no such differences were observed when binding to Galanthus nivalis was assessed. The 134mut form of gp120 also exhibited decreased binding to DC-SIGN in the context of native envelope spikes on a virion, and virus bearing 134mut exhibited less efficient DC-SIGN-mediated infection in trans. Significantly, 124mut and 134mut differed by only one glycosylation site mutation in each construct, and both 124mut and 134mut viruses exhibited wild-type levels of infectivity when used in a direct infection assay. In summary, while DC-SIGN can bind to a flexible combination of N-glycans on gp120, its optimal binding site overlaps with specific N-glycans within the 2G12 epitope. Conformationally intact envelopes that are DC-SIGN binding deficient can be used to probe the in vivo biological functions of DC-SIGN. [Abstract/Link to Full Text]

Donlin MJ, Cannon NA, Yao E, Li J, Wahed A, Taylor MW, Belle SH, Di Bisceglie AM, Aurora R, Tavis JE
Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy.
J Virol. 2007 Aug;81(15):8211-24.
Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients. [Abstract/Link to Full Text]

Madisch I, Hofmayer S, Moritz C, Grintzalis A, Hainmueller J, Pring-Akerblom P, Heim A
Phylogenetic analysis and structural predictions of human adenovirus penton proteins as a basis for tissue-specific adenovirus vector design.
J Virol. 2007 Aug;81(15):8270-81.
The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV and closely related simian adenoviruses were studied. Adjacent sequences and predicted overall secondary structure were conserved. Phylogenetic analysis revealed clustering corresponding to the HAdV species and recombination events in the origin of HAdV prototypes. All HAdV except serotypes 40 and 41 of species F exhibited an integrin binding RGD motif in the second loop. The lengths of the loops (HVR1 and RGD loops) varied significantly between HAdV species with the longest RGD loop observed in species C and the longest HVR1 in species B. Long loops may permit the insertion of motifs that modify tissue tropism. Genetic analysis of HAdV prime strain p17'H30, a neutralization variant of HAdV-D17, indicated the significance of nonhexon neutralization epitopes for HAdV immune escape. Fourteen highly conserved motifs of the penton base were analyzed by site-directed mutagenesis of HAdV-D8 and tested for sustained induction of early cytopathic effects. Thus, three new motifs essential for penton base function were identified additionally to the RGD site, which interacts with a secondary cellular receptor responsible for internalization. Therefore, our penton primary structure data and secondary structure modeling in combination with the recently published fiber knob sequences may permit the rational design of tissue-specific adenoviral vectors. [Abstract/Link to Full Text]

Williams JV, Chen Z, Cseke G, Wright DW, Keefer CJ, Tollefson SJ, Hessell A, Podsiad A, Shepherd BE, Sanna PP, Burton DR, Crowe JE, Williamson RA
A recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo.
J Virol. 2007 Aug;81(15):8315-24.
Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 mug/ml and bound to hMPV F with an affinity of 9.8 x10(-10) M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection. [Abstract/Link to Full Text]

Twu KY, Kuo RL, Marklund J, Krug RM
The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells.
J Virol. 2007 Aug;81(15):8112-21.
The NS1A proteins of human influenza A viruses bind CPSF30, a cellular factor required for the processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including beta interferon mRNA. Here we show that the NS1A protein of the pathogenic H5N1 influenza A/Hong Kong/483/97 (HK97) virus isolated from humans has an intrinsic defect in CPSF30 binding. It does not bind CPSF30 in vitro and causes high beta interferon mRNA production and reduced virus replication in MDCK cells when expressed in a recombinant virus in which the other viral proteins are encoded by influenza A/Udorn/72. We traced this defect to the identities of amino acids 103 and 106 in the HK97 NS1A protein, which differ from the consensus amino acids, F and M, respectively, found in the NS1A proteins of almost all human influenza A virus strains. X-ray crystallography has shown that F103 and M106, which are not part of the CPSF30 binding pocket of the NS1A protein, stabilize the NS1A-CPSF30 complex. In contrast to the HK97 NS1A protein, the NS1A proteins of H5N1 viruses isolated from humans after 1998 contain F103 and M106 and hence bind CPSF30 in vitro and do not attenuate virus replication. The HK97 NS1A protein is less attenuating when expressed in a virus that also encodes the other internal HK97 proteins and under these conditions binds to CPSF30 to a substantial extent in vivo. Consequently, these internal HK97 proteins largely compensate for the absence of F103 and M106, presumably by stabilizing the NS1A-CPSF30 complex. [Abstract/Link to Full Text]

Helle F, Goffard A, Morel V, Duverlie G, McKeating J, Keck ZY, Foung S, Penin F, Dubuisson J, Voisset C
The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein.
J Virol. 2007 Aug;81(15):8101-11.
Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response. [Abstract/Link to Full Text]

Solé M, Perkins EM, Frisancho A, Huang E, Desai P
The N terminus of the herpes simplex virus type 1 triplex protein, VP19C, cannot be detected on the surface of the capsid shell by using an antibody (hemagglutinin) epitope tag.
J Virol. 2007 Aug;81(15):8367-70.
The herpes simplex virus (HSV) triplex is a complex of three protein subunits, VP19C and a dimer of VP23 that is essential for capsid assembly. We have derived HSV-1 recombinant viruses that contain monomeric red fluorescent protein (mRFP1), a Flu hemagglutinin (HA) epitope, and a six-histidine tag fused to the amino terminus of VP19C. These viruses were capable of growth on Vero cells, indicating that the amino terminus of VP19C could tolerate these fusions. By use of immunoelectron microscopy methods, capsids that express VP19C-mRFP but not VP19C-HA were labeled with gold particles when incubated with the corresponding antibody. Our conclusion from the data is that a large tag at the N terminus of VP19C was sufficiently exposed on the capsid surface for polyclonal antibody reactivity, while the small HA epitope was inaccessible to the antibody. These data indicate that an epitope tag at the amino terminus of VP19C is not exposed at the capsid surface for reactivity to its antibody. [Abstract/Link to Full Text]

Russell RA, Pathak VK
Identification of two distinct human immunodeficiency virus type 1 Vif determinants critical for interactions with human APOBEC3G and APOBEC3F.
J Virol. 2007 Aug;81(15):8201-10.
Human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) inhibit replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their proteasomal degradation. The Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development because inhibiting the interactions could allow the host defense mechanism to control HIV-1 replication. It was recently reported that the Vif amino acids D(14)RMR(17) are important for functional interaction and degradation of the previously identified Vif-resistant mutant of A3G (D128K-A3G). However, the Vif determinants important for functional interaction with A3G and A3F have not been fully characterized. To identify these determinants, we performed an extensive mutational analysis of HIV-1 Vif. Our analysis revealed two distinct Vif determinants, amino acids Y(40)RHHY(44) and D(14)RMR(17), which are essential for binding to A3G and A3F, respectively. Interestingly, mutation of the A3G-binding region increased Vif's ability to suppress A3F. Vif binding to D128K-A3G was also dependent on the Y(40)RHHY(44) region but not the D(14)RMR(17) region. Consistent with previous observations, subsequent neutralization of the D128K-A3G antiviral activity required substitution of Vif determinant D(14)RMR(17) with SEMQ, similar to the SERQ amino acids in simian immunodeficiency virus SIV(AGM) Vif, which is capable of neutralizing D128K-A3G. These studies are the first to clearly identify two distinct regions of Vif that are critical for independent interactions with A3G and A3F. Pharmacological interference with the Vif-A3G or Vif-A3F interactions could result in potent inhibition of HIV-1 replication by the APOBEC3 proteins. [Abstract/Link to Full Text]

Gaudreault E, Fiola S, Olivier M, Gosselin J
Epstein-Barr virus induces MCP-1 secretion by human monocytes via TLR2.
J Virol. 2007 Aug;81(15):8016-24.
Epstein-Barr virus (EBV) is a gammaherpesvirus infecting the majority of the human adult population in the world. TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune responses to different viruses including members of the herpesvirus family, such as human cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. In this report, we demonstrate that infectious and UV-inactivated EBV virions lead to the activation of NF-kappaB through TLR2 using HEK293 cells cotransfected with TLR2-expressing vector along with NF-kappaB-Luc reporter plasmid. NF-kappaB activation in HEK293-TLR2 cells (HEK293 cells transfected with TLR2) by EBV was not enhanced by the presence of CD14. The effect of EBV was abrogated by pretreating HEK293-TLR2 cells with blocking anti-TLR2 antibodies or by preincubating viral particles with neutralizing anti-EBV antibodies 72A1. In addition, EBV infection of primary human monocytes induced the release of MCP-1 (monocyte chemotactic protein 1), and the use of small interfering RNA targeting TLR2 significantly reduced such a chemokine response to EBV. Taken together, these results indicate that TLR2 may be an important pattern recognition receptor in the immune response directed against EBV infection. [Abstract/Link to Full Text]

Ackermann A, Staeheli P, Schneider U
Adaptation of Borna disease virus to new host species attributed to altered regulation of viral polymerase activity.
J Virol. 2007 Aug;81(15):7933-40.
Borna disease virus (BDV) can persistently infect the central nervous system of a broad range of mammalian species. Mice are resistant to infections with primary BDV isolates, but certain laboratory strains can be adapted to replicate in mice. We determined the molecular basis of adaptation by studying mutations acquired by a cDNA-derived BDV strain during one brain passage in rats and three passages in mice. The adapted virus propagated efficiently in mouse brains and induced neurological disease. Its genome contained seven point mutations, three of which caused amino acid changes in the L polymerase (L1116R and N1398D) and in the polymerase cofactor P (R66K). Recombinant BDV carrying these mutations either alone or in combination all showed enhanced multiplication speed in Vero cells, indicating improved intrinsic viral polymerase activity rather than adaptation to a mouse-specific factor. Mutations R66K and L1116R, but not N1398D, conferred replication competence of recombinant BDV in mice if introduced individually. Virus propagation in mouse brains was substantially enhanced if both L mutations were present simultaneously, but infection remained mostly nonsymptomatic. Only if all three amino acid substitutions were combined did BDV replicate vigorously and induce early disease in mice. Interestingly, the virulence-enhancing effect of the R66K mutation in P could be attributed to reduced negative regulation of polymerase activity by the viral X protein. Our data demonstrate that BDV replication competence in mice is mediated by the polymerase complex rather than the viral envelope and suggest that altered regulation of viral gene expression can favor adaptation to new host species. [Abstract/Link to Full Text]

---

Recent Articles in Medical Immunology

---

No recent articles are currently available.

---

Recent Articles in Microbiology and Immunology

---

Sunden Y, Semba S, Suzuki T, Okada Y, Orba Y, Nagashima K, Umemura T, Sawa H
DDX1 promotes proliferation of the JC virus through transactivation of its promoter.
Microbiol Immunol. 2007;51(3):339-47.
Recently, we demonstrated that the DEAD box protein 1 (DDX1), an RNA helicase, and the cleavage stimulation factor (CstF) form a complex that binds to the JC virus transcriptional control region (JCV-TCR). Here, we examined the function of DDX1, which is expressed at much higher levels in the JCV-susceptible cell line IMR-32 than in non-susceptible cell lines. DDX1 had no effect on the replication efficiency of JCV, but overexpression of DDX1 significantly increased transactivation of the JCV promoter. Furthermore, DDX1 enhanced the expression of JCV proteins in JCV infected cells, and knockdown of DDX1 using small interfering (si) RNA suppressed the expression of JCV proteins. Our results clearly demonstrate that DDX1 regulates proliferation of JCV in vitro through transcriptional activation. [Abstract/Link to Full Text]

Sunden Y, Semba S, Suzuki T, Okada Y, Orba Y, Nagashima K, Umemura T, Sawa H
Identification of DDX1 as a JC virus transcriptional control region-binding protein.
Microbiol Immunol. 2007;51(3):327-37.
To investigate the mechanism behind JC virus (JCV) cell specificity we performed electrophoretic mobility shift assays (EMSA) using probes derived from the JCV transcriptional control region (JCV-TCR). Using nuclear extracts from the JCV-susceptible neuroblastoma cell line IMR-32, EMSA revealed a 670 kDa JCV-TCR-binding protein complex designated as #3-bp. This complex could not be detected in nuclear extracts from non-susceptible cell lines. Using column chromatographic purifi-cation and microsequencing, we identified cleavage stimulation factor (CstF) as a component of #3-bp. However, as CstF is present in many cell types, we speculated that the IMR-32-specific component(s) of #3-bp bind CstF. We performed a yeast two-hybrid assay using CstF-77 as the bait against a HeLa cDNA-subtracted IMR-32 cDNA library. This analysis detected binding between CstF-77 and the RNA helicase DDX1. Subsequently, biotinylated DNA affinity precipitation and chromatin immunoprecipitation assays also confirmed that DDX1 binds specifically to JCV-TCR. Our findings indicate that an association between DDX1 and the JCV-TCR may play a significant role in JCV infection in IMR-32 cells. [Abstract/Link to Full Text]

Maruyama T, Shiba T, Iizuka H, Matsuda T, Kurohane K, Imai Y
Effects of phthalate esters on dendritic cell subsets and interleukin-4 production in fluorescein isothiocyanate-induced contact hypersensitivity.
Microbiol Immunol. 2007;51(3):321-6.
Phthalate esters with short alkyl chains, such as di-ethyl (DEP), di-n-propyl (DPP), and di-butyl phthalate (DBP), have adjuvant effects on an FITC-induced contact hypersensitivity mouse model. The adjuvant effects of DPP and DBP are associated with enhanced trafficking of FITC-presenting CD11b(+) dendritic cells (DC). DEP has relatively weak activity as to FITC-positive cell migration. Here we demonstrated that DBP and DPP also increased the number of FITC-positive CD8alpha(+) DC in draining lymph nodes. We also found enhanced production of interleukin-4 in draining lymph nodes after FITC sensitization with DEP, DPP, or DBP, suggesting an additional adjuvant mechanism of phthalate esters. [Abstract/Link to Full Text]

Pagamjav O, Yamada S, Ibrahim el-SM, Crandell RA, Matsumura T, Yamaguchi T, Fukushi H
Molecular characterization of equine herpesvirus 1 (EHV-1) isolated from cattle indicating no specific mutations associated with the interspecies transmission.
Microbiol Immunol. 2007;51(3):313-9.
Interspecies trasmission of equine herpesvirus 1 (EHV-1) from horse to cattle was shown by Crandell et al. (1988). Specific mutations related to the transmission were studied by comparison of five EHV-1 isolates in cattle (BH1247, 3M20-3, G118, G1753, and 9BSV4) using polymerase chain reaction and restriction fragment length polymorphism analysis with added sequencing. G118 and 3M20-3 were the genome type EHV-1 P, while G1753 was the genome type EHV-1 B. BH1247 and 9BSV4 might be other genome types. We could not identify specific mutations related to the interspecies transmission. [Abstract/Link to Full Text]

Choi YJ, Lee EM, Park JM, Lee KM, Han SH, Kim JK, Lee SH, Song HJ, Choi MS, Kim IS, Park KH, Jang WJ
Molecular detection of various rickettsiae in mites (acari: trombiculidae) in southern Jeolla Province, Korea.
Microbiol Immunol. 2007;51(3):307-12.
This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission. [Abstract/Link to Full Text]

Nomi H, Tashiro-Yamaji J, Miura-Takeda S, Shimizu T, Azuma H, Ueda H, Katsuoka Y, Kubota T, Yoshida R
Infiltration of H-2d-specific cytotoxic macrophage with unique morphology into rejection site of allografted meth A (H-2d) tumor cells in C57BL/6 (H-2b) mice.
Microbiol Immunol. 2007;51(3):297-306.
It is assumed that CD8(+) cytotoxic T lymphocytes (CTLs) mediate direct lysis of allografts and that their growth, differentiation, and activation are dependent upon cytokine production by CD4(+) helper T lymphocytes. In the present study, the effector cells responsible for the rejection of i.p. allografted, CTL-resistant Meth A tumor cells from C57BL/6 mice were characterized. The cytotoxic activity was associated exclusively with peritoneal exudate cells and not with the cells in lymphoid organs or blood. On day 8, when the cytotoxic activity reached a peak, 3 types of cells (i.e., lymphocytes, granulocytes, and macrophages) infiltrated into the rejection site; and allograft-induced macrophages (AIM) were cytotoxic against the allograft. Bacterially-elicited macrophages also exhibited cytotoxic activity (approximately 1/2 of that of AIM) against Meth A cells, whereas the cytotoxic activity of AIM against these cells but not that of bacterially-elicited macrophages was completely inhibited by the addition of donor (H-2(d))-type lymphoblasts, suggesting H-2(d)-specific cytotoxicity of AIM against Meth A cells. In contrast, resident macrophages were inactive toward Meth A cells. Morphologically, the three-dimensional appearance of AIM showed them to be unique large elongated cells having radiating peripheral filopodia and long cord-like extensions arising from their cytoplasmic surfaces. The ultrastructural examination of AIM revealed free ribosomes in their cytoplasm, which was often deformed by numerous large digestive vacuoles. These results indicate that AIM are the H-2(d)-specific effector cells for allografted Meth A cells and are a more fully activated macrophage with unique morphological features. [Abstract/Link to Full Text]

Tavares RC, Salgado J, Moreira VB, Ferreira MA, Mello FC, Leung JW, Fonseca Lde S, Spallek R, Singh M, Saad MH
Interferon gamma response to combinations 38 kDa/CFP-10, 38 kDa/MPT-64, ESAT-6/MPT-64 and ESAT-6/CFP-10, each related to a single recombinant protein of Mycobacterium tuberculosis in individuals from tuberculosis endemic areas.
Microbiol Immunol. 2007;51(3):289-96.
Several antigens of Mycobacterium tuberculosis have been identified and specificity to one or multiple antigens could determine the distinction between protective and pathogenic host reaction. Therefore T cell immune response to combinations 38 kDa/CFP-10, 38 kDa/MPT-64, ESAT-6/MPT-64 and ESAT-6/CFP-10 (each related to a single protein of Mycobacterium tuberculosis) in individuals from tuberculosis endemic areas have been examined. ELISA was used to detect IFN-gamma production in PBMC priming with single proteins and combinations in a panel of 105 individuals: 38 tuberculosis patients (6 untreated and 32 treated) and 67 healthy controls with tuberculin skin test positive or negative (TST). Brazilian TB patients highly recognized ESAT-6 (66%), but combinations improved response in the following order: ESAT-6/MPT-64 (89%) > ESAT-6/CFP-10 (73%) > 38 kDa/CFP-10 (70%), the last combination showing the highest specificity (TST(/) = 42% and TST(-) = 83%). Average IFN-gamma production in TB patients was signifi-cantly higher for 38 kDa/CFP-10 (P = 0.012) and 38 kDa/MPT-64 (P <0.035), when compared to single antigens. None of the combinations was able to discriminate TB patients from TST(+) controls; however, 38 kDa/CFP-10 displayed a borderline significance (P = 0.053). Similar to the ESAT-6/CFP-10 combination, IFN-gamma response to 38 kDa/CFP-10 showed an increased tendency in treated patients, although not signifi-cant (P = 0.16). We demonstrated for the first time that 38 kDa/CFP-10 had prediction sensitivity for TB patients similar to the ESAT-6/CFP-10 combination and also significant response improvement related to the single proteins with more selective reactivity among TST-positive individuals, which could be of potential interest for diagnostic evaluation for tuberculosis infection. [Abstract/Link to Full Text]

Akamine M, Higa F, Haranaga S, Tateyama M, Mori N, Heuner K, Fujita J
Interferon-gamma reverses the evasion of Birc1e/Naip5 gene mediated murine macrophage immunity by Legionella pneumophila mutant lacking flagellin.
Microbiol Immunol. 2007;51(3):279-87.
Legionella pneumophila is the etiologic agent of Legionnaires' disease. This bacterium contains a single monopolar flagellum, of which the FlaA subunit is a major protein constituent. The murine macrophage resistance against this bacterium is controlled by the Birc1e/Naip5 gene, which belongs to the NOD family. We evaluated the intracellular growth of the flaA mutant bacteria as well as another aflagellated fliA mutant, within bone marrow-derived macrophages from mice with an intact (C57BL/6, BALB/c) or mutated (A/J) Birc1e/Naip5 gene. The flaA mutant L. pneumophila multiplied within C57BL/6 and BALB/c macrophages while the wild-type strain did not. Cell viability was not impaired until 3 days after infection when the flaA mutant bacteria replicated 10(2-3)-fold in macrophages, implying that L. pneumophila inhibited host cell death during the early phase of intracellular replication. The addition of recombinant interferon-gamma (IFN-gamma) to the infected macrophages restricted replication of the flaA mutant within macrophages; these treated cells also showed enhanced nitric oxide production, although inhibition of nitric oxide production did not affect the IFN-gamma induced inhibition of Legionella replication. These findings suggested that IFN-gamma activated macrophages to restrict the intracellular growth of the L. pneumophila flaA mutant by a NO independent pathway. [Abstract/Link to Full Text]

Ohara M, Kouda S, Onodera M, Fujiue Y, Sasaki M, Kohara T, Kashiyama S, Hayashida S, Kadono M, Komatsuzawa H, Gotoh N, Usui T, Itaha H, Kuwabara M, Yokoyama T, Sugai M
Molecular characterization of imipenem-resistant Pseudomonas aeruginosa in Hiroshima, Japan.
Microbiol Immunol. 2007;51(3):271-7.
Pseudomonas aeruginosa showing resistance to imipenem were found in 100 of 1,058 strains (9.5%) from six hospitals (a-f) in Hiroshima City, Japan. Of the 100 strains, 14 (14%) were double disk synergy test positive using sodium mercaptoacetic acid disks, and 18 (18%) were bla(IMP-1) or bla(VIM-2) allele positive by polymerase chain reaction (PCR). Among 100 imipenem-resistant strains, 32 were categorized into multi-drug resistant strains, in which 13 were positive for the metallo-beta-lactamase gene. Fifty-one strains (51%) among the 100 imipenem-resistant strains had elevated RND efflux pump activity against levofloxacin. But only 6 of 51 strains were classified as multi-drug resistant strains. The pulsed field gel electrophoresis analysis of the Spe I-digested DNA from the 100 isolates suggested not only clonal spread but spread of heterogeneous clones started to contribute to the prevalence of metallo-beta-lactamase producing P. aeruginosa strains in Japanese hospitals. [Abstract/Link to Full Text]

Ito H, Ura A, Oyamada Y, Yoshida H, Yamagishi J, Narita S, Matsuyama S, Tokuda H
A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target.
Microbiol Immunol. 2007;51(3):263-70.
As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in-house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system. [Abstract/Link to Full Text]

Kohwiwattanagun J, Kawamura I, Fujimura T, Mitsuyama M
Mycobacterial mammalian cell entry protein 1A (Mce1A)-mediated adherence enhances the chemokine production by A549 alveolar epithelial cells.
Microbiol Immunol. 2007;51(2):253-61.
Mycobacterial mammalian cell entry protein 1A (Mce1A) is involved in the uptake of bacteria in non-phagocytic cells and also possibly in granuloma formation. However, it has not been clarified whether the interaction between mycobacterial Mce1A and epithelial cell induces chemokine and cytokine production which is required for granuloma formation. To this end, we infected A549 alveolar epithelial cells in vitro with E. coli expressing Mce1A on the cell surface and examined the resultant chemokine/cytokine production. Mce1A promoted bacterial adherence and internalization of E. coli into A549 cells, and these recombinant bacteria induced high levels of MCP-1 and IL-8 production, compared to E. coli harboring the plasmid vector alone. Chemokine production was enhanced by the internalization of recombinant E. coli expressing Mce1A because cytochalasin D treatment partially inhibited MCP-1 and IL-8 production. However, Mce1A-coated latex beads did not induce the chemokine production. These results suggest that although Mce1A does not induce production of chemokines, it may promote chemokine induction by augmenting the interaction between bacteria and epithelial cells. [Abstract/Link to Full Text]

Yoshida M, Gotoh K, Fujii M, Shimada H, Touma M, Hosono M
Adrenal participation in thymocyte death by anti-CD3 antibodies in vivo.
Microbiol Immunol. 2007;51(2):243-51.
The deletion of CD4- and CD8-double-positive (DP) cells in the thymus after treatment with anti-CD3 antibodies has long been considered as a useful model for clonal deletion during T cell development, although it was reported that DP cell death was not observed in neonates where self-tolerance should be developing. We dealt with the cellular basis of this enigmatic phenomenon in this report. Due to the similar susceptibility to the antibody-treatment in vitro between neonatal and adult thymocytes, critical factors may be outside rather than within the thymus. Indeed, newborn thymus lobes transplanted into recipients of different ages showed an increased susceptibility to the thymo-toxicity as the age of the recipient increased. The thymo-toxicity seems to be based on the adrenal function of glucocorticoid (GC) synthesis, because administration of an inhibitor of GC synthesis significantly reduced the DP cell death by the antibody-treatment. Finally, adrenalectomy completely prevented DP cell death by anti-CD3 antibodies in adult mice. Therefore, the thymocyte death by anti-CD3 antibodies in vivo may not be due to the T cellreceptor mediated selection in the thymus. [Abstract/Link to Full Text]

Miyano-Kurosaki N, Kira J, Barnor JS, Maeda N, Misawa N, Kawano Y, Tanaka Y, Yamamoto N, Koyanagi Y
Autonomous proliferation of HTLV-CD4+ T cell clones derived from human T cell leukemia virus type I (HTLV-I)-associated myelopathy patients.
Microbiol Immunol. 2007;51(2):235-42.
That HTLV-I infects CD4(+) T cells and enhances their cell growth has been shown as successful long-term in vitro proliferation in the presence of IL-2. It is known that T cells isolated from HAM patients possess strong ability for cell proliferation in vitro and mRNA of various cytokines are abundantly expressed in CNS tissues of HAM patients. Hence, the cytokine-induced proliferation could have an important role in pathogenesis and immune responses of HAM. In this study, we examined the relationship between cell proliferation and ability of in vitro cytokine production of CD4(+) T cell clones isolated from HAM patients. We started a culture from a single cell to isolate cell clones immediately after drawing blood from the patients using limiting dilution method, which could allow the cell to avoid in vitro HTLV-I infection after initiation of culture. Many cell clones were obtained and the rate of proliferation efficiency from a single cell was as high as 80%, especially in the 4 weeks' culture cells from HAM patients. These cells were classified as mainly Th0 phenotype that produce both IFN-gamma and IL-4 after CD3-stimulation. However, the frequency of proviral DNA in these cloned cells was significantly low. Our results indicate that the ability of cell proliferation in HAM patients is not restricted in HTLV-I-infected T cells. HTLV-Iuninfected CD4(+) T cells, mainly Th0 cells, also have a strong ability to respond to IL-2-stimulation, showing that unusual immune activation on T cells has been observed in HAM patients. [Abstract/Link to Full Text]

Das S, Das SC, Verma R
Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis.
Microbiol Immunol. 2007;51(2):231-4.
The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no ampli-fication product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis. [Abstract/Link to Full Text]

Yokota S, Harimaya A, Sato K, Somekawa Y, Himi T, Fujii N
Colonization and turnover of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in otitis-prone children.
Microbiol Immunol. 2007;51(2):223-30.
Recurrent otitis media are frequently intractable during childhood. It is unclear whether recurrent otitis media is caused by etiological bacteria colonization or by new infections. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were isolated from the nasopharynx of 7 otitisprone and 2 non-prone children with recurrent otitis media. Plural bacterial species and strains were found in all children while affected by otitis media. The same strain was repeatedly isolated from all otitisprone children even after administration of antibiotics but was not from the non-prone children. Antibiotic susceptibility did not differ significantly among the same repeatedly isolated strains. This pilot study suggests that the etiological bacteria tend to colonize and is hard to eliminate in otitis-prone children. [Abstract/Link to Full Text]

Cho YJ, Ahn BY, Song ES, Park SA, Lee DH, Kim DS, Lee NG
Bacterial DNA containing methylated CpG motifs retains immunostimulatory activity in synergy with modified lipopolysaccharides.
Microbiol Immunol. 2007;51(2):211-22.
We previously described the immunostimulatory activity of CIA07, a combination of bacterial DNA fragments and modified LPS, and demonstrated that CIA07 has antitumor activity in a mouse bladder cancer model. In this study, we investigated whether methylation of the CpG motifs on the bacterial DNA fragments affects the immunostimulatory potential of CIA07. E. coli DNA fragments were methylated with CpG methylase, and then combined with modified LPS for experiments. Our results revealed that methylated CIA07 (mCIA07) and unmethylated CIA07 were equally active in inducing cytokine secretion from human whole blood cells. In addition, both methylated DNA fragments and mCIA07 retained the ability to activate expression and nuclear translocation of NF-kappaB in RAW 264.7 cells. Finally, methylated DNA fragments and mCIA07 exhibited an antitumor activity comparable to those of their unmethylated counterparts in our mouse bladder cancer model. These data demonstrate that CpG methylation of E. coli DNA does not abrogate the immunostimulatory activity of DNA fragments or CIA07, suggesting that the synergistic activity by bacterial DNA in combination with LPS may be independent of the methylation status of CpG motifs. [Abstract/Link to Full Text]

Rajavelu P, Das SD
A correlation between phagocytosis and apoptosis in THP-1 cells infected with prevalent strains of Mycobacterium tuberculosis.
Microbiol Immunol. 2007;51(2):201-10.
The innate ability of infected macrophages to undergo programmed cell death (apoptosis) and curtail the infection is crucial for the host defense. Although phagocytosis and intracellular killing mechanisms leading to apoptosis in macrophages are highly effective in eliminating the infecting tuberculous bacilli, some Mycobacterium tuberculosis(Mtb) strains have evolved strategies to inhibit this microbicidal function and make use of macrophage for its successful and prolonged survival. Two clinical strains of Mtb (S7 and S10) found to be prevalent and primitive, based on molecular epidemiological studies, were used to study the magnitude in induction of apoptosis in THP-1 cells at various time points of infection and to correlate it with phagocytosis. The percentage of phagocytosis did not show any strain-specific association with differentiated THP-1 cells. But in the phagocytic index, the clinical strains showed a low dose of infection in the 1-10 bacilli category thereby exerting less burden on the cells. The induction of apoptosis was strain dependent. The THP-1 cells infected with H37Ra and S10 showed an increase in apoptosis at all time points while the S7 strain induced minimum apoptosis. A negative correlation between apoptosis and phagocytic index was observed in the 1-10 category and a positive correlation in the > 20 category of the phagocytic index. This novel observation indicates that the magnitude of THP-1 cell apoptosis is a function of the number of internalized mycobacteria. These results indicated a differential mode of infection by clinical strains and their adaptation to different survival strategies that may lead to immune suppression and pathogenesis of the disease. [Abstract/Link to Full Text]

Hattori J, Okumura N, Yamazaki Y, Uchiyama M, Hamaguchi M, Nishiyama Y, Kaneda T
Beneficial effect of GB virus C co-infection in Human Immunodeficiency Virus type 1-infected individuals.
Microbiol Immunol. 2007;51(2):193-200.
Several reports have documented a better prognosis for HIV-1-infected patients co-infected with GBV-C, while other reports have contradicted such findings with the result that this issue remains controversial. We attempted to clarify the complicated status of the effect of GBV-C co-infection on HIV-1-infected patients. GBV-C RNA was detected in 37 samples in 182 HIV-1-infected patients (20.3%) using RT/nested PCR. Of these, 3 were determined to be GBV-C genotype 1, 12 were genotype 2, and the remaining 22 were genotype 3. The GBV-C viral load quantified by real-time PCR ranged from 7.8x10(3) to 3.3x10(6) copies/ml. Weakly negative correlation was observed between GBV-C viral load and HIV-1 viral load in 19 HAART-naďve patients, indicating that a higher GBV-C viral load is associated with a greater suppression of HIV-1 replication. A previously published in vitro study suggested that GBV-C infection would induce up-regulation of RANTES, leading to suppression of HIV-1 replication. However, in our present study, the blood RANTES level was significantly lower in the GBV-C co-infected group than in the uninfected group (190-9,959 vs. 264-31,038 pg/ml, P=0.004). Our results suggested that a suppression of HIV-1 replication by GBV-C co-infection is not mediated by up-regulated RANTES, and thus call for another as yet unknown factor. [Abstract/Link to Full Text]

Kiyohara T, Sato T, Totsuka A, Miyamura T, Ito T, Yoneyama T
Shifting seroepidemiology of hepatitis A in Japan, 1973-2003.
Microbiol Immunol. 2007;51(2):185-91.
BACKGROUND: Hepatitis A infection is caused by hepatitis A virus (HAV) contracted through fecal-oral transmission. Life-long immunity is conferred after infection. Improved sanitary conditions have generally resulted in a significant decline in the incidence of hepatitis A. However, a low incidence of infection results in increased HAV susceptibility. The present study investigates the prevalence of anti-HAV antibody and clarifies the current HAV status and HAV susceptibility in Japan at 2003. METHODS: A total of 2,430 serum specimens collected during 2003 from Japanese individuals ranging in age from 0-92 years, were tested for anti-HAV antibody using an inhibition enzyme linked immunosorbent assay. All specimens were obtained from the WHO and the National Serum Reference Bank/National Institute of Infectious Diseases, Tokyo, Japan. RESULTS: The overall seroprevalence was 12.2%. Anti-HAV antibodies were rarely detected in individuals between 0-44 years of age. Starting from the age of 45-49 years, seropositivity gradually increased through age 65 years and above. Seroprevalence was not affected by gender, and geographic distribution did not affect age-specific seroprevalence until the age of 60 years. CONCLUSIONS: HAV susceptibility in Japan is increasing annually. Particularly, the prevalence of anti-HAV antibody in individuals older than 50 years in 2003 was 50.3%, which is significantly lower than that of corresponding studies in 1994 (74.3%), 1984 (96.9%) and 1973 (96.9%). The growing susceptible population of advanced age results in more frequent HAV infection among them. The surveillance of anti-HAV antibody prevalence is useful for implementing preventive measures and for controlling the spread of HAV. [Abstract/Link to Full Text]

Nishida T, Nishio O, Kato M, Chuma T, Kato H, Iwata H, Kimura H
Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan.
Microbiol Immunol. 2007;51(2):177-84.
Norovirus (NV) is a causative agent of acute gastroenteritis in humans, and shellfishes including oysters act as major vehicles of the virus. To investigate the genetic characteristics of NVs, we collected 1,512 oysters for raw consumption between October 2002 and March 2005 from two distinct areas (area A: the Sanriku Sea area; area B: the Setouchi Sea area). We detected the capsid gene and subjected it to phylogenetic analysis. By further quantification of the copy number of the genome by using real-time PCR, the NV capcid gene was detected in approximately 5% of the oysters, and they showed wide diversity. Two percent of the oysters from area B showed relatively large number of NVs, i.e., over 100 copies of capsid gene/oyster, whereas this was not observed in area A. Most of the detected NVs from oysters and humans were genetically related when the capsid region was compared. These results suggested that NVs obtained from humans and those obtained from oysters showed a potential relationship to each other and that some populations of Japanese oysters accumulated a relatively large number of NVs. [Abstract/Link to Full Text]

Zaraket H, Otsuka T, Saito K, Dohmae S, Takano T, Higuchi W, Ohkubo T, Ozaki K, Takano M, Reva I, Baranovich T, Yamamoto T
Molecular characterization of methicillin-resistant Staphylococcus aureus in hospitals in Niigata, Japan: divergence and transmission.
Microbiol Immunol. 2007;51(2):171-6.
The major methicillin-resistant Staphylococcus aureus(MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCC mec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCC mec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst(+) sec(+) strains represented a majority of 86.5% and 9.4% were tst(-) sec(-). SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCC mec IV and seemed to originate in the community, while ST8 strains exhibited SCC mec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type. [Abstract/Link to Full Text]

Fujinami Y, Hirai Y, Sakai I, Yoshino M, Yasuda J
Sensitive detection of Bacillus anthracis using a binding protein originating from gamma-phage.
Microbiol Immunol. 2007;51(2):163-9.
Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Therefore, there is a pressing need to develop novel methods for rapid, simple, and precise detection of B. anthracis. Here, we report that the C-terminal region of gamma-phage lysin protein (PlyG) binds specifically to the cell wall of B. anthracis and the recombinant protein corresponding to this region (positions, 156-233), PlyGB, is available as a bioprobe for detection of B. anthracis. Our detection method, based on a membrane direct blot assay using recombinant PlyGB, was more rapid and sensitive than the gamma-phage test and was simpler and more inexpensive than genetic methods such as PCR, or immunological methods using specific antibodies. Furthermore, its specificity was comparable to the gamma-phage test. PlyGB is applicable in conventional methods instead of antibodies and could be a potent tool for detection of B. anthracis. [Abstract/Link to Full Text]

Horie K, Ohashi M, Satoh Y, Sairenji T
The role of p38 mitogen-activated protein kinase in regulating interleukin-10 gene expression in Burkitt's lymphoma cell lines.
Microbiol Immunol. 2007;51(1):149-61.
In malignant B lymphoma cells interleukin-10 (IL-10) expression is frequently upregulated. This effect is thought to support to the malignant transformation of these cells and to be a potential target for pharmacotherapy. To define better the mechanism for upregulation of the IL-10 gene, we tested the association between IL-10 and p38 mitogen-activated protein kinase (MAPK) in several Epstein-Barr virus (EBV) infected and non-infected Burkitt's lymphoma (BL) cell lines. The all BL cell lines expressed IL-10 and IL-10 receptor mRNAs, and produced IL-10. p38 MAPK was constitutively phosphorylated in the cytoplasm of the BL cell lines. We further analyzed molecular effects of p38 MAPK on IL-10 expression in Akata cells. Exogenous IL-10 lead rapidly to phosphorylation of Jak1 and Tyk2 as transducers of signals of IL-10, and promoted growth of Akata cells in a dose-dependent manner. The phosphorylation of cytoplasmic p38 MAPK in Akata cells was reduced by the serine/threonine kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). A specific inhibitor of p38 MAPK, SB203580, blocked simultaneously STAT3 DNA-binding activity, and IL-10 mRNA expression, IL-10 production, and then the cell growth was inhibited. These results indicate that the p38 MAPK pathway is functionally linked to IL-10 gene expression and supports the view that the constitutive activation of cytoplasmic p38 MAPK in BL cells is a step in the upregulation of IL-10 gene expression and lymphomagenesis. [Abstract/Link to Full Text]

Begum MD, Umemura M, Kon S, Yahagi A, Hamada S, Oshiro K, Gotoh K, Nishizono A, Uede T, Matsuzaki G
Suppression of the bacterial antigen-specific T cell response and the dendritic cell migration to the lymph nodes by osteopontin.
Microbiol Immunol. 2007;51(1):135-47.
Osteopontin (OPN) has been reported to enhance the interferon (IFN)-gamma-producing Th1-type T cell response through the induction of interleukin (IL)-12 and the suppression of IL-10. We therefore investigated whether OPN could enhance Th1 induction by vaccination against bacterial antigen in vivo. Unexpectedly, the co-inoculation of OPN suppressed the induction of IFN-gamma-producing CD4(+) T cells and T cell proliferative response after the subcutaneous heat-killed Listeria monocytogenes(HKLM) immunization. These results suggest that OPN down-regulates T cell priming. Since dendritic cells (DC) play a pivotal role in T cell priming, we next analyzed the effects of OPN on DC. The addition of OPN into the culture of either bone marrow-derived immature DC or an immature DC line JAWSII showed no effects on the expression of MHC class II, CD80, and CD86 molecules before and after HKLM stimulation. Consistently, in vitro OPN-treated DC showed a normal antigen-presenting function to an established Listeria-specific Th1-type T cells. However, when the DC were transferred into the footpad with HKLM and OPN, the migration of the transferred DC into the regional LN was suppressed in comparison to the DC transferred with HKLM alone. Furthermore, the addition of OPN into the culture of the DC line and HKLM severely suppressed the HKLM-induced expression of CCR7 chemokine receptor which is an important factor in the migration of DC into LN. All the results suggest the existence of an OPN-mediated negative feedback mechanism in the T cell immune response through the regulation of DC migration. [Abstract/Link to Full Text]

El-Farrash MA, Aly HH, Watashi K, Hijikata M, Egawa H, Shimotohno K
In vitro infection of immortalized primary hepatocytes by HCV genotype 4a and inhibition of virus replication by cyclosporin.
Microbiol Immunol. 2007;51(1):127-33.
Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma worldwide. We previously reported that cyclosporin A (CsA) inhibits HCV-1b replication. However, its inhibition of JFH-1 (HCV-2a) was much less. Since HCV genotype clearly affects the in vitro and in vivo response to anti-viral therapy, we wished to examine the effect of CsA and its non-immunosuppressive derivative NIM811 on HCV genotype 4a replication. We first established an in vitro system supporting HCV-4a infection and replication using immortalized human hepatocytes, HuS-E7/DN24 (HuS) cells, and these cells were infected with sera obtained from Egyptian patients with chronic HCV-4a infection. HuS cells supported more robust HCV-4a replication than both HuH-7.5 and PH5CH8 cells, and HCV-4a infection and replication were completely inhibited by 3 mug/ml CsA and 0.5 mug/ml NIM811. Thus, HuS cells are a good model system supporting the infection and high-level replication of HCV-4a, and both CsA and NIM811 effectively inhibit HCV-4a replication in this system. [Abstract/Link to Full Text]

Abe K, Nozaki A, Tamura K, Ikeda M, Naka K, Dansako H, Hoshino HO, Tanaka K, Kato N
Tandem repeats of lactoferrin-derived anti-hepatitis C virus peptide enhance antiviral activity in cultured human hepatocytes.
Microbiol Immunol. 2007;51(1):117-25.
Previously, we found that bovine and human lactoferrin (LF) specifically inhibited hepatitis C virus (HCV) infection in cultured non-neoplastic human hepatocyte-derived PH5CH8 cells, and we identified 33 amino acid residues (termed C-s3-33; amino acid 600-632) from human LF that were primarily responsible for the binding activity to the HCV E2 envelope protein and for the inhibiting activity against HCV infection. Since the anti-HCV activity of C-s3-33 was weaker than that of human LF, we speculated that an increase of E2 protein-binding activity might contribute to the enhancement of anti-HCV activity. To test this possibility, we made two repeats [(C-s3-33)(2)] and three repeats [(C-s3-33)(3)] of C-s3-33 and characterized them. Far-Western blot analysis revealed that the E2 protein-binding activities of (C-s3-33)(2) and (C-s3-33)(3) became stronger than that of the C-s3-33, and that the binding activity of (C-s3-33)(3) was stronger than that of (C-s3-33)(2). Using an HCV infection system in PH5CH8 cells, we demonstrated that the anti-HCV activities of (C-s3-33)(2) and (C-s3-33)(3) became stronger than that of the C-s3-33. Furthermore, using a recently developed infection system with a VSV pseudotype harboring the green fluorescent protein gene and the native E1 and E2 genes, we demonstrated that the antiviral activities of (C-s3-33)(2) and (C-s3-33)(3) were stronger than that of C-s3-33. These results suggest that tandem repeats of LF-derived anti-HCV peptide are useful as anti-HCV reagents. [Abstract/Link to Full Text]

Asai T, Ishihara K, Harada K, Kojima A, Tamura Y, Sato S, Takahashi T
Long-term prevalence of antimicrobial-resistant Salmonella enterica subspecies enterica Serovar infantis in the broiler chicken industry in Japan.
Microbiol Immunol. 2007;51(1):111-5.
Eleven broiler isolates of Salmonella Infantis obtained between 1989 and 1998 were examined for antimicrobial susceptibility and pulse field gel electrophoresis (PFGE) profiles. Seven strains of S. Infantis isolated after 1993 harbored similar antimicrobial susceptibilities to the recent isolates between 2001 and 2003. In comparison of PFGE profile with 22 isolates obtained from 22 apparently healthy broiler chickens between 2001 and 2003, the predominant cluster included the seven strains isolated after 1993. We could not clarify the reasons why the serovar has been prevalent in the broiler industry for a long time, but current antimicrobial usage is not always linked to its prevalence. [Abstract/Link to Full Text]

Mun HS, Kim HJ, Oh EJ, Kim H, Park YG, Bai GH, Do J, Cha CY, Kook YH, Kim BJ
Direct application of AvaII PCR restriction fragment length polymorphism analysis (AvaII PRA) targeting 644 bp heat shock protein 65 (hsp65) gene to sputum samples.
Microbiol Immunol. 2007;51(1):105-10.
To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS 6110 PCR. Although this method showed a sensitivity slightly lower than IS 6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS 6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens. [Abstract/Link to Full Text]

Kida N, Mochizuki Y, Taguchi F
Sporicidal Activity of the KMT reagent in its vapor phase against Geobacillus stearothermophilus Spores.
Microbiol Immunol. 2007;51(1):99-103.
In an investigation of the sporicidal activity of the KMT reagent, a vapor phase study was performed using five kinds of carriers contaminated with Geobacillus stearothermophilus spores. When 25 ml of the KMT reagent was vaporized in a chamber (capacity; approximately 95 liters), the 2-step heating method (vaporization by a combination of low temperature and high temperature) showed the most effective sporicidal activity in comparison with the 1-step heating method (rapid vaporization). The 2-step heating method appeared to be related to the sporicidal activity of vaporized KMT reagent, i.e., ethanol and iodine, which vaporized mainly when heated at a low temperature such as 55 C, and acidic water, which vaporized mainly when heated at a high temperature such as 300 C. We proposed that the KMT reagent can be used as a new disinfectant not only in the liquid phase but also in the vapor phase in the same way as peracetic acid and hydrogen peroxide. [Abstract/Link to Full Text]

Shimada S, Nakamura M, Tanaka Y, Tsutsumi K, Katano M, Masuko K, Yudoh K, Koizuka I, Kato T
Crosslinking of the CD69 molecule enhances S100A9 production in activated neutrophils.
Microbiol Immunol. 2007;51(1):87-98.
Expression of CD69 on neutrophils and generation of anti-CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti-CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint-composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte-macrophage colony stimulating factor (GM-CSF)-activated neutrophils was crosslinked by anti-CD69 monoclonal antibodies, and then intracellular proteins were detected using 2-dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up-regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS-27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS-27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL-1beta production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti-CD69 autoantibodies possibly affect joint-composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA. [Abstract/Link to Full Text]

---

Recent Articles in International Microbiology

---

Benítez T, Rincón AM, Limón MC, Codón AC
Biocontrol mechanisms of Trichoderma strains.
Int Microbiol. 2004 Dec;7(4):249-60.
The genus Trichoderma comprises a great number of fungal strains that act as biological control agents, the antagonistic properties of which are based on the activation of multiple mechanisms. Trichoderma strains exert biocontrol against fungal phytopathogens either indirectly, by competing for nutrients and space, modifying the environmental conditions, or promoting plant growth and plant defensive mechanisms and antibiosis, or directly, by mechanisms such as mycoparasitism. These indirect and direct mechanisms may act coordinately and their importance in the biocontrol process depends on the Trichoderma strain, the antagonized fungus, the crop plant, and the environmental conditions, including nutrient availability, pH, temperature, and iron concentration. Activation of each mechanism implies the production of specific compounds and metabolites, such as plant growth factors, hydrolytic enzymes, siderophores, antibiotics, and carbon and nitrogen permeases. These metabolites can be either overproduced or combined with appropriate biocontrol strains in order to obtain new formulations for use in more efficient control of plant diseases and postharvest applications. [Abstract/Link to Full Text]

Llorca J
Organic matter in meteorites.
Int Microbiol. 2004 Dec;7(4):239-48.
Some primitive meteorites are carbon-rich objects containing a variety of organic molecules that constitute a valuable record of organic chemical evolution in the universe prior to the appearance of microorganisms. Families of compounds include hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids, amino acids, amines, amides, heterocycles, phosphonic acids, sulfonic acids, sugar-related compounds and poorly defined high-molecular weight macromolecules. A variety of environments are required in order to explain this organic inventory, including interstellar processes, gas-grain reactions operating in the solar nebula, and hydrothermal alteration of parent bodies. Most likely, substantial amounts of such organic materials were delivered to the Earth via a late accretion, thereby providing organic compounds important for the emergence of life itself, or that served as a feedstock for further chemical evolution. This review discusses the organic content of primitive meteorites and their relevance to the build up of biomolecules. [Abstract/Link to Full Text]

Guerrero R
Year's comments for 2004.
Int Microbiol. 2004 Dec;7(4):235-7. [Abstract/Link to Full Text]

Frette L, Johnsen K, Jřrgensen NO, Nybroe O, Kroer N
Functional characteristics of culturable bacterioplankton from marine and estuarine environments.
Int Microbiol. 2004 Sep;7(3):219-27.
Information on the structure of bacterioplankton communities is continuously increasing, while knowledge of their metabolic capabilities remains limited. In this study, the metabolic capacity of bacterioplankton was investigated, as such information is necessary to fully understand carbon cycling and other biogeochemical processes. The diversity of dominant culturable chemoorganotrophic bacteria from one estuarine and three marine environments was analyzed by random isolation of colony-forming units on solid media, taxonomical identification by partial 16S rRNA gene sequence analysis, and functional characterization of the isolates. A total of 76 16S rRNA gene sequences, representing 19 different genotypes, were obtained from the four sampling localities, including Bacillus, Pseudomonas, Pseudoalteromonas, Vibrio, and Erythrobacter as the most frequently isolated genera. The range of metabolic functions possessed by the cultured bacterial assemblages differed significantly between sites. Similarly, the percentage at each sampling station of bacteria capable of performing a specific function was significantly different for 18 of the 25 investigated metabolic functions. At two localities, the bacterial assemblages were dominated by a single genus (Pseudoalteromonas or Erythrobacter) and appeared to be functionally specialized. More than 95% of the isolates were capable of utilizing dissolved free amino acids and protein as their sole nitrogen sources, and all isolates of the specialized assemblages expressed beta-glucosidase. Furthermore, only some of the isolates were able to utilize NH4+, while up to two thirds of the isolates of the two marine sites were able to grow on NO3-. [Abstract/Link to Full Text]

Cardonha AM, Vieira RH, Rodrigues DP, Macrae A, Peirano G, Teophilo GN
Fecal pollution in water from storm sewers and adjacent seashores in Natal, Rio Grande do Norte, Brazil.
Int Microbiol. 2004 Sep;7(3):213-8.
A study on the distribution patterns of enteropathogenic bacteria polluting the shoreline in Natal, Rio Grande do Norte, Brazil, was carried out based on 72 samples obtained from three storm sewers and adjoining beach locations, Praia do Meio (PM), Areia Preta (AP) and Ponta Negra (PN). From each location, 12 water samples were taken and analyzed for fecal coliforms (FC) and Escherichia coli. In AP, two (16.7%) of the seawater samples and five (41.7%) of the storm sewer samples yielded values above 1.1 x 107 FC/100 ml, whereas only one (8.3%) of the samples from PM reached this level. There was no correlation (p > 0.05) between rainfall indices and FC values. A total of 64 E. coli isolates were obtained: 37 from the storm sewer samples and 27 from the seawater samples. Of these isolates, four (O143, two O112ac, and O124) were enteroinvasive and two (O111 and O125) were enteropathogenic. Resistance to antibiotics and to heavy metals was also analyzed. Almost 36% of the E. coli strains isolated were resistant to more than one antibiotic. All strains were resistant to zinc and copper at the highest concentration tested (250 microg/ml), and several (23.4%) were resistant to mercury at 50 microg/ml. Our results agreed with previous reports that antibiotic resistance is commonly associated with heavy-metal resistance in pathogens. [Abstract/Link to Full Text]

Saavedra MJ, Guedes-Novais S, Alves A, Rema P, Tacăo M, Correia A, Martínez-Murcia A
Resistance to beta-lactam antibiotics in Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss).
Int Microbiol. 2004 Sep;7(3):207-11.
Bacterial infections caused by members of the genus Aeromonas, with a relatively high antibiotic resistance, are among the most common and troublesome diseases of fish raised in ponds with recirculation systems. In this study, carried out at an experimental aquaculture station in northern Portugal, 51 strains identified as belonging to the genus Aeromonas were isolated from 20 rainbow trout (Oncorhynchus mykiss) skin and kidney samples, as well as from raceway water samples. Macro- and microscopic examination of the fish tissues revealed lesions or cellular alterations in skin and kidney that seemed to correlate with the presence of those isolates. The sensitivity of all isolated strains to different groups of beta-lactam antibiotics (penicillins, cephalosporins, monobactams and carbapenems) was evaluated using the disc diffusion method. The highest rates of resistance were to amoxicillin, carbenicillin and ticarcillin. Unexpected resistance to imipenem, an antibiotic of clinical usage, was also detected, which suggests that resistance may have been transferred to the Aeromonas population from the environment. [Abstract/Link to Full Text]

Alberola TM, García-Martínez J, Antúnez O, Viladevall L, Barceló A, Arińo J, Pérez-Ortín JE
A new set of DNA macrochips for the yeast Saccharomyces cerevisiae: features and uses.
Int Microbiol. 2004 Sep;7(3):199-206.
The yeast Saccharomyces cerevisiae has been widely used for the implementation of DNA chip technologies. For this reason and due to the extensive use of this organism for basic and applied studies, yeast DNA chips are being used by many laboratories for expression or genomic analyses. While membrane arrays (macroarrays) offer several advantages, for many laboratories they are not affordable. Here we report that a cluster of four Spanish molecular-biology yeast laboratories, with relatively small budgets, have developed a complete set of probes for the genome of S. cerevisiae. These have been used to produce a new type of macroarray on a nylon surface. The macroarrays have been evaluated and protocols for their use have been optimized. [Abstract/Link to Full Text]

Belkova NL, Zakharova JR, Tazaki K, Okrugin VM, Parfenova VV
Fe-Si biominerals in the Vilyuchinskie hot springs, Kamchatka Peninsula, Russia.
Int Microbiol. 2004 Sep;7(3):193-8.
The micromorphological structure of microbial mats (biomats) from the hot springs of the Vilyuchinskaya hydrothermal system, Kamchatka Peninsula, Russia, were investigated. The Vilyuchinskie hot springs had a discharge temperature of 55-56 degrees C and Na-Ca-HCO3-type waters rich in silicic and boric acids. Water and biomats had high concentrations of Fe, Mn, Sr, and As. Enumeration of total bacterial abundance (TBA) demonstrated a low density of bacterial populations. However, the fractions of metabolically active bacteria and respiring iron-oxidizing bacteria in the hot-spring water were high, comprising 68 and 21% of TBA, respectively. Scanning electron microscopy equipped with an energy dispersive X-ray spectrometer (SEM-EDX) showed that unicellular rod-shaped bacteria about 5-microm long predominated in the brown biomats. The mineral capsules of these bacteria contained large amounts of Fe and Si. Extracellular and intracellular particles were observed by transmission electron microscopy. Fe-oxidizing bacteria were isolated from the biomats on agar plates with selective medium. Therefore, it can be concluded that microorganisms inhabiting the biomats of the Vilyuchinskie hot springs are essential for the deposition of Fe-minerals at neutral pH. [Abstract/Link to Full Text]

Alonso A, García-del Portillo F
Hijacking of eukaryotic functions by intracellular bacterial pathogens.
Int Microbiol. 2004 Sep;7(3):181-91.
Intracellular bacterial pathogens have evolved as a group of microorganisms endowed with weapons to hijack many biological processes of eukaryotic cells. This review discusses how these pathogens perturb diverse host cell functions, such as cytoskeleton dynamics and organelle vesicular trafficking. Alteration of the cytoskeleton is discussed in the context of the bacterial entry process (invasion), which occurs either by activation of membrane-located host receptors ("zipper" mechanism) or by injection of bacterial proteins into the host cell cytosol ("trigger" mechanism). In addition, the two major types of intracellular lifestyles, cytosolic versus intravacuolar (phagosomal), which are the consequence of alterations in the phagosome-lysosome maturation route, are compared. Specific examples illustrating known mechanisms of mimicry or hijacking of the host target are provided. Finally, recent advances in phagosome proteomics and genome expression in intracellular bacteria are described. These new technologies are yielding valuable clues as to how these specialized bacterial pathogens manipulate the mammalian host cell. [Abstract/Link to Full Text]

Díaz E
Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility.
Int Microbiol. 2004 Sep;7(3):173-80.
Although most organisms have detoxification abilities (i.e mineralization, transformation and/or immobilization of pollutants), microorganisms, particularly bacteria, play a crucial role in biogeochemical cycles and in sustainable development of the biosphere. Next to glucosyl residues, the benzene ring is the most widely distributed unit of chemical structure in nature, and many of the aromatic compounds are major environmental pollutants. Bacteria have developed strategies for obtaining energy from virtually every compound under oxic or anoxic conditions (using alternative final electron acceptors such as nitrate, sulfate, and ferric ions). Clusters of genes coding for the catabolism of aromatic compounds are usually found in mobile genetic elements, such as transposons and plasmids, which facilitate their horizontal gene transfer and, therefore, the rapid adaptation of microorganisms to new pollutants. A successful strategy for in situ bioremediation has been the combination, in a single bacterial strain or in a syntrophic bacterial consortium, of different degrading abilities with genetic traits that provide selective advantages in a given environment. The advent of high-throughput methods for DNA sequencing and analysis of gene expression (genomics) and function (proteomics), as well as advances in modelling microbial metabolism in silico, provide a global, rational approach to unravel the largely unexplored potentials of microorganisms in biotechnological processes thereby facilitating sustainable development. [Abstract/Link to Full Text]

López R
Streptococcus pneumoniae and its bacteriophages: one long argument.
Int Microbiol. 2004 Sep;7(3):163-71.
Infectious diseases currently kill more than 15 million people annually, and the WHO estimates that every year 1.6 million people die from pneumococcal diseases. Streptococcus pneumoniae (pneumococcus), a bacterium with a long biological pedigree, best illustrates the rapid evolution of antibiotic resistance, which has led to major public health concern. This article discusses the molecular basis of the two main virulence factors of pneumococcus, the capsule and cell-wall hydrolases, as well as new approaches to developing medicinal weapons for preventing pneumococcal infections. In addition, current knowledge regarding pneumococcal phages as potential contributors to virulence and the use of lytic enzymes encoded by these phages as therapeutic tools is reviewed. [Abstract/Link to Full Text]

Guerrero R, Piqueras M
Open access. A turning point in scientific publication.
Int Microbiol. 2004 Sep;7(3):157-61. [Abstract/Link to Full Text]

Marusic M, Misak A, Kljakovic-Gaspic M, Fister K, Hren D, Marusic A
Producing a scientific journal in a small scientific community: an author-helpful policy.
Int Microbiol. 2004 Jun;7(2):143-7. [Abstract/Link to Full Text]

Brorson O, Brorson SH
An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole.
Int Microbiol. 2004 Jun;7(2):139-42.
The susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole (TZ) was examined. The minimal bactericidal concentration (MBC) of TZ against the mobile spirochetes was >128 microg/ml at 37 degrees C in micro-oxic atmosphere when incubated for 14 days. TZ significantly reduced the conversion of mobile spirochetes to cystic forms during incubation. The MBC for older (10-months-old) cysts at 37 degrees C in a micro-oxic atmosphere was >0.5 microg/ml, but >0.125 microg/ml for young (1-day-old) cysts. Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that, when the concentration of TZ was > or = MBC, the contents of the cysts were partly degraded, core structures did not develop inside the young cysts, and the amount of RNA in these cysts decreased significantly. When cysts were exposed to TZ, both the spirochetal structures and core structures inside the cysts dissolved, and the production of blebs was significantly reduced. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of TZ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi. [Abstract/Link to Full Text]

Obregón V, García JL, García E, López R, García P
Peculiarities of the DNA of MM1, a temperate phage of Streptococcus pneumoniae.
Int Microbiol. 2004 Jun;7(2):133-7.
The abundant presence of temperate phages in the chromosomes of clinical isolates of Streptococcus pneumoniae has been well documented. The genome of MM1, a temperate phage of pneumococcus, has been isolated as a DNA-protein complex. The protein is covalently bound to the DNA, was iodinated in vitro with Na125I, and has an Mr of 22,000. Electron microscopy and enzymatic analyses revealed that the MM1 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules packaged via a headful mechanism. The location of the pac site appears to be downstream of the terminase, between orf32 and orf34 of the MM1 genome. [Abstract/Link to Full Text]

Oeltjen A, Marquardt J, Rhiel E
Differential circadian expression of genes fcp2 and fcp6 in Cyclotella cryptica.
Int Microbiol. 2004 Jun;7(2):127-31.
The steady-state mRNA concentrations of two fcp genes encoding fucoxanthin chlorophyll a/c light-harvesting polypeptides of the centric diatom Cyclotella cryptica were investigated over a 4-day period by RNA dot-blotting experiments. Before and during the first day of the experiment, the cultures were grown under a 12-h light/12-h dark regime. On the following 3 days, the algae were kept in darkness. On the first day, the steady-state mRNA concentration of fcp2 followed a diurnal pattern, with a maximum occurring around noon, approximately 6 h after the onset of light. The gene fcp6 also had a diurnal pattern on the first day. Its maximum, however, occurred immediately after the onset of light. During the subsequent incubation period in darkness, the diurnal pattern of expression of both fcp genes continued, thus demonstrating that their steady-state mRNA concentrations oscillated in a circadian manner. [Abstract/Link to Full Text]

García-Rosado E, Castro D, Cano I, Alonso MC, Pérez-Prieto SI, Borrego JJ
Protein and glycoprotein content of lymphocystis disease virus (LCDV).
Int Microbiol. 2004 Jun;7(2):121-6.
The polypeptide and glycoprotein composition of eight strains of the fish-pathogenic lymphocystis disease virus (LCDV) isolated from gilt-head seabream (Sparus aurata), blackspot seabream (Pagellus bogaraveo), and sole (Solea senegalensis) were determined. The protein electrophoretic patterns of all LCDV isolates were quite similar regardless of the host fish, showing two major proteins (79.9 and 55.6 kDa) and a variable number of minor proteins. Three groups of LCDV isolates were distinguished according to the number and molecular masses of the minor proteins. Eight glycoproteins were detected inside viral particles of LCDV 2, LCDV 3 and LCDV 5 isolates, but only seven glycoproteins were found inside viral particles of LCDV 1, LCDV 4, LCDV 6, LCDV 7, and LCDV 11 isolates and the reference virus ATCC VR 342 by using five lectins. LCDV glycoproteins were mainly composed of mannose and sialic acid. These glycoproteins could be part of an external viral envelope probably derived from the host cell membrane. [Abstract/Link to Full Text]

Urbina D, Rodríguez JG, Arzuza O, Parra E, Young G, Castro R, del-Portillo P
G and P genotypes of rotavirus circulating among children with diarrhea in the Colombian northern coast.
Int Microbiol. 2004 Jun;7(2):113-20.
A study on the prevalence of rotavirus G and P genotypes was carried out based on 253 stool specimens obtained from children living in the Colombia northern coast region who were less than 3-years-old and who suffered from acute diarrhea. A previous study had detected the presence of rotavirus A in 90 (36.5%) of the 246 samples tested by enzyme immunoassay (EIA), and these strains were investigated in the present study. Of these, 50 strains yielded an RNA electropherotype, most of which (80.0%) had long profiles and 20.0% of which had short profiles. Genotyping of 84 positive samples indicated that 67.9% of the strains could be typed. G1 (57.9%), was the most predominant VP7 genotype, followed by G3 (21.1%), G9 (15.8%) and G2 (5.3%). Among the VP4 genotypes, P[4] (49.1%) was the most prevalent, followed by P[6] 36.4% and P[8] (14.5%). Neither G4 nor G8 nor P[9] types were detected. The most common G-P combinations were G3 P[4] (8.8%) and G9 P[6] (7.0%), followed by G1 P[4] and G1 P[8] (5.3% each). All G1 P[8] strains showed long RNA profiles, whereas G3 P[4] and G9 P[6] displayed both long and short patterns. Mixed infections involved 21.0% of strains. There was a marked diversity among strains collected, and novel strains, including G9, as well as other atypical combinations of G and P genotypes, such as G9 P[6] and G3 P[4], were found. [Abstract/Link to Full Text]

González-Novo A, Jiménez J, García MJ, Ríos-Serrano I, Pla J, Jiménez A, Sánchez-Pérez M
Dynamics of CaCdc10, a septin of Candida albicans, in living cells and during infection.
Int Microbiol. 2004 Jun;7(2):105-12.
The morphogenetic program in the pathogenic fungus Candida albicans, including the dimorphic transition, is an interesting field of study, not only because it is absent in the commonly used model yeast Saccharomyces cerevisiae, but because of the close relationship between hyphal development and virulence of C. albicans. We studied one of the most important aspects of fungal morphogenesis--the septin ring--in C. albicans. By using a fusion construct to green fluorescent protein (GFP), the subcellular localization and dynamics of C. albicans Cdc10 in the different morphologies that this fungus is able to adopt was identified. The localization features reached were contrasted and compared with the results obtained from Candida cells directly extracted from an animal infection model under environmental conditions as similar as possible to the physiological conditions encountered by C. albicans during host infection. [Abstract/Link to Full Text]

Jiménez-Gasco MM, Navas-Cortés JA, Jiménez-Díaz RM
The Fusarium oxysporum f. sp. ciceris/Cicer arietinum pathosystem: a case study of the evolution of plant-pathogenic fungi into races and pathotypes.
Int Microbiol. 2004 Jun;7(2):95-104.
The use of resistant cultivars is one of the most practical and cost-efficient strategies for managing plant diseases. However, the efficiency of resistant cultivars in disease management is limited by pathogenic variability in pathogen populations. Knowledge of the evolutionary history and potential of the pathogen population may help to optimize the management of disease-resistance genes, irrespective of the breeding strategy used for their development. In this review, we examine the diversity in virulence phenotypes of Fusarium oxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpeas, analyze the genetic variability existing within and among those phenotypes, and infer a phylogenetic relationship among the eight known pathogenic races of this fungus. The inferred intraspecific phylogeny shows that each of those races forms a monophyletic lineage. Moreover, virulence of races to resistant chickpea cultivars has been acquired in a simple stepwise pattern, with few parallel gains or losses. Although chickpea cultivars resistant to Fusarium wilt are available, they have not yet been extensively deployed, so that the stepwise acquisition of virulence is still clearly evident. [Abstract/Link to Full Text]

Rocha CD, Caetano BC, Machado AV, Bruna-Romero O
Recombinant viruses as tools to induce protective cellular immunity against infectious diseases.
Int Microbiol. 2004 Jun;7(2):83-94.
Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms. Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen. However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes. In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools. Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes. This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development. Thus, this review will focus in particular on the development of new vaccination protocols against these diseases. [Abstract/Link to Full Text]

Mir J
Industrial microbiology. A new challenge.
Int Microbiol. 2004 Jun;7(2):81-2. [Abstract/Link to Full Text]

Rubio V
Happy microbes in hostile niches. A symposium on extremophiles.
Int Microbiol. 2004 Mar;7(1):71-6. [Abstract/Link to Full Text]

Villanueva JR, García-Acha I
In memory of Federico Uruburu (1934-2003).
Int Microbiol. 2004 Mar;7(1):67-70. [Abstract/Link to Full Text]

Plummer S, Weaver MA, Harris JC, Dee P, Hunter J
Clostridium difficile pilot study: effects of probiotic supplementation on the incidence of C. difficile diarrhoea.
Int Microbiol. 2004 Mar;7(1):59-62.
Colonic infection with Clostridium difficile, leading to pseudomembranous colitis, is a common complication of antibiotic therapy, especially in elderly patients. It has been suggested that non-pathogenic probiotic bacteria might prevent the development and recurrence of C. difficile infection. This double-blind, placebo-controlled study examines the role of probiotic administration in the prevention of C. difficile-associated diarrhoea (CDAD) in elderly patients receiving antibiotic therapy. Consecutive patients (150) receiving antibiotic therapy were randomised to receive either a probiotic containing both Lactobacillus and Bifidobacterium or placebo for 20 days. Upon admission to hospital, bowel habit was recorded and a faecal sample taken. Trial probiotic or placebo was taken within 72 h of prescription of antibiotics, and a second stool sample was taken in the event of development of diarrhoea during hospitalisation or after discharge. Of the randomised patients, 138 completed the study, 69 with probiotics in conjunction with antibiotics and 69 with antibiotics alone. On the basis of development of diarrhoea, the incidence of samples positive for C. difficile-associated toxins was 2.9% in the probiotic group compared with 7.25% in the placebo-control group. When samples from all patients were tested (rather than just those developing diarrhoea) 46% of probiotic patients were toxin-positive compared with 78% of the placebo group. [Abstract/Link to Full Text]

Serra J, Vińas M
Laboratory diagnosis of brucellosis in a rural endemic area in northeastern Spain.
Int Microbiol. 2004 Mar;7(1):53-8.
Sera obtained from 62 patients from four mountain counties in Catalonia (Northeastern Spain), in whom brucellosis had been diagnosed on the basis of clinical evidence and/or personal history, were analyzed using the rose Bengal test, standard serum agglutination test (SAT), Coombs' test, ELISA, and complement fixation. The diagnosis was further confirmed through blood cultures. Clinical evidence, epidemiology, and the results from serologic tests were used to assign patients to one of two groups: group 1 (n = 38) patients had primary infections, whereas group 2 (n = 24) patients had been previously exposed to the microorganism, i.e. re-infection of group 2 individuals occurred after long periods of time during which no active infection by Brucella had been detected. Receiving-operating charts (ROC) were used to determine the diagnostic value of the different tests and to establish discriminant values. Blood culture was a valuable diagnostic tool in group 1 (0.92 sensitivity) but was inappropriate in group 2 (0.08). The combination of positive rose Bengal test and agglutination >/=1/160 was valid for diagnosis in group 1. In group 2, agglutination <1/160 (including negative agglutination) did not rule out brucellosis. The combination of positive rose Bengal test and Coombs' test >/=1/320 was the best diagnostic criterion (0.8 specificity; 1 sensitivity). ELISA (for IgG, IgM, or both) did not improve diagnostic accuracy. [Abstract/Link to Full Text]

Griffiths E, Gupta RS
Signature sequences in diverse proteins provide evidence for the late divergence of the Order Aquificales.
Int Microbiol. 2004 Mar;7(1):41-52.
The Aquificales species are presently believed to be the earliest branching lineage within Bacteria. However, the branching order of this group in different phylogenetic trees is highly variable and not resolved. In the present work, the phylogenetic placement of Aquificales was examined by means of a cladistic approach based on the shared presence or absence of definite signature sequences (consisting of conserved inserts or deletions) in many highly conserved and important proteins, e.g. RNA polymerase beta (RpoB), RNA polymerase beta (RpoC), alanyl-tRNA synthetase (AlaRS), CTP synthase, inorganic pyrophosphatase (PPase), Hsp70 and Hsp60. For this purpose, fragments of the above genes that contained the signature regions were cloned from different Aquificales species (Calderobacterium hydrogenophilum, Hydrogenobacter marinus, and Thermocrinis ruber) and the sequence data were compared with those available from all other species. The presence in Aquificales species of distinctive inserts in Hsp70 and Hsp60 that are not found in any Firmicutes, Actinobacteria, or Thermotoga-Clostridium species excluded them from these groups of Bacteria. The shared presence of prominent indels in the RpoB (>100 amino acids), RpoC (>100 amino acids) and AlaRS (4 amino acids) proteins, which are only found in the various Aquificales species, the Chlamydiae, the CFBG (Cytophaga-Flavobacteria-Bacteroides-green sulfur bacteria) group, and Proteobacteria, strongly suggests their placement within these groups of Bacteria. A specific relationship between Proteobacteria and Aquificales is suggested by the presence in inorganic pyrophosphatase of a 2-amino-acid insert that is uniquely found in these phyla. However, the Aquificales species lacked a number of other protein signatures (e.g. indels in CTP synthase and Hsp70) that are characteristic of Proteobacteria, indicating that they constitute a distinct phylum related to Proteobacteria. These results provide strong and consistent evidence that the Aquificales diverged after the branching of Firmicutes, Actinobacteria, Thermotoga, Deinococcus-Thermus, green nonsulfur bacteria, Cyanobacteria, Spirochetes, Chlamydiae, and CFBG group, but before the emergence of the Proteobacteria. [Abstract/Link to Full Text]

Trueba G, Zapata S, Madrid K, Cullen P, Haake D
Cell aggregation: a mechanism of pathogenic Leptospira to survive in fresh water.
Int Microbiol. 2004 Mar;7(1):35-40.
Transmission of leptospirosis is facilitated by the survival of pathogenic leptospires in moist environments outside their mammalian host. In the present study, the survival mechanisms of Leptospira interrogans serovar Canicola in aqueous conditions and lack of nutrients were investigated. In distilled water, leptospires were able to remain motile for 110 days (pH 7.2). However, when incubated in a semi-solid medium composed of distilled water and 0.5% purified agarose (pH 7.2), they survived 347 days. In this viscous environment, aggregates of live spirochetes were observed. Neither antibiotics (e.g. tetracycline and ampicillin) nor nutrients inhibited leptospiral aggregation. Immunoblot analysis suggested that cells incubated in water down-regulate the expression of LipL31, an inner-membrane protein, but retain expression of other membrane proteins. These studies provide insights into the mechanisms by which pathogenic Leptospira survives for prolonged periods of time in natural aqueous environments, a key stage in the leptospiral lifecycle. [Abstract/Link to Full Text]

Park JE, Schlegel HG, Rhie HG, Lee HS
Nucleotide sequence and expression of the ncr nickel and cobalt resistance in Hafnia alvei 5-5.
Int Microbiol. 2004 Mar;7(1):27-34.
The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced. The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX. The predicted amino acid sequences of ncrA were homologous to the amino acid sequences of nreB of Achromobacter xylosoxidans 31A. Expression of ncr with the T7 RNA polymerase-promoter system allowed Escherichia coli BL21 (DE3) to overexpress NcrA, NcrB, and NcrC but not NcrY, and NcrX. The apparent molecular masses of NcrA, NcrB, and NcrC were 30, 33, and 17 kDa, respectively. Primer-extension analysis showed that ncr mRNA started at nucleotide position 23 upstream from ncrA. The promoter region of the ncr operon possessed a strong, putative -35 element of sigma(32)-type promoter sequence, and transcriptional 'lacZ fusion studies indicated that the -35 element influenced sigma(32)-specific transcription. [Abstract/Link to Full Text]

Martínez-Alonso M, Mir J, Caumette P, Gaju N, Guerrero R, Esteve I
Distribution of phototrophic populations and primary production in a microbial mat from the Ebro Delta, Spain.
Int Microbiol. 2004 Mar;7(1):19-25.
Microbial mats arising in the sand flats of the Ebro Delta (Tarragona, Spain) were investigated during the summer season, when the community was highly developed. These mats are composed of three pigmented layers of phototrophic organisms, an upper brown layer mainly composed of Lyngbya aestuarii and diatoms, an intermediate green layer of the cyanobacterium Microcoleus chthonoplastes, and an underlying pink layer of a so-far unidentified purple sulfur bacterium. In the photic zone, oxygenic phototrophs constitute about 58% of total photosynthetic biomass, measured as biovolume, and anoxygenic phototrophs represent 42%. Diatoms constitute 11.8% of the oxygenic biomass, M. chthonoplastes 61.2%, and L. aestuarii and coccoid cyanobacteria 20.6 and 6.4%, respectively. In this laminated community, organic matter has an autochthonous origin, and photosynthesis is the most important source of organic carbon. Oxygen production reaches up to 27.2 mmol O(2) m(-2) h(-1), measured at 1000 microE m(-2) s(-1) light intensity, whereas oxidation of sulfide in the light has been calculated to be 18.6 mmol S m(-2) h(-1). This amount represents 26% of the total photosynthetic production in terms of photoassimilated carbon, demonstrating the important role of anoxygenic phototrophs as primary producers in the pink layer of Ebro Delta microbial mats. [Abstract/Link to Full Text]

---

Recent Articles in Retrovirology

---

Whitney JB, Wainberg MA
Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIVmac239.
Retrovirology. 2006;396.
BACKGROUND: The 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIVmac239) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). We have constructed a series of deletions in the SIVmac239 leader sequence in order to determine the involvement of this region in both the packaging and dimerization of viral genomic RNA. We also assessed the impact of these deletions upon viral infectiousness, replication kinetics and gene expression in cell lines and monkey peripheral blood mononuclear cells (PBMC). RESULTS: Regions on both sides of the major splice donor (SD) were found to be necessary for the efficiency and specificity of viral genome packaging. However, stem-loop1 is critical for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the initiation site of SIV-Gag have additive effects on RNA packaging and contribute to a lesser degree to RNA dimerization. The targeted disruption of structures on both sides of the SD also severely impacts viral infectiousness, gene expression and replication in both CEMx174 cells and rhesus PBMC. CONCLUSION: In the leader region of SIVmac239, stem-loop1 functions as the primary determinant for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the translational initiation site of SIV-Gag are classified as secondary determinants and play a role in dimerization. Collectively, these data signify a linkage between the primary encapsidation determinant of SIVmac239 and RNA dimerization. [Abstract/Link to Full Text]

Bennasser Y, Jeang KT
HIV-1 Tat interaction with Dicer: requirement for RNA.
Retrovirology. 2006;395.
Dicer is an RNase III which processes two classes of cellular small RNAs: the microRNAs (miRNA) and short interfering RNAs (siRNA). Previously, we observed that over-expressed HIV-1 Tat protein can suppress the processing of small RNAs inside cells. Here, we have investigated the requirements for Tat interaction with Dicer. We report that Tat-Dicer interaction depends on RNA, requires the helicase domain of Dicer, and is independent of Tat's transactivation domain. [Abstract/Link to Full Text]

Wootton SK, Metzger MJ, Hudkins KL, Alpers CE, York D, DeMartini JC, Miller AD
Lung cancer induced in mice by the envelope protein of jaagsiekte sheep retrovirus (JSRV) closely resembles lung cancer in sheep infected with JSRV.
Retrovirology. 2006;394.
BACKGROUND: Jaagsiekte sheep retrovirus (JSRV) causes a lethal lung cancer in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse lung, by using a replication-defective adeno-associated virus type 6 (AAV6) vector, induces tumors resembling those seen in sheep. However, the mouse and sheep tumors have not been carefully compared to determine if Env expression alone in mice can account for the disease features observed in sheep, or whether additional aspects of virus replication in sheep are important, such as oncogene activation following retrovirus integration into the host cell genome. RESULTS: We have generated mouse monoclonal antibodies (Mab) against JSRV Env and have used these to study mouse and sheep lung tumor histology. These Mab detect Env expression in tumors in sheep infected with JSRV from around the world with high sensitivity and specificity. Mouse and sheep tumors consisted mainly of well-differentiated adenomatous foci with little histological evidence of anaplasia, but at long times after vector exposure some mouse tumors did have a more malignant appearance typical of adenocarcinoma. In addition to epithelial cell tumors, lungs of three of 29 sheep examined contained fibroblastic cell masses that expressed Env and appeared to be separate neoplasms. The Mab also stained nasal adenocarcinoma tissue from one United States sheep, which we show was due to expression of Env from ovine enzootic nasal tumor virus (ENTV), a virus closely related to JSRV. Systemic administration of the AAV6 vector encoding JSRV Env to mice produced numerous hepatocellular tumors, and some hemangiomas and hemangiosarcomas, showing that the Env protein can induce tumors in multiple cell types. CONCLUSION: Lung cancers induced by JSRV infection in sheep and by JSRV Env expression in mice have similar histologic features and are primarily characterized by adenomatous proliferation of peripheral lung epithelial cells. Thus it is unnecessary to invoke a role for insertional mutagenesis, gene activation, viral replication, or expression of other viral gene products in sheep lung tumorigenesis, although these processes may play a role in other clinically less important sequelae of JSRV infection such as metastasis observed with variable frequency in sheep. [Abstract/Link to Full Text]

Invernizzi CF, Xie B, Richard S, Wainberg MA
PRMT6 diminishes HIV-1 Rev binding to and export of viral RNA.
Retrovirology. 2006;393.
BACKGROUND: The HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNA through interaction with the Rev response element (RRE) by means of an arginine rich motif that is similar to the one found in Tat. Since Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its arginine rich motif, we investigated whether the Rev protein could act as a substrate for this enzyme. RESULTS: Here, we report the methylation of Rev due to a single arginine dimethylation in the N-terminal portion of its arginine rich motif and the association of Rev with PRMT6 in vivo. Further analysis demonstrated that the presence of increasing amounts of wild-type PRMT6, as well as a methylation-inactive mutant PRMT6, dramatically down-regulated Rev protein levels in concentration-dependent fashion, which was not dependent on the methyltransferase activity of PRMT6. Quantification of Rev mRNA revealed that attenuation of Rev protein levels was due to a posttranslational event, carried out by a not yet defined activity of PRMT6. However, no relevant protein attenuation was observed in subsequent chloramphenicol acetyltransferase (CAT) expression experiments that screened for RNA export and interaction with the RRE. Binding of the Rev arginine rich motif to the RRE was reduced in the presence of wild-type PRMT6, whereas mutant PRMT6 did not exert this negative effect. In addition, diminished interactions between viral RNA and mutant Rev proteins were observed, due to the introduction of single arginine to lysine substitutions in the Rev arginine rich motif. More importantly, wild-type PRMT6, but not mutant methyltransferase, significantly decreased Rev-mediated viral RNA export from the nucleus to the cytoplasm in a dose-dependent manner. CONCLUSION: These findings indicate that PRMT6 severely impairs the function of HIV-1 Rev. [Abstract/Link to Full Text]

Willemsen NM, Hitchen EM, Bodetti TJ, Apolloni A, Warrilow D, Piller SC, Harrich D
Protein methylation is required to maintain optimal HIV-1 infectivity.
Retrovirology. 2006;392.
BACKGROUND: Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. In this study, adenosine periodate (AdOx) was used to globally inhibit protein methyltransferase activity so that the effect of protein methylation on HIV-1 infectivity could be assessed. RESULTS: Two cell culture models were used: HIV-1-infected CEM T-cells and HEK293T cells transfected with a proviral DNA plasmid. In both models, AdOx treatment of cells increased the levels of virion in culture supernatant. However, these viruses had increased levels of unprocessed or partially processed Gag-Pol, significantly increased diameter, and displayed reduced infectivity in a MAGI X4 assay. AdOx reduced infectivity equally in both dividing and non-dividing cells. However, infectivity was further reduced if Vpr was deleted suggesting virion proteins, other than Vpr, were affected by protein methylation. Endogenous reverse transcription was not inhibited in AdOx-treated HIV-1, and infectivity could be restored by pseudotyping HIV with VSV-G envelope protein. These experiments suggest that AdOx affects an early event between receptor binding and uncoating, but not reverse transcription. CONCLUSION: Overall, we have shown for the first time that protein methylation contributes towards maximal virus infectivity. Furthermore, our results also indicate that protein methylation regulates HIV-1 infectivity in a complex manner most likely involving the methylation of multiple viral or cellular proteins and/or multiple steps of replication. [Abstract/Link to Full Text]

Baird HA, Gao Y, Galetto R, Lalonde M, Anthony RM, Giacomoni V, Abreha M, Destefano JJ, Negroni M, Arts EJ
Influence of sequence identity and unique breakpoints on the frequency of intersubtype HIV-1 recombination.
Retrovirology. 2006;391.
BACKGROUND: HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population. RESULTS: Increased sequence identity between viruses or RNA templates resulted in increased recombination frequencies, with the exception of the 115-A virus or RNA template. Analyses of the recombination breakpoints and mechanistic studies revealed that the presence of a recombination hotspot in the C3/V4 env region, unique to 115-A as donor RNA, could account for the higher recombination frequencies with the 115-A virus/template. Single-cycle infections supported proportionally less recombination than the in vitro reverse transcription assay but both systems still had significantly higher recombination frequencies than observed in the multiple-cycle virus replication system. In the multiple cycle assay, increased replicative fitness of one HIV-1 over the other in a dual infection dramatically decreased recombination frequencies. CONCLUSION: Sequence variation at specific sites between HIV-1 isolates can introduce unique recombination hotspots, which increase recombination frequencies and skew the general observation that decreased HIV-1 sequence identity reduces recombination rates. These findings also suggest that the majority of intra- or intersubtype A/D HIV-1 recombinants, generated with each round of infection, are not replication-competent and do not survive in the multiple-cycle system. Ability of one HIV-1 isolate to outgrow the other leads to reduced co-infections, heterozygous virus production, and recombination frequencies. [Abstract/Link to Full Text]

Gallo SA, Reeves JD, Garg H, Foley B, Doms RW, Blumenthal R
Kinetic studies of HIV-1 and HIV-2 envelope glycoprotein-mediated fusion.
Retrovirology. 2006;390.
BACKGROUND: HIV envelope glycoprotein (Env)-mediated fusion is driven by the concerted coalescence of the HIV gp41 N-helical and C-helical regions, which results in the formation of 6 helix bundles. Kinetics of HIV Env-mediated fusion is an important determinant of sensitivity to entry inhibitors and antibodies. However, the parameters that govern the HIV Env fusion cascade have yet to be fully elucidated. We address this issue by comparing the kinetics HIV-1IIIB Env with those mediated by HIV-2 from two strains with different affinities for CD4 and CXCR4. RESULTS: HIV-1 and HIV-2 Env-mediated cell fusion occurred with half times of about 60 and 30 min, respectively. Binding experiments of soluble HIV gp120 proteins to CD4 and co-receptor did not correlate with the differences in kinetics of fusion mediated by the three different HIV Envs. However, escape from inhibition by reagents that block gp120-CD4 binding, CD4-induced CXCR4 binding and 6-helix bundle formation, respectively, indicated large difference between HIV-1 and HIV-2 envelope glycoproteins in their CD4-induced rates of engagement with CXCR4. CONCLUSION: The HIV-2 Env proteins studied here exhibited a significantly reduced window of time between the engagement of gp120 with CD4 and exposure of the CXCR4 binding site on gp120 as compared with HIV-1IIIB Env. The efficiency with which HIV-2 Env undergoes this CD4-induced conformational change is the major cause of the relatively rapid rate of HIV-2 Env mediated-fusion. [Abstract/Link to Full Text]

Kammler S, Otte M, Hauber I, Kjems J, Hauber J, Schaal H
The strength of the HIV-1 3' splice sites affects Rev function.
Retrovirology. 2006;389.
BACKGROUND: The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function. RESULTS: In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression. CONCLUSION: Under the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs. [Abstract/Link to Full Text]

Kondo R, Higuchi M, Takahashi M, Oie M, Tanaka Y, Gejyo F, Fujii M
Human T-cell leukemia virus type 2 Tax protein induces interleukin 2-independent growth in a T-cell line.
Retrovirology. 2006;388.
BACKGROUND: While human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia, HTLV type 2 (HTLV-2) is not associated with this malignancy. Accumulating evidence suggests that Tax, a transforming protein of HTLV-1 or HTLV-2, plays a crucial role in the distinctive pathogenesis of these two infections. We herein examined whether Tax2 by itself has a growth promoting activity in a mouse T-cell line CTLL-2, and compared the activity with that of Tax1. RESULTS: We found that Tax2 converts the cell growth of CTLL-2 from an interleukin(IL)-2-dependent growth into an independent one. Cyclosporine A, an inhibitor of transcription factor NFAT, inhibited the growth of two out of four Tax2-transformed CTLL-2 cells, but it had little effect on two Tax1-transformed cells. While the HTLV-2-transformed human T-cell lines produce a significant amount of IL-2, Tax2-transformed CTLL-2 cells only produced a minimal amount of IL-2. These results thus suggest that NFAT-inducible gene(s) other than IL-2 play a role in the cell growth of Tax2-transformed CTLL-2 cells. CONCLUSION: These results show that HTLV-2 Tax2 by itself has a growth promoting activity toward a T-cell line CTLL-2, and the CTLL-2 assay used in this study may therefore be a useful tool for comparing the activity of Tax2 with that of Tax1 in T-cells, thereby elucidating the mechanism of HTLV-1 specific leukemogenesis. [Abstract/Link to Full Text]

Shao Y
AIDS epidemic at age 25 and control efforts in China.
Retrovirology. 2006;387.
In the first 10 years of AIDS epidemic in China, intravenous drug users (IDUs) and Former Plasma Donors (FPDs) were hardly hit in the late 1980s and mid 1990s respectively. In the last 10 years, while IDU epidemic keeps at a fast pace, sexual transmitted cases of HIV have been steadily increasing. All signs indicate that the HIV epidemic in China is at a turning point, spreading from high risk groups to the general population. Learning from the SARS epidemic, China has recently launched an impressive AIDS campaign by making serious political commitments, and by strengthening the public health system and implementing an aggressive Four Free One Care Policy. There remains huge challenges both at the societal level which form the roots of the AIDS epidemic and at increasing the capabilities of the implementation teams. In addition to other needed efforts, enhancing AIDS research through international collaborations will strengthen China's ability to conduct her huge control program efficiently. Only with a scientific approach and evidence-based strategy, can China seize the opportunity to stop AIDS at an early stage. [Abstract/Link to Full Text]

Lewin SR, Kaldor JM, Cooper DA
HIV research in Australia: linking basic research findings with clinical and public health outcomes.
Retrovirology. 2006;386.
Despite a population of only 20 million and sustained low prevalence of HIV infection in Australia, Australian researchers have provided many substantial original findings to the fields of HIV pathogenesis, treatment and prevention. More recently, Australian clinicians and scientists have turned their attention to assisting other countries in developing effective responses, particularly within the Asia-Pacific region. It is therefore fitting that the 4th International AIDS Society (IAS) Conference on HIV Pathogenesis, Treatment and Prevention will be held in Sydney in July 2007. The meeting is expected to attract over 5000 participants and will have a dynamic and innovative programme within the three major themes of HIV basic science, clinical research and biomedical prevention. [Abstract/Link to Full Text]

Cahn P, McClure C
Beyond the first 25 years: The International AIDS Society and its role in the global response to AIDS.
Retrovirology. 2006;385.
Dr. Pedro Cahn, International AIDS Society (IAS) President and Mr. Craig McClure, IAS Executive Director, provide their thoughts and analysis on the current and future role of the IAS as part of the global response to HIV/AIDS. [Abstract/Link to Full Text]

Baldwin CE, Berkhout B
Second site escape of a T20-dependent HIV-1 variant by a single amino acid change in the CD4 binding region of the envelope glycoprotein.
Retrovirology. 2006;384.
BACKGROUND: We previously described the selection of a T20-dependent human immunodeficiency virus type-1 (HIV-1) variant in a patient on T20 therapy. The fusion inhibitor T20 targets the viral envelope (Env) protein by blocking a conformational switch that is critical for viral entry into the host cell. T20-dependent viral entry is the result of 2 mutations in Env (GIA-SKY), creating a protein that undergoes a premature conformational switch, and the presence of T20 prevents this premature switch and rescues viral entry. In the present study, we performed 6 independent evolution experiments with the T20-dependent HIV-1 variant in the absence of T20, with the aim to identify second site compensatory changes, which may provide new mechanistic insights into Env function and the T20-dependence mechanism. RESULTS: Escape variants with improved replication capacity appeared within 42 days in 5 evolution cultures. Strikingly, 3 cultures revealed the same single amino acid change in the CD4 binding region of Env (glycine at position 431 substituted for arginine: G431R). This mutation was sufficient to abolish the T20-dependence phenotype and restore viral replication in the absence of T20. The GIA-SKY-G431R escape variant produces an Env protein that exhibits reduced syncytia formation and reduced cell-cell fusion activity. The escape variant was more sensitive to an antibody acting on an early gp41 intermediate, suggesting that the G431R mutation helps preserve a pre-fusion Env conformation, similar to T20 action. The escape variant was also less sensitive to soluble CD4, suggesting a reduced CD4 receptor affinity. CONCLUSION: The forced evolution experiments indicate that the premature conformational switch of the T20-dependent HIV-1 Env variant (GIA-SKY) can be corrected by a second site mutation in Env (GIA-SKY-G431R) that affects the interaction with the CD4 receptor. [Abstract/Link to Full Text]

Urcuqui-Inchima S, Castańo ME, Hernandez-Verdun D, St-Laurent G, Kumar A
Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function.
Retrovirology. 2006;383.
BACKGROUND: The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. RESULTS: Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. CONCLUSION: The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv. [Abstract/Link to Full Text]

Zhou X, Vink M, Berkhout B, Das AT
Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control.
Retrovirology. 2006;382.
BACKGROUND: We have previously constructed a doxycycline (dox)-dependent HIV-1 variant by incorporating the Tet-On gene regulatory system into the viral genome. Replication of this HIV-rtTA virus is driven by the dox-inducible transactivator protein rtTA, and can be switched on and off at will. We proposed this conditional-live virus as a novel vaccine approach against HIV-1. Upon vaccination, replication of HIV-rtTA can be temporarily activated by transient dox administration and controlled to the extent needed for optimal induction of the immune system. However, subsequent dox-withdrawal may impose a selection for virus variants with reduced dox-dependence. RESULTS: We simulated this on/off switching of virus replication in multiple, independent cultures and could indeed select for HIV-rtTA variants that replicated without dox. Nearly all evolved variants had acquired a typical amino acid substitution at position 56 in the rtTA protein. We developed a novel rtTA variant that blocks this undesired evolutionary route and thus prevents HIV-rtTA from losing dox-control. CONCLUSION: The loss of dox-control observed upon evolution of the dox-dependent HIV-1 variant was effectively blocked by modification of the Tet-On regulatory system. [Abstract/Link to Full Text]

Sáez-Cirión A, Versmisse P, Truong LX, Chakrabarti LA, Carpentier W, Barré-Sinoussi F, Scott-Algara D, Pancino G
Persistent resistance to HIV-1 infection in CD4 T cells from exposed uninfected Vietnamese individuals is mediated by entry and post-entry blocks.
Retrovirology. 2006;381.
BACKGROUND: We have previously reported that CD4 T cells from some exposed uninfected (EU) Vietnamese intravenous drug users are relatively resistant to HIV infection in vitro. Here, we further characterized the restriction of viral replication in CD4 T cells from five EUs and assessed its persistence in serial samples. RESULTS: CD4 T cells and/or PBMC sampled during a period of between 2 and 6 years were challenged with replication-competent HIV-1 and other retroviral particles pseudotyped with envelope proteins of various tropisms. CCR5 expression and function in resistant CD4 T cells was evaluated. The step at which HIV-1 replication is restricted was investigated by real-time PCR quantification of HIV-1 reverse transcripts.We identified three patterns of durable HIV-1 restriction in EU CD4 T cells. CD4 T cells from four of the five EU subjects were resistant to HIV-1 R5 infection. In two cases this resistance was associated with low CCR5 surface expression, which was itself associated with heterozygous CCR5 mutations. In the other two cases, CD4 T cells were resistant to HIV-1 R5 infection despite normal CCR5 expression and signaling function, and normal beta-chemokine secretion upon CD4 T cell activation. Instead, restriction appeared to be due to enhanced CD4 T cell sensitivity to beta-chemokines in these two subjects. In the fifth EU subject the restriction involved post-entry steps of viral replication and affected not only HIV-1 but also other lentiviruses. The restriction was not overcome by a high viral inoculum, suggesting that it was not mediated by a saturable inhibitory factor. CONCLUSION: Various constitutive mechanisms of CD4 T cell resistance to HIV-1 infection, affecting entry or post-entry steps of viral replication, are associated with resistance to HIV-1 in subjects who remain uninfected despite long-term high-risk behavior. [Abstract/Link to Full Text]

Ludwig LB, Ambrus JL, Krawczyk KA, Sharma S, Brooks S, Hsiao CB, Schwartz SA
Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products.
Retrovirology. 2006;380.
BACKGROUND: While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). RESULTS: Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. CONCLUSION: HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development. [Abstract/Link to Full Text]

Kaumanns P, Hagmann I, Dittmar MT
Human TRIM5alpha mediated restriction of different HIV-1 subtypes and Lv2 sensitive and insensitive HIV-2 variants.
Retrovirology. 2006;379.
In order to characterize the antiviral activity of human TRIM5alpha in more detail human derived indicator cell lines over expressing wild type human TRIM5alpha were generated and challenged with HIV-1 and HIV-2 viruses pseudotyped with HIV envelope proteins in comparison to VSV-G pseudotyped particles. HIV envelope protein pseudotyped particles (HIV-1[NL4.3], HIV-1[BaL]) showed a similar restriction to infection (12 fold inhibition) compared to VSV-G pseudotyped viruses after challenging TZM-huTRIM5alpha cells. For HIV-2 a stronger restriction to infection was observed when the homologous envelope protein Env42S was pseudotyped onto these particles compared to VSV-G pseudotyped HIV-2 particles (8.6 fold inhibition versus 3.4 fold inhibition). It has been shown that HIV-2 is restricted by the restriction factor Lv2, acting on capsid like TRIM5alpha. A mutation of amino acid 73 (I73V) of HIV-2 capsid renders this virus Lv2-insensitive. Lv2-insensitive VSV-G pseudotyped HIV-2/I73V particles showed a similar restriction to infection as did HIV-2[VSV-G] particles (4 fold inhibition). HIV-2 envelope protein (Env42S)-pseudotyped HIV-2/I73V particles revealed a 9.3 fold increase in infection in TZM cells but remained restricted in TZM-huTRIM5alpha cells (80.6 fold inhibition) clearly indicating that at least two restriction factors, TRIM5alpha and Lv2, act on incoming HIV-2 particles. Further challenge experiments using primary isolates from different HIV-1 subtypes and from HIV-1 group O showed that wild type human TRIM5alpha restricted infection independent of coreceptor use of the infecting particle but to variable degrees (between 1.2 and 19.6 fold restriction). [Abstract/Link to Full Text]

Ammosova T, Berro R, Jerebtsova M, Jackson A, Charles S, Klase Z, Southerland W, Gordeuk VR, Kashanchi F, Nekhai S
Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription.
Retrovirology. 2006;378.
BACKGROUND: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription. RESULTS: We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease. CONCLUSION: Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription. [Abstract/Link to Full Text]

Freed EO, Mouland AJ
The cell biology of HIV-1 and other retroviruses.
Retrovirology. 2006;377.
In recognition of the growing influence of cell biology in retrovirus research, we recently organized a Summer conference sponsored by the American Society for Cell Biology (ASCB) on the Cell Biology of HIV-1 and other Retroviruses (July 20-23, 2006, Emory University, Atlanta, Georgia). The meeting brought together a number of leading investigators interested in the interplay between cell biology and retrovirology with an emphasis on presentation of new and unpublished data. The conference was arranged from early to late events in the virus replication cycle, with sessions on viral fusion, entry, and transmission; post-entry restrictions to retroviral infection; nuclear import and integration; gene expression/regulation of retroviral Gag and genomic RNA; and assembly/release. In this review, we will attempt to touch briefly on some of the highlights of the conference, and will emphasize themes and trends that emerged at the meeting. MEETING REPORT: The conference began with a keynote address from W. Sundquist on the biochemistry of HIV-1 budding. This presentation will be described in the section on Assembly and Release of Retroviruses. [Abstract/Link to Full Text]

Berges BK, Wheat WH, Palmer BE, Connick E, Akkina R
HIV-1 infection and CD4 T cell depletion in the humanized Rag2-/-gamma c-/- (RAG-hu) mouse model.
Retrovirology. 2006;376.
BACKGROUND: The currently well-established humanized mouse models, namely the hu-PBL-SCID and SCID-hu systems played an important role in HIV pathogenesis studies. However, despite many notable successes, several limitations still exist. They lack multi-lineage human hematopoiesis and a functional human immune system. These models primarily reflect an acute HIV infection with rapid CD4 T cell loss thus limiting pathogenesis studies to a short-term period. The new humanized Rag2-/-gamma c-/- mouse model (RAG-hu) created by intrahepatic injection of CD34 hematopoietic stem cells sustains long-term multi-lineage human hematopoiesis and is capable of mounting immune responses. Thus, this model shows considerable promise to study long-term in vivo HIV infection and pathogenesis. RESULTS: Here we demonstrate that RAG-hu mice produce human cell types permissive to HIV-1 infection and that they can be productively infected by HIV-1 ex vivo. To assess the capacity of these mice to sustain long-term infection in vivo, they were infected by either X4-tropic or R5-tropic HIV-1. Viral infection was assessed by PCR, co-culture, and in situ hybridization. Our results show that both X4 and R5 viruses are capable of infecting RAG-hu mice and that viremia lasts for at least 30 weeks. Moreover, HIV-1 infection leads to CD4 T cell depletion in peripheral blood and thymus, thus mimicking key aspects of HIV-1 pathogenesis. Additionally, a chimeric HIV-1 NL4-3 virus expressing a GFP reporter, although capable of causing viremia, failed to show CD4 T cell depletion possibly due to attenuation. CONCLUSION: The humanized RAG-hu mouse model, characterized by its capacity for sustained multi-lineage human hematopoiesis and immune response, can support productive HIV-1 infection. Both T cell and macrophage tropic HIV-1 strains can cause persistent infection of RAG-hu mice resulting in CD4 T cell loss. Prolonged viremia in the context of CD4 T cell depletion seen in this model mirrors the main features of HIV infection in the human. Thus, the RAG-hu mouse model of HIV-1 infection shows great promise for future in vivo pathogenesis studies, evaluation of new drug treatments, vaccines and novel gene therapy strategies. [Abstract/Link to Full Text]

Groot F, Kuijpers TW, Berkhout B, de Jong EC
Dendritic cell-mediated HIV-1 transmission to T cells of LAD-1 patients is impaired due to the defect in LFA-1.
Retrovirology. 2006;375.
BACKGROUND: Dendritic cells (DC) have been proposed to mediate sexual HIV-1 transmission by capturing the virus in the mucosa and subsequently presenting it to CD4+ T cells. We have demonstrated before that DC subsets expressing higher levels of intercellular adhesion molecule-1 (ICAM-1) are better HIV-1 transmitters. ICAM-1 binds leukocyte function-associated molecule-1 (LFA-1) on T cells, an integrin responsible for adhesion and signaling at the immunological synapse. To corroborate the importance of the ICAM-1- LFA-1 interaction, we performed transmission experiments to LFA-1 negative leukocytes from Leukocyte Adhesion Deficiency type 1 (LAD-1) patients. RESULTS: We clearly show that DC-mediated HIV-1 transmission to LAD-1 T cells is impaired in comparison to healthy controls. Furthermore, HIV-1 transmission to T cells from a unique LAD-1 patient with a well characterized LFA-1 activation defect was impaired as well, demonstrating that activation of LFA-1 is crucial for efficient transmission. Decreased cell adhesion between DC and LAD-1 T cells could also be illustrated by significantly smaller DC-T cell clusters after HIV-1 transmission. CONCLUSION: By making use of LFA-1 defect cells from unique patients, this study provides more insight into the mechanism of HIV-1 transmission by DC. This may offer new treatment options to reduce sexual transmission of HIV-1. [Abstract/Link to Full Text]

Fassati A
HIV infection of non-dividing cells: a divisive problem.
Retrovirology. 2006;374.
Understanding how lentiviruses can infect terminally differentiated, non-dividing cells has proven a very complex and controversial problem. It is, however, a problem worth investigating, for it is central to HIV-1 transmission and AIDS pathogenesis. Here I shall attempt to summarise what is our current understanding for HIV-1 infection of non-dividing cells. In some cases I shall also attempt to make sense of controversies in the field and advance one or two modest proposals. [Abstract/Link to Full Text]

Zhang H, Hoffmann F, He J, He X, Kankasa C, West JT, Mitchell CD, Ruprecht RM, Orti G, Wood C
Characterization of HIV-1 subtype C envelope glycoproteins from perinatally infected children with different courses of disease.
Retrovirology. 2006;373.
BACKGROUND: The causal mechanisms of differential disease progression in HIV-1 infected children remain poorly defined, and much of the accumulated knowledge comes from studies of subtype B infected individuals. The applicability of such findings to other subtypes, such as subtype C, remains to be substantiated. In this study, we longitudinally characterized the evolution of the Env V1-V5 region from seven subtype C HIV-1 perinatally infected children with different clinical outcomes. We investigated the possible influence of viral genotype and humoral immune response on disease progression in infants. RESULTS: Genetic analyses revealed that rapid progressors (infants that died in the first year of life) received and maintained a genetically homogeneous viral population throughout the disease course. In contrast, slow progressors (infants that remained clinically asymptomatic for up to four years) also exhibited low levels variation initially, but attained higher levels of diversity over time. Genetic assessment of variation, as indicated by dN/dS, showed that particular regions of Env undergo selective changes. Nevertheless, the magnitude and distribution of these changes did not segregate slow and rapid progressors. Longitudinal trends in Env V1-V5 length and the number of potential N-glycosylation sites varied among patients but also failed to discriminate between fast and slow progressors. Viral isolates from rapid progressors and slow progressors displayed no significant growth properties differences in vitro. The neutralizing activity in maternal and infant baseline plasma also varied in its effectiveness against the initial virus from the infants but did not differentiate rapid from slow progressors. Quantification of the neutralization susceptibility of the initial infant viral isolates to maternal baseline plasma indicated that both sensitive and resistant viruses were transmitted, irrespective of disease course. We showed that humoral immunity, whether passively acquired or developed de novo in the infected children, varied but was not predictive of disease progression. CONCLUSION: Our data suggest that neither genetic variation in env, or initial maternal neutralizing activity, or the level of passively acquired neutralizing antibody, or the level of the de novo neutralization response appear to be linked to differences in disease progression in subtype C HIV-1 infected children. [Abstract/Link to Full Text]

Gallo RC
A reflection on HIV/AIDS research after 25 years.
Retrovirology. 2006;372.
Dr. Robert C. Gallo provides a personal reflection on the 25 year history of AIDS. [Abstract/Link to Full Text]

Ishioka K, Higuchi M, Takahashi M, Yoshida S, Oie M, Tanaka Y, Takahashi S, Xie L, Green PL, Fujii M
Inactivation of tumor suppressor Dlg1 augments transformation of a T-cell line induced by human T-cell leukemia virus type 1 Tax protein.
Retrovirology. 2006;371.
BACKGROUND: The interaction of human T-cell leukemia virus type 1 (HTLV-1) Tax1 protein with the tumor suppressor Dlg1 is correlated with cellular transformation. RESULTS: Here, we show that Dlg1 knockdown by RNA interference increases the ability of Tax1 to transform a mouse T-cell line (CTLL-2), as measured interleukin (IL)-2-independent growth. A Tax1 mutant defective for the Dlg1 interaction showed reduced transformation of CTLL-2 compared to wild type Tax1, but the transformation was minimally affected by Dlg1 reduction. The few Tax1DeltaC-transduced CTLL-2 cells that became transformed expressed less Dlg1 than parental cells, suggesting that Dlg1-low cells were selectively transformed by Tax1DeltaC. Moreover, all human T-cell lines immortalized by HTLV-1, including the recombinant HTLV-1-containing Tax1DeltaC, expressed less Dlg1 than control T-cell lines. CONCLUSION: These results suggest that inactivation of Dlg1 augments Tax1-mediated transformation of CTLL-2, and PDZ protein(s) other than Dlg1 are critically involved in the transformation. [Abstract/Link to Full Text]

Lin TY, Emerman M
Cyclophilin A interacts with diverse lentiviral capsids.
Retrovirology. 2006;370.
BACKGROUND: The capsid (CA) protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA). This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay. RESULTS: We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA. CONCLUSION: These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection. [Abstract/Link to Full Text]

Ishida T, Hamano A, Koiwa T, Watanabe T
5' long terminal repeat (LTR)-selective methylation of latently infected HIV-1 provirus that is demethylated by reactivation signals.
Retrovirology. 2006;369.
We previously described selective hypermethylation of the 5'-long terminal repeat (LTR) of HTLV-1 provirus in vivo and in vitro. This prompted us to analyze CpG methylation of the two LTRs of the HIV provirus in chronically infected cell lines. The results demonstrate selective hypermethylation of the 5' LTR of the HIV provirus in ACH-2 cells. Moreover, induction of viral gene expression by TNF-alpha resulted in demethylation of the 5'-LTR. These results suggest that selective epigenetic modification of the 5'LTR of the HIV-1 provirus may be an important mechanism by which proviral activity is suppressed. [Abstract/Link to Full Text]

Scaria V, Hariharan M, Maiti S, Pillai B, Brahmachari SK
Host-virus interaction: a new role for microRNAs.
Retrovirology. 2006;368.
MicroRNAs (miRNAs) are a new class of 18-23 nucleotide long non-coding RNAs that play critical roles in a wide spectrum of biological processes. Recent reports also throw light into the role of microRNAs as critical effectors in the intricate host-pathogen interaction networks. Evidence suggests that both virus and hosts encode microRNAs. The exclusive dependence of viruses on the host cellular machinery for their propagation and survival also make them highly susceptible to the vagaries of the cellular environment like small RNA mediated interference. It also gives the virus an opportunity to fight and/or modulate the host to suite its needs. Thus the range of interactions possible through miRNA-mRNA cross-talk at the host-pathogen interface is large. These interactions can be further fine-tuned in the host by changes in gene expression, mutations and polymorphisms. In the pathogen, the high rate of mutations adds to the complexity of the interaction network. Though evidence regarding microRNA mediated cross-talk in viral infections is just emerging, it offers an immense opportunity not only to understand the intricacies of host-pathogen interactions, and possible explanations to viral tropism, latency and oncogenesis, but also to develop novel biomarkers and therapeutics. [Abstract/Link to Full Text]

Weiss RA
The discovery of endogenous retroviruses.
Retrovirology. 2006;367.
When endogenous retroviruses (ERV) were discovered in the late 1960s, the Mendelian inheritance of retroviral genomes by their hosts was an entirely new concept. Indeed Howard M Temin's DNA provirus hypothesis enunciated in 1964 was not generally accepted, and reverse transcriptase was yet to be discovered. Nonetheless, the evidence that we accrued in the pre-molecular era has stood the test of time, and our hypothesis on ERV, which one reviewer described as 'impossible', proved to be correct. Here I recount some of the key observations in birds and mammals that led to the discovery of ERV, and comment on their evolution, cross-species dispersion, and what remains to be elucidated. [Abstract/Link to Full Text]

---

Recent Articles in Virology Journal

---

Lambert C, Prange R
Posttranslational N-glycosylation of the hepatitis B virus large envelope protein.
Virol J. 2007;445.
BACKGROUND: The addition of N-linked glycans to proteins is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. Here, we report on an exception to this rule occurring on the hepatitis B virus (HBV) large L envelope protein that is a subject to co-plus posttranslational N-glycosylation. RESULTS: By using an improved detection system, we identified so far unrecognized, novel isoforms of L. Based on mutational analyses, the use of N-glycosylation inhibitors, and pulse-chase studies, we showed that these isoforms are due to posttranslational N-glycan addition to the asparagines 4 and 112 within the preS domain of L. While an inhibition of N-glycosylation and glycan trimming profoundly blocked virus assembly and release, the posttranslational N-glycosylation of L itself was found to be dispensable for HBV morphogenesis. CONCLUSION: These data together with previous results implicate that the N-glycosylation requirements of virion release are due to functional inhibition of cell glycoproteins engaged by HBV. [Abstract/Link to Full Text]

Freistadt MS, Vaccaro JA, Eberle KE
Biochemical characterization of the fidelity of poliovirus RNA-dependent RNA polymerase.
Virol J. 2007;444.
BACKGROUND: Putative high mutation rates of RNA viruses are believed to mediate undesirable phenomena, such as emergence of drug resistance. However, very little is known about biochemical fidelity rates for viral RNA-dependent RNA polymerases. Using a recently developed in vitro polymerase assay for poliovirus polymerase 3Dpol [Arnold and Cameron (2000) JBC 275:5329], we measured fidelity for each possible mismatch. Polymerase fidelity, in contrast to sequence error rate, is biochemically defined as kpol/Kd of {(correct plus incorrect) divided by incorrect} incorporations, such that a larger value connotes higher fidelity. RESULTS: To derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1. Pre-steady state measurements for incorrect base incorporation were less straightforward: several anomalous phenomena interfered with data collection. To obtain pre-steady state kinetic data for incorrect base incorporation, three strategies were employed. (1) For some incorrect bases, a conventional approach was feasible, although care was taken to ensure that only single turnovers were being assessed. (2) Heparin or unlabeled RNA traps were used to simulate single turnover conditions. (3) Finally, for some incorrect bases, incorporation was so poor that single datapoints were used to provide kinetic estimates. Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations. CONCLUSION: These values are unexpectedly high showing that high RNA virus sequence variation is not due to intrinsically low polymerase fidelity. Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation. If so, high sequence variation of RNA viruses may be due to template-switch RNA recombination and/or unknown fitness/selection phenomena. These findings may lead to a mechanistic understanding of RNA virus error catastrophe and improved anti-viral strategies. [Abstract/Link to Full Text]

Bragstad K, Jřrgensen PH, Handberg K, Hammer AS, Kabell S, Fomsgaard A
First introduction of highly pathogenic H5N1 avian influenza A viruses in wild and domestic birds in Denmark, Northern Europe.
Virol J. 2007;443.
BACKGROUND: Since 2005 highly pathogenic (HP) avian influenza A H5N1 viruses have spread from Asia to Africa and Europe infecting poultry, humans and wild birds. HP H5N1 virus was isolated in Denmark for the first time in March 2006. A total of 44 wild birds were found positive for the HP H5N1 infection. In addition, one case was reported in a backyard poultry flock. RESULTS: Full-genome characterisation of nine isolates revealed that the Danish H5N1 viruses were highly similar to German H5N1 isolates in all genes from the same time period. The haemagglutinin gene grouped phylogenetically in H5 clade 2 subclade 2 and closest relatives besides the German isolates were isolates from Croatia in 2005, Nigeria and Niger in 2006 and isolates from Astrakhan in Russia 2006. The German and Danish isolates shared unique substitutions in the NA, PB1 and NS2 proteins. CONCLUSION: The first case of HP H5N1 infection of wild and domestic birds in Denmark was experienced in March 2006. This is the first full genome characterisation of HP H5N1 avian influenza A virus in the Nordic countries. The Danish viruses from this time period have their origin from the wild bird strains from Qinghai in 2005. These viruses may have been introduced to the Northern Europe through unusual migration due to the cold weather in Eastern Europe at that time. [Abstract/Link to Full Text]

Kumari K, Gulati S, Smith DF, Gulati U, Cummings RD, Air GM
Receptor binding specificity of recent human H3N2 influenza viruses.
Virol J. 2007;442.
BACKGROUND: Human influenza viruses are known to bind to sialic acid linked alpha2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection. RESULTS: Viruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of alpha2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for alpha2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of alpha2-6 and alpha2-3 sialic acids, including human A549 and HeLa cells with high levels of alpha2-6 sialic acid, and CHO cells that have only alpha2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells. CONCLUSION: Agglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan Array, and may result from increased avidity due to density of hemagglutinin and not increased affinity. Absence of alpha2-6 sialic acid does not protect a cell from influenza infection and the presence of high levels of alpha2-6-sialic acids on a cell surface does not guarantee productive replication of a virus with alpha2-6 receptor specificity. [Abstract/Link to Full Text]

Bennett RS, Ton DR, Hanson CT, Murphy BR, Whitehead SS
Genome sequence analysis of La Crosse virus and in vitro and in vivo phenotypes.
Virol J. 2007;441.
BACKGROUND: La Crosse virus (LACV), family Bunyaviridae, is a mosquito-borne virus recognized as a major cause of pediatric encephalitis in North America with 70-130 symptomatic cases each year. The virus was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl who suffered encephalitis and died in La Crosse, Wisconsin. The majority of LACV infections are mild and never reported, however, serologic studies estimate infection rates of 10-30/100,000 in endemic areas. RESULTS: In the present study, sequence analysis of the complete LACV genomes of low-passage LACV/human/1960, LACV/mosquito/1978, and LACV/human/1978 strains and of biologically cloned derivatives of each strain, indicates that circulating LACVs are genetically stable over time and geographic distance with 99.6-100%, 98.9-100%, 97.8-99.6%, and 99.2-99.7% amino acid identity for N, NsS, M polyprotein, and L proteins respectively. We identified 5 amino acid differences in the RNA polymerase and 4 nucleotide differences in the non-coding region of the L segment specific to the human virus isolates, which may result in altered disease outcomes. CONCLUSION: All three wild type viruses had similar in vitro growth kinetics and phenotypes in mosquito C6/36 and Vero cells, and similar levels of neurovirulence and neuroinvasiveness in Swiss Webster mice. The biologically cloned derivative of LACV/human/1960 was significantly less neuroinvasive than its uncloned parent and differed in sequence at one amino acid position in the GN glycoprotein, identifying this residue as an attenuating mutation. [Abstract/Link to Full Text]

Kistler AL, Webster DR, Rouskin S, Magrini V, Credle JJ, Schnurr DP, Boushey HA, Mardis ER, Li H, DeRisi JL
Genome-wide diversity and selective pressure in the human rhinovirus.
Virol J. 2007;440.
BACKGROUND: The human rhinoviruses (HRV) are one of the most common and diverse respiratory pathogens of humans. Over 100 distinct HRV serotypes are known, yet only 6 genomes are available. Due to the paucity of HRV genome sequence, little is known about the genetic diversity within HRV or the forces driving this diversity. Previous comparative genome sequence analyses indicate that recombination drives diversification in multiple genera of the picornavirus family, yet it remains unclear if this holds for HRV. RESULTS: To resolve this and gain insight into the forces driving diversification in HRV, we generated a representative set of 34 fully sequenced HRVs. Analysis of these genomes shows consistent phylogenies across the genome, conserved non-coding elements, and only limited recombination. However, spikes of genetic diversity at both the nucleotide and amino acid level are detectable within every locus of the genome. Despite this, the HRV genome as a whole is under purifying selective pressure, with islands of diversifying pressure in the VP1, VP2, and VP3 structural genes and two non-structural genes, the 3C protease and 3D polymerase. Mapping diversifying residues in these factors onto available 3-dimensional structures revealed the diversifying capsid residues partition to the external surface of the viral particle in statistically significant proximity to antigenic sites. Diversifying pressure in the pleconaril binding site is confined to a single residue known to confer drug resistance (VP1 191). In contrast, diversifying pressure in the non-structural genes is less clear, mapping both nearby and beyond characterized functional domains of these factors. CONCLUSION: This work provides a foundation for understanding HRV genetic diversity and insight into the underlying biology driving evolution in HRV. It expands our knowledge of the genome sequence space that HRV reference serotypes occupy and how the pattern of genetic diversity across HRV genomes differs from other picornaviruses. It also reveals evidence of diversifying selective pressure in both structural genes known to interact with the host immune system and in domains of unassigned function in the non-structural 3C and 3D genes, raising the possibility that diversification of undiscovered functions in these essential factors may influence HRV fitness and evolution. [Abstract/Link to Full Text]

Di Trani L, Savarino A, Campitelli L, Norelli S, Puzelli S, D'Ostilio D, Vignolo E, Donatelli I, Cassone A
Different pH requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza A viruses.
Virol J. 2007;439.
Chloroquine is a 4-aminoquinoline previously used in malaria therapy and now becoming an emerging investigational antiviral drug due to its broad spectrum of antiviral activities. To explore whether the low pH-dependency of influenza A viruses might affect the antiviral effects of chloroquine at clinically achievable concentrations, we tested the antiviral effects of this drug on selected human and avian viruses belonging to different subtypes and displaying different pH requirements. Results showed a correlation between the responses to chloroquine and NH4Cl, a lysosomotropic agent known to increase the pH of intracellular vesicles. Time-of-addition experiments showed that the inhibitory effect of chloroquine was maximal when the drug had been added at the time of infection and was lost after 2 h post-infection. This timing approximately corresponds to that of virus/cell fusion. Moreover, there was a clear correlation between the EC50 of chloroquine in vitro and the electrostatic potential of the HA subunit (HA2) mediating the virus/cell fusion process. Overall, the present study highlights the critical importance of a host cell factor such as intravesicular pH in determining the anti-influenza activity of chloroquine and other lysosomotropic agents. [Abstract/Link to Full Text]

Lubicz JV, Rush CM, Payton M, Colberg T
Beet necrotic yellow vein virus accumulates inside resting spores and zoosporangia of its vector Polymyxa betae BNYVV infects P. betae.
Virol J. 2007;437.
BACKGROUND: Plasmodiophorids and chytrids are zoosporic parasites of algae and land plant and are distributed worldwide. There are 35 species belonging to the order Plasmodiophorales and three species, Polymyxa betae, P. graminis, and Spongospora subterranea, are plant viral vectors. Plasmodiophorid transmitted viruses are positive strand RNA viruses belonging to five genera. Beet necrotic yellow vein virus (BNYVV) and its vector, P. betae, are the causal agents for rhizomania. RESULTS: Evidence of BNYVV replication and movement proteins associating with P. betae resting spores was initially obtained using immunofluorescence labeling and well characterized antisera to each of the BNYVV proteins. Root cross sections were further examined using immunogold labeling and electron microscopy. BNYVV proteins translated from each of the four genomic and subgenomic RNAs accumulate inside P. betae resting spores and zoospores. Statistical analysis was used to determine if immunolabelling detected viral proteins in specific subcellular domains and at a level greater than in control samples. CONCLUSION: Virus-like particles were detected in zoosporangia. Association of BNYVV replication and movement proteins with sporangial and sporogenic stages of P. betae suggest that BNYVV resides inside its vector during more than one life cycle stage. These data suggest that P. betae might be a host as well as a vector for BNYVV. [Abstract/Link to Full Text]

Rong Q, Huang J, Su E, Li J, Li J, Zhang L, Cao K
Infection of hepatitis B virus in extrahepatic endothelial tissues mediated by endothelial progenitor cells.
Virol J. 2007;436.
BACKGROUND: Hepatitis B virus (HBV) replication has been reported to be involved in many extrahepatic viral disorders; however, the mechanism by which HBV is trans-infected into extrahepatic tissues such as HBV associated myocarditis remains largely unknown. RESULTS: In this study, we showed that human cord blood endothelial progenitor cells (EPCs), but not human umbilical vein endothelial cells (HUVECs) could be effectively infected by uptake of HBV in vitro. Exposure of EPCs with HBV resulted in HBV DNA and viral particles were detected in EPCs at day 3 after HBV challenge, which were peaked around day 7 and declined in 3 weeks. Consistently, HBV envelope surface and core antigens were first detected in EPCs at day 3 after virus challenge and were retained to be detectable for 3 weeks. In contrast, HBV covalently closed circular DNA was not detected in EPCs at any time after virus challenge. Intravenous transplantation of HBV-treated EPCs into myocardial infarction and acute renal ischemia mouse model resulted in incorporation of HBV into injured heart, lung, and renal capillary endothelial tissues. CONCLUSION: These results strongly support that EPCs serve as virus carrier mediating HBV trans-infection into the injured endothelial tissues. The findings might provide a novel mechanism for HBV-associated myocarditis and other HBV-related extrahepatic diseases as well. [Abstract/Link to Full Text]

Fournier C, Duverlie G, François C, Schnuriger A, Dedeurwaerder S, Brochot E, Capron D, Wychowski C, Thibault V, Castelain S
A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies.
Virol J. 2007;435.
BACKGROUND/AIM: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. METHODS: The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. RESULTS: The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively). The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134) for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85) for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles system. CONCLUSION: This study presents a simple, specific and reproducible cell culture-based assay for determination of HCV-neutralizing antibodies in human sera. The assay should be an important tool for gauging the relationship between the neutralizing antibodies response and viral load kinetics in acutely or chronically infected patients and for investigating the possible eradication or prevention of HCV infection by neutralizing antibodies. [Abstract/Link to Full Text]

Kibenge FS, Xu H, Kibenge MJ, Qian B, Joseph T
Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus.
Virol J. 2007;434.
BACKGROUND: Infectious salmon anaemia (ISA) virus (ISAV), an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE) protein encoded on segment 6 and fusion (F) protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs) of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV. RESULTS: In the present study we show that ISAV segment 7 encodes 3 proteins with estimated molecular masses of 32, 18, and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on removal of an intron each from the primary transcript (7-ORF1) so that the translation continues in the +2 and +3 reading frames, respectively. The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids. This antiserum detected a single 35-kDa protein in Western blots of purified virus, and immunoprecipitated a 32-kDa protein in ISAV-infected TO cells. Immunofluorescence staining of infected cells with the same antiserum revealed the protein(s) to be localized in the cytoplasm. Vaccination of farmed Atlantic salmon with the 32-kDa protein resulted in a higher survival rate than what was attainable with the HE protein, albeit a moderate protection against the low ISAV challenge. CONCLUSION: Collectively, our observations suggest that the product of ISAV segment 7 primary transcript (7-ORF1) is a structural protein. The 18-kDa (7-ORF1/2) protein is identified as the putative ISAV nuclear export protein based on the presence of nuclear export signals. The function of the 9.5-kDa (7-ORF1/3) protein is not presently known. [Abstract/Link to Full Text]

Angulo M, Carvajal-Rodríguez A
Evidence of recombination within human alpha-papillomavirus.
Virol J. 2007;433.
BACKGROUND: Human papillomavirus (HPV) has a causal role in cervical cancer with almost half a million new cases occurring each year. Presence of the carcinogenic HPV is necessary for the development of the invasive carcinoma of the genital tract. Therefore, persistent infection with carcinogenic HPV causes virtually all cervical cancers. Some aspects of the molecular evolution of this virus, as the putative importance of recombination in its evolutionary history, are an opened current question. In addition, recombination could also be a significant issue nowadays since the frequency of co-infection with more than one HPV type is not a rare event and, thus, new recombinant types could be currently being generated. RESULTS: We have used human alpha-PV sequences from the public database at Los Alamos National Laboratory to report evidence that recombination may exist in this virus. A model-based population genetic approach was used to infer the recombination signal from the HPV DNA sequences grouped attending to phylogenetic and epidemiological information, as well as to clinical manifestations. Our results agree with recently published ones that use a different methodology to detect recombination associated to the gene L2. In addition, we have detected significant recombination signal in the genes E6, E7, L2 and L1 at different groups, and importantly within the high-risk type HPV16. The method used has recently been shown to be one of the most powerful and reliable procedures to detect the recombination signal. CONCLUSION: We provide new support to the recent evidence of recombination in HPV. Additionally, we performed the recombination estimation assuming the best-fit model of nucleotide substitution and rate variation among sites, of the HPV DNA sequence sets. We found that the gene with recombination in most of the groups is L2 but the highest values were detected in L1 and E6. Gene E7 was recombinant only within the HPV16 type. The topic deserves further study because recombination is an important evolutionary mechanism that could have high impact both in pharmacogenomics (i.e. on the influence of genetic variation on the response to drugs) and for vaccine development. [Abstract/Link to Full Text]

He Z, Zhuang H, Zhao C, Dong Q, Peng G, Dwyer DE
Using patient-collected clinical samples and sera to detect and quantify the severe acute respiratory syndrome coronavirus (SARS-CoV).
Virol J. 2007;432.
BACKGROUND: Severe acute respiratory syndrome (SARS) caused a large outbreak of pneumonia in Beijing, China, in 2003. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect and quantify SARS-CoV in 934 sera and self-collected throat washes and fecal samples from 271 patients with laboratory-confirmed SARS managed at a single institution. RESULTS: SARS-CoV detection rates in sera were highest in the first 9 days of illness, whereas detection was highest in throat washes 5-14 days after onset of symptoms. The highest SARS-CoV RT-PCR rates (70.4-86.3%) and viral loads (log10 4.5-6.1) were seen in fecal samples collected 2-4 weeks after the onset of clinical illness. Fecal samples were frequently SARS-CoV RT-PCR positive beyond 40 days, and occasional sera still had SARS-CoV detected after 3 weeks of illness. CONCLUSION: In the context of an extensive outbreak with major pressure on hospital resources, patient self-collected samples are an alternative to nasopharyngeal aspirates for laboratory confirmation of SARS-CoV infection. [Abstract/Link to Full Text]

Lorrot M, Vasseur M
How do the rotavirus NSP4 and bacterial enterotoxins lead differently to diarrhea?
Virol J. 2007;431.
Rotavirus is the major cause of infantile gastroenteritis and each year causes 611,000 deaths worldwide. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur with no visible tissue damage and, conversely, the histological lesions can be asymptomatic. Rotavirus impairs activities of intestinal disaccharidases and Na+-solute symports coupled with water transport. Maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption can lead to a malabsorption component of diarrhea. Since the discovery of the NSP4 enterotoxin, diverse hypotheses have been proposed in favor of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of diarrhea, but the mechanisms appear to be quite different from those used by bacterial enterotoxins that cause pure secretory diarrhea. Rotavirus failed to stimulate Cl- secretion in crypt, whereas it stimulated Cl- reabsorption in villi, questioning, therefore, the origin of net Cl- secretion. A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection. Also, the overall chloride secretory response is regulated by a phospholipase C-dependent calcium signaling pathway induced by NSP4. However, the overall response is weak, suggesting that NSP4 may exert both secretory and subsequent anti-secretory actions, as did carbachol, hence limiting Cl- secretion. All these characteristics provide the means to make the necessary functional distinction between viral NSP4 and bacterial enterotoxins. [Abstract/Link to Full Text]

Serwer P
Evolution and the complexity of bacteriophages.
Virol J. 2007;430.
BACKGROUND: The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. HYPOTHESIS: Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. TESTING THE HYPOTHESIS: I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the presence of cellular gene homologs, (3) determine which, if any, bacteriophage genes were selected for maintaining the homologs and (4) determine the dynamics of homolog evolution. IMPLICATIONS OF THE HYPOTHESIS: This hypothesis is an explanation of evolutionary leaps in general. If accurate, it will assist both understanding and influencing the evolution of microbes and their communities. Analysis of evolutionary complexity increase for at least prokaryotes should include analysis of genomes of long-genome bacteriophages. [Abstract/Link to Full Text]

Sallie R
Replicative homeostasis III: implications for antiviral therapy and mechanisms of response and non-response.
Virol J. 2007;429.
While improved drug regimens have greatly enhanced outcomes for patients with chronic viral infection, antiviral therapy is still not ideal due to drug toxicities, treatment costs, primary drug failure and emergent resistance. New antiviral agents, alternative treatment strategies and a better understanding of viral pathobiology, host responses and drug action are desperately needed. Interferon (IFN) and ribavirin, are effective drugs used to treat hepatitis C (HCV), but the mechanism(s) of their action are uncertain. Error catastrophe (EC), or precipitous loss of replicative fitness caused by genomic mutation, is postulated to mediate ribavirin action, but is a deeply flawed hypothesis lacking empirical confirmation. Paradoxically ribavirin, a proven RNA mutagen, has no impact on HCV viraemia long term, suggesting real viruses, replicating in-vitro, as opposed to mathematical models, replicating in-silico, are likely to resist EC by highly selective replication of fit (~consensus sequence) genomes mediated, in part, by replicative homeostasis (RH), an epicyclic mechanism that dynamically links RNApol fidelity and processivity and other viral protein functions. Replicative homeostasis provides a rational explanation for the various responses seen during treatment of HCV, including genotype-specific and viral load-dependent differential response rates, as well as otherwise unexplained phenomena like the transient inhibition and rebound of HCV viraemia seen during ribavirin monotherapy. Replicative homeostasis also suggests a primarily non-immunological mechanism that mediates increased immune responsiveness during treatment with ribavirin (and other nucleos(t)ide analogues), explicating the enhanced second-phase clearance of HCV ribavirin promotes and, thus, the apparent immunomodulatory action of ribavirin. More importantly, RH suggests specific new antiviral therapeutic strategies. [Abstract/Link to Full Text]

Freistadt MS, Eberle KE
Conserved aspartic acid 233 and alanine 231 are not required for poliovirus polymerase function in replicons.
Virol J. 2007;428.
Nucleic acid polymerases have similar structures and motifs. The function of an aspartic acid (conserved in all classes of nucleic acid polymerases) in motif A remains poorly understood in RNA-dependent RNA polymerases. We mutated this residue to alanine in a poliovirus replicon. The resulting mutant could still replicate, although at a reduced level. In addition, mutation A231C (also in motif A) yielded high levels of replication. Taken together these results show that poliovirus polymerase conserved residues D233 and A231 are not essential to poliovirus replicon function. [Abstract/Link to Full Text]

Mistry N, Simonsson M, Evander M
Transcriptional activation of the human papillomavirus type 5 and 16 long control region in cells from cutaneous and mucosal origin.
Virol J. 2007;427.
Human papillomavirus type-16 (HPV-16) infects mucosal epithelium and is the most common type found in cervical cancer. HPV-5 infects cornified epithelium and is the most common type found on normal skin and belongs to the types frequently associated with skin cancers of Epidermodysplasia verruciformis patients. One factor by which this anatomical tropism could be determined is the regulation of HPV gene expression in the host cell. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. Since HPV-16 and other types infecting the mucosa differ in host cell from HPV types infecting skin, it has been hypothesized that it is the combination of ubiquitous transcription factors working in concert in the host cell that determines the cell-type-specific expression. To study if HPV tropism could be determined by differences in transcriptional regulation we have cloned the transcriptional regulating region, LCR, from HPV-16 and HPV-5 and studied the activation of a reporter gene in cell lines with different origin. To analyse promoter activity we transfected the plasmids into four different cell lines; HaCaT, C33A, NIKS and W12E and the efficiency of HPV-5 and HPV-16 LCR in the different cell lines was compared. In HaCaT cells, with a skin origin, the HPV-5 LCR was two-fold more efficient in transcriptional activation compared to the HPV-16 LCR. In cervical W12E cells the HPV-16 LCR was almost 2-fold more effective in activating transcription compared to the HPV-5 LCR. The ability to initiate transcription in the other cell lines was independent on cell origin and HPV-type. [Abstract/Link to Full Text]

Woolf NK, Jaquish DV, Koehrn FJ
Transplacental murine cytomegalovirus infection in the brain of SCID mice.
Virol J. 2007;426.
BACKGROUND: Congenital cytomegalovirus (CMV) infection is the most common congenital viral infection in humans and the major nonhereditary cause of central nervous system (CNS) developmental disorders. Previous attempts to develop a murine CMV (MCMV) model of natural congenital human CMV (HCMV) infection have failed because MCMV does not cross the placenta in immunocompetent mice. RESULTS: In marked contrast with immunocompetent mice, C.B-17 SCID (severe combined immunodeficient) mice were found to be highly susceptible to natural MCMV transplacental transmission and congenital infection. Timed-pregnant SCID mice were intraperitoneally (IP) injected with MCMV at embryonic (E) stages E0-E7, and vertical MCMV transmission was evaluated using nested polymerase chain reaction (nPCR), in situ hybridization (ISH) and immunohistochemical (IHC) assays. SCID mouse dams IP injected at E0 with 102 PFU of MCMV died or resorbed their fetuses by E18. Viable fetuses collected at E18 from SCID mice IP injected with 102-104 PFU of MCMV at E7 did not demonstrate vertical MCMV transmission. Notably, transplacental MCMV transmission was confirmed in E18 fetuses from SCID mice IP injected with 103 PFU of MCMV at stages E3-E5. The maximum rate of transplacental MCMV transmission (53%) at E18 occurred when SCID mouse dams were IP injected with 103 PFU of MCMV at E4. Congenital infection was confirmed by IHC immunostaining of MCMV antigens in 26% of the MCMV nPCR positive E18 fetuses. Transplacental MCMV transmission was associated with intrauterine growth retardation and microcephaly. Additionally, E18 fetuses with MCMV nPCR positive brains had cerebral interleukin-1alpha (IL-1alpha) expression significantly upregulated and cerebral IL-1 receptor II (IL-1RII) transcription significantly downregulated. However, MCMV-induced changes in cerebral cytokine expression were not associated with any histological signs of MCMV infection or inflammation in the brain. CONCLUSION: Severe T- and B-cell immunodeficiencies in SCID mice significantly enhance the rate of natural MCMV transplacental transmission and congenital infection. During gestation MCMV exhibits a tissue tropism for the developing brain, and vertical MCMV transmission is correlated with fetal growth retardation and abnormal cerebral proinflammatory cytokine expression. These data confirm that natural vertical MCMV infection in SCID mice constitutes a useful new experimental rodent model of congenital HCMV infection. [Abstract/Link to Full Text]

van Hemert FJ, Lukashov VV, Berkhout B
Different rates of (non-)synonymous mutations in astrovirus genes; correlation with gene function.
Virol J. 2007;425.
BACKGROUND: Complete genome sequences of the Astroviridae include human, non-human mammalian and avian species. A consensus topology of astroviruses has been derived from nucleotide substitutions in the full-length genomes and from non-synonymous nucleotide substitutions in each of the three ORFs. Analyses of synonymous substitutions displayed a loss of tree structure, suggesting either saturation of the substitution model or a deviant pattern of synonymous substitutions in certain virus species. RESULTS: We analyzed the complete Astroviridae family for the inference of adaptive molecular evolution at sites and in branches. High rates of synonymous mutations are observed among the non-human virus species. Deviant patterns of synonymous substitutions are found in the capsid structural genes. Purifying selection is a dominant force among all astrovirus genes and only few codon sites showed values for the dN/dS ratio that may indicate site-specific molecular adaptation during virus evolution. One of these sites is the glycine residue of a RGD motif in ORF2 of human astrovirus serotype 1. RGD or similar integrin recognition motifs are present in nearly all astrovirus species. CONCLUSION: Phylogenetic analysis directed by maximum likelihood approximation allows the inclusion of significantly more evolutionary history and thereby, improves the estimation of dN and dS. Sites with enhanced values for dN/dS are prominent at domains in charge of environmental communication (f.i. VP27 and domain 4 in ORF1a) more than at domains dedicated to intrinsic virus functions (f.i. VP34 and ORF1b (the virus polymerase)). Integrin recognition may play a key role in astrovirus to target cell attachment. [Abstract/Link to Full Text]

Grinde B, Gayorfar M, Hoddevik G
Modulation of gene expression in a human cell line caused by poliovirus, vaccinia virus and interferon.
Virol J. 2007;424.
BACKGROUND: The project was initiated to describe the response of a human embryonic fibroblast cell line to the replication of two different viruses, and, more specifically, to look for candidate genes involved in viral defense. For this purpose, the cells were synchronously infected with poliovirus in the absence or presence of interferon-alpha, or with vaccinia virus, a virus that is not inhibited by interferon. By comparing the changes in transcriptosome due to these different challenges, it should be possible to suggest genes that might be involved in defense. RESULTS: The viral titers were sufficient to yield productive infection in a majority of the cells. The cells were harvested in triplicate at various time-points, and the transcriptosome compared with mock infected cells using oligo-based, global 35 k microarrays. While there was very limited similarities in the response to the different viruses, a large proportion of the genes up-regulated by interferon-alpha were also up-regulated by poliovirus. Interferon-alpha inhibited poliovirus replication, but there were no signs of any interferons being induced by poliovirus. The observations suggest that the cells do launch an antiviral response to poliovirus in the absence of interferon. Analyses of the data led to a list of candidate antiviral genes. Functional information was limited, or absent, for most of the candidate genes. CONCLUSION: The data are relevant for our understanding of how the cells respond to poliovirus and vaccinia virus infection. More annotations, and more microarray studies with related viruses, are required in order to narrow the list of putative defence-related genes. [Abstract/Link to Full Text]

Blaney JE, Sathe NS, Hanson CT, Firestone CY, Murphy BR, Whitehead SS
Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the structural genes of rDEN4 and rDEN4Delta30 with those of DEN1.
Virol J. 2007;423.
BACKGROUND: Antigenic chimeric viruses have previously been generated in which the structural genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2/4Delta30(ME) and rDEN3/4Delta30(ME), which contain the membrane (M) precursor and envelope (E) genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Delta30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Delta30(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME structural genes of DEN1 Puerto Rico/94 strain. RESULTS: Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN1/4(CME), rDEN1/4Delta30(CME), rDEN1/4(ME), and rDEN1/4Delta30(ME). With the exception of rDEN1/4(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the Delta30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1/4Delta30(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1/4Delta30(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the Delta30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1/4Delta30(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal. CONCLUSION: The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4Delta30(ME) should be considered for evaluation in a clinical trial. [Abstract/Link to Full Text]

Hayasaka D, Ennis FA, Terajima M
Pathogeneses of respiratory infections with virulent and attenuated vaccinia viruses.
Virol J. 2007;422.
BACKGROUND: Respiratory infection with the neurovirulent vaccinia virus (VV) strain Western Reserve (WR) results in an acute infection of the lung followed by dissemination of the virus to other organs and causes lethality in mice. The mechanisms of lethality are not well-understood. In this study, we analyzed virus replication and host immune responses after intranasal infection with lethal and non-lethal doses of VV using the WR strain and the less virulent Wyeth strain. RESULTS: The WR strain replicated more vigorously in the lung and in the brain than the Wyeth strain. There were, however, no differences between the virus titers in the brains of mice infected with the higher lethal dose and the lower non-lethal dose of WR strain, suggesting that the amount of virus replication in the brain is unlikely to be the sole determining factor of lethality. The WR strain grew better in primary mouse lung cells than the Wyeth strain. Lethal infection with WR strain was associated with a reduced number of lymphocytes and an altered phenotype of the T cells in the lung compared to non-lethal infections with the WR or Wyeth strains. Severe thymus atrophy with a reduction of CD4 and CD8 double positive T cells was also observed in the lethal infection. CONCLUSION: These results suggest that the lethality induced by intranasal infection with a high dose of the WR strain is caused by the higher replication of virus in lung cells and immune suppression during the early phase of the infection, resulting in uncontrolled virus replication in the lung. [Abstract/Link to Full Text]

Serwer P, Hayes SJ, Thomas JA, Hardies SC
Propagating the missing bacteriophages: a large bacteriophage in a new class.
Virol J. 2007;421.
The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly). As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 x 26 nm), corkscrew-like tail fibers (187 x 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305phi8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage), has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305phi8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305phi8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305phi8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion. [Abstract/Link to Full Text]

Chu VC, McElroy LJ, Aronson JM, Oura TJ, Harbison CE, Bauman BE, Whittaker GR
Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus.
Virol J. 2007;420.
BACKGROUND: Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS), and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV). Feline aminopeptidase N (fAPN) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV), canine coronavirus, transmissible gastroenteritis virus (TGEV), and human coronavirus 229E (HCoV-229E). A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047-2056. RESULTS: Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41), but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 (<0.01% efficiency), but this level of infection is not increased by fAPN expression. CONCLUSION: We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation. [Abstract/Link to Full Text]

Hindmarsh PL, Laimins LA
Mechanisms regulating expression of the HPV 31 L1 and L2 capsid proteins and pseudovirion entry.
Virol J. 2007;419.
Human papillomaviruses (HPV) infect stratified epithelia and restrict expression of late capsid genes to highly differentiated cells. In order to begin to understand the processes regulating HPV 31 infection we examined the synthesis of the HPV 31 capsid proteins, L1 and L2, using heterologous expression systems. Similar to studies in HPV 16, expression of wild type HPV 31 L1 and L2 from heterologous promoters resulted in very low levels of synthesis. In contrast, modification of the codons in the capsid genes to ones more commonly used in cellular genes resulted in high-level synthesis. Through the use of chimeric proteins that fused fragments of wild type L1 to Green Fluorescent Protein (GFP) coding sequences, a short region was identified that was sufficient to inhibit high level synthesis and similar elements were detected in L2. One element was localized to the 3' end of the L1 gene while a series of elements were localized at the 3' end of the L2 coding sequences. These observations are most consistent with negative RNA regulatory elements controlling the levels of L1 and L2 synthesis that are distinct from those identified in HPV 16. Expression vectors for the codon modified HPV 31 capsid proteins were then transfected together with GFP reporter plasmids to generate HPV 31 pseudoviruses. Infection of cells with HPV 31 pseudoviruses in the presence of the inhibitors, chlorpromazine, nystatin or methyl-beta-cyclodextrin, demonstrated that HPV 31, like HPV 16, enters human and monkey cells through a clathrin-mediated pathway rather than through caveolae as previously reported. This suggests that high-risk HPV types may enter cells through common mechanisms. [Abstract/Link to Full Text]

de la Cruz-Hernández E, Pérez-Cárdenas E, Contreras-Paredes A, Cantú D, Mohar A, Lizano M, Dueńas-González A
The effects of DNA methylation and histone deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study.
Virol J. 2007;418.
BACKGROUND: The methylation status at the human papilloma virus (HPV) genome found in pre-invasive and invasive cervical lesions suggests that neoplastic transformation can be suppressed by gene hypermethylation, whereas hypomethylation accompanies or causes cancer progression; hence, epigenetic therapy aimed at reactivating cellular suppressor-gene expression has the potential to act as a tumor promoter by enhancing HPV oncoprotein expression in HPV-related malignancies. The objective of this study was to determine the influence of hydralazine and valproate on HPV oncogene expression in cervical cancer cell lines and the primary tumors of patients undergoing treatment with hydralazine and valproate. RESULTS: Overall, hydralazine and valproate either alone or combined exerted a growth inhibitory effect on cervical cancer cell lines. A cell line-specific up-regulating effect was observed on E6/E7 gene expression, which in general correlated with DNA hypomethylation and histone acetylation at the long control region (LCR). Nonetheless, E6/E7 expression was unchanged or decreased in the majority of patients with cervical cancer treated with hydralazine, valproate, or both. In some cervical cancer cell lines, these drugs led to increased transcription of p53, and increased its stabilization due to acetylation at lysines 273 and 282, which allowed a higher bax-protein transactivating effect. CONCLUSION: The results of this study demonstrate that hydralazine and valproate can be safely administered to HPV-related malignancies such as cervical cancer because they do not increase viral oncoprotein expression. Most importantly, the antitumor effect of hydralazine and valproate in cervical cancer may at least partially depend on an up-regulating effect on p53 gene and on the valproate-induced hyperacetylation of p53 protein, protecting it from degradation by E6. [Abstract/Link to Full Text]

Zhang F, Ma W, Zhang L, Aasa-Chapman M, Zhang H
Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line.
Virol J. 2007;417.
BACKGROUND: Japanese encephalitis virus (JEV), a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E) is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system. RESULTS: The coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line. CONCLUSION: The Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV. [Abstract/Link to Full Text]

Zekri AR, El-Din HM, Bahnassy AA, Khaled MM, Omar A, Fouad I, El-Hefnewi M, Thakeb F, El-Awady M
Genetic distance and heterogenecity between quasispecies is a critical predictor to IFN response in Egyptian patients with HCV genotype-4.
Virol J. 2007;416.
BACKGROUND: HCV is one of the major health problems in Egypt, where it is highly prevalent. Genotype 4 is the most common genotype of HCV and its response to treatment is still a controversy. METHODS: HCV genotype 4 quasispecies diversity within the 5' untranslated region (5'UTR) was studied in a series of 22 native Egyptian patients with chronic hepatitis C virus with no previous treatment who satisfied all NIH criteria for combined treatment of pegylated IFN and ribavirine and was correlated with the outcome of treatment. The study also included 7 control patients with no antiviral treatment. HCV sequencing was done using the TRUGENE HCV 5-NC genotyping kit. RESULTS: At the 48th week of treatment, 15 patients (68%) showed virological response. Whereas HCV-RNA was still detected in 7 patients (32%) in this period; of those, 6 experienced a partial virological response followed by viral breakthrough during treatment. Only one patient did not show any virological or chemical response. The four females included in this study were all responders. There was a significant correlation between the response rate and lower fibrosis (p = 0.026) as well as the total number of mutation spots (including all the insertions, deletions, transitions and transversions) (p = 0.007, p = 0.035). CONCLUSION: Patients who responded to interferon treatment had statistically significant less number in both transitions (p = 0.007) and the genetic distances between the quasispecies (p = 0.035). So, viral genetic complexity and variability may play a role in the response to IFN treatment. The consensus alignment of all three groups revealed no characteristic pattern among the three groups. However, the G to A transitions at 160 was observed among non responders who need further study to confirm this observation. [Abstract/Link to Full Text]

Kareem KT, Taiwo MA
Interactions of viruses in Cowpea: effects on growth and yield parameters.
Virol J. 2007;415.
The study was carried out to investigate the effects of inoculating three cowpea cultivars: "OLO II", "OLOYIN" and IT86D-719 with three unrelated viruses: Cowpea aphid-borne mosaic virus (CABMV), genus Potyvirus, Cowpea mottle virus (CMeV), genus Carmovirus and Southern bean mosaic virus (SBMV), genus Sobemovirus singly and in mixture on growth and yield of cultivars at 10 and 30 days after planting (DAP). Generally, the growth and yield of the buffer inoculated control plants were significantly higher than those of the virus inoculated plants. Inoculation of plants at an early age of 10 DAP resulted in more severe effect than inoculations at a later stage of 30 DAP. The average values of plant height and number of leaves produced by plants inoculated 30 DAP were higher than those produced by plants inoculated 10 DAP. Most of the plants inoculated 10 DAP died and did not produce seeds. However, " OLOYIN" cultivar was most tolerant and produced reasonable yields when infected 30 DAP. The effect of single viruses on growth and yield of cultivars showed that CABMV caused more severe effects in IT86D-719, SBMV had the greatest effect on "OLO II" while CMeV induced the greatest effect on "OLOYIN". Yield was greatly reduced in double infections involving CABMV in combination with either CMeV or SBMV in "OLOYIN" and "OLO II", however, there was complete loss in yield of IT86D-719. Triple infection led to complete yield loss in all the three cultivars. [Abstract/Link to Full Text]

---

Recent Articles in Microbial Cell Factories

---

Glenting J, Wessels S
Ensuring safety of DNA vaccines.
Microb Cell Fact. 2005 Sep 6;426.
In 1990 a new approach for vaccination was invented involving injection of plasmid DNA in vivo, which elicits an immune response to the encoded protein. DNA vaccination can overcome most disadvantages of conventional vaccine strategies and has potential for vaccines of the future. However, today 15 years on, a commercial product still has not reached the market. One possible explanation could be the technique's failure to induce an efficient immune response in humans, but safety may also be a fundamental issue. This review focuses on the safety of the genetic elements of DNA vaccines and on the safety of the microbial host for the production of plasmid DNA. We also propose candidates for the vaccine's genetic elements and for its microbial production host that can heighten the vaccine's safety and facilitate its entry to the market. [Abstract/Link to Full Text]

Warnecke T, Gill RT
Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications.
Microb Cell Fact. 2005 Aug 25;425.
Organic acids are valuable platform chemicals for future biorefining applications. Such applications involve the conversion of low-cost renewable resources to platform sugars, which are then converted to platform chemicals by fermentation and further derivatized to large-volume chemicals through conventional catalytic routes. Organic acids are toxic to many of the microorganisms, such as Escherichia coli, proposed to serve as biorefining platform hosts at concentrations well below what is required for economical production. The toxicity is two-fold including not only pH based growth inhibition but also anion-specific effects on metabolism that also affect growth. E. coli maintain viability at very low pH through several different tolerance mechanisms including but not limited to the use of decarboxylation reactions that consume protons, ion transporters that remove protons, increased expression of known stress genes, and changing membrane composition. The focus of this mini-review is on organic acid toxicity and associated tolerance mechanisms as well as several examples of successful organic acid production processes for E. coli. [Abstract/Link to Full Text]

Qureshi N, Annous BA, Ezeji TC, Karcher P, Maddox IS
Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reaction rates.
Microb Cell Fact. 2005 Aug 25;424.
This article describes the use of biofilm reactors for the production of various chemicals by fermentation and wastewater treatment. Biofilm formation is a natural process where microbial cells attach to the support (adsorbent) or form flocs/aggregates (also called granules) without use of chemicals and form thick layers of cells known as "biofilms." As a result of biofilm formation, cell densities in the reactor increase and cell concentrations as high as 74 gL(-1) can be achieved. The reactor configurations can be as simple as a batch reactor, continuous stirred tank reactor (CSTR), packed bed reactor (PBR), fluidized bed reactor (FBR), airlift reactor (ALR), upflow anaerobic sludge blanket (UASB) reactor, or any other suitable configuration. In UASB granular biofilm particles are used. This article demonstrates that reactor productivities in these reactors have been superior to any other reactor types. This article describes production of ethanol, butanol, lactic acid, acetic acid/vinegar, succinic acid, and fumaric acid in addition to wastewater treatment in the biofilm reactors. As the title suggests, biofilm reactors have high potential to be employed in biotechnology/bioconversion industry for viable economic reasons. In this article, various reactor types have been compared for the above bioconversion processes. [Abstract/Link to Full Text]

Ignatova Z
Monitoring protein stability in vivo.
Microb Cell Fact. 2005 Aug 24;423.
Reduced protein stability in vivo is a prerequisite to aggregation. While this is merely a nuisance factor in recombinant protein production, it holds a serious impact for man. This review focuses on specific approaches to selectively determine the solubility and/or stability of a target protein within the complex cellular environment using different detection techniques. Noninvasive techniques mapping folding/misfolding events on a fast time scale can be used to unravel the complexity and dynamics of the protein aggregation process and factors altering protein solubility in vivo. The development of approaches to screen for folding and solubility in vivo should facilitate the identification of potential components that improve protein solubility and/or modulate misfolding and aggregation and may provide a therapeutic benefit. [Abstract/Link to Full Text]

Orchard SS, Goodrich-Blair H
An encoded N-terminal extension results in low levels of heterologous protein production in Escherichia coli.
Microb Cell Fact. 2005 Jul 21;422.
BACKGROUND: The tdk gene (encoding deoxythymidine kinase) of the gamma-proteobacterium Xenorhabdus nematophila has two potential translation start sites. The promoter-distal start site was predicted to be functional based on amino acid sequence alignment with closely related Tdk proteins. However, to experimentally determine if either of the two possible start codons allows production of a functional Tdk, we expressed the "long-form" (using the promoter-proximal start codon) and "short-form" (using the promoter-distal start codon) X. nematophila tdk genes from the T7 promoter of the pET-28a(+) vector. We assessed Tdk production and activity using a functional assay in an Escherichia coli tdk mutant, which, since it lacks functional Tdk, is able to grow in 5-fluorodeoxyuridine (FUdR)-containing medium. RESULTS: Short-form Tdk complemented the E. coli tdk mutant strain, resulting in FUdR sensitivity of the strain. However, the E. coli tdk mutant expressing the long form of tdk remained FUdR resistant, indicating it did not have a functional deoxythymidine kinase enzyme. We report that long-form Tdk is at least 13-fold less abundant than short-form Tdk, the limited protein produced was as stable as short-form Tdk and the long-form transcript was 1.7-fold less abundant than short-form transcript. Additionally, we report that the long-form extension was sufficient to decrease heterologous production of a different X. nematophila protein, NilC. CONCLUSION: We conclude that the difference in the FUdR growth phenotype between the E. coli tdk mutant carrying the long-or short-form X. nematophila tdk is due to a difference in Tdk levels. The lower long-form protein level does not result from protein instability, but instead from reduced transcript levels possibly combined with reduced translation efficiency. Because the observed effect of the encoded N-terminal extension is not specific to Tdk production and can be overcome with induction of gene expression, these results may have particular relevance to researchers attempting to limit production of toxic proteins under non-inducing conditions. [Abstract/Link to Full Text]

Büssow K, Scheich C, Sievert V, Harttig U, Schultz J, Simon B, Bork P, Lehrach H, Heinemann U
Structural genomics of human proteins--target selection and generation of a public catalogue of expression clones.
Microb Cell Fact. 2005 Jul 5;421.
BACKGROUND: The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells. RESULTS AND CONCLUSION: Protein expression and sequence data of 1414 E. coli expression clones representing 537 different proteins are presented. 139 human proteins (18%) could be expressed and purified in soluble form and with the expected size. All E. coli expression clones are publicly available to facilitate further functional characterisation of this set of human proteins. [Abstract/Link to Full Text]

Centeno NB, Planas-Iglesias J, Oliva B
Comparative modelling of protein structure and its impact on microbial cell factories.
Microb Cell Fact. 2005 Jun 30;420.
Comparative modeling is becoming an increasingly helpful technique in microbial cell factories as the knowledge of the three-dimensional structure of a protein would be an invaluable aid to solve problems on protein production. For this reason, an introduction to comparative modeling is presented, with special emphasis on the basic concepts, opportunities and challenges of protein structure prediction. This review is intended to serve as a guide for the biologist who has no special expertise and who is not involved in the determination of protein structure. Selected applications of comparative modeling in microbial cell factories are outlined, and the role of microbial cell factories in the structural genomics initiative is discussed. [Abstract/Link to Full Text]

Bansal AK
Bioinformatics in microbial biotechnology--a mini review.
Microb Cell Fact. 2005 Jun 28;4(1):19.
The revolutionary growth in the computation speed and memory storage capability has fueled a new era in the analysis of biological data. Hundreds of microbial genomes and many eukaryotic genomes including a cleaner draft of human genome have been sequenced raising the expectation of better control of microorganisms. The goals are as lofty as the development of rational drugs and antimicrobial agents, development of new enhanced bacterial strains for bioremediation and pollution control, development of better and easy to administer vaccines, the development of protein biomarkers for various bacterial diseases, and better understanding of host-bacteria interaction to prevent bacterial infections. In the last decade the development of many new bioinformatics techniques and integrated databases has facilitated the realization of these goals. Current research in bioinformatics can be classified into: (i) genomics--sequencing and comparative study of genomes to identify gene and genome functionality, (ii) proteomics--identification and characterization of protein related properties and reconstruction of metabolic and regulatory pathways, (iii) cell visualization and simulation to study and model cell behavior, and (iv) application to the development of drugs and anti-microbial agents. In this article, we will focus on the techniques and their limitations in genomics and proteomics. Bioinformatics research can be classified under three major approaches: (1) analysis based upon the available experimental wet-lab data, (2) the use of mathematical modeling to derive new information, and (3) an integrated approach that integrates search techniques with mathematical modeling. The major impact of bioinformatics research has been to automate the genome sequencing, automated development of integrated genomics and proteomics databases, automated genome comparisons to identify the genome function, automated derivation of metabolic pathways, gene expression analysis to derive regulatory pathways, the development of statistical techniques, clustering techniques and data mining techniques to derive protein-protein and protein-DNA interactions, and modeling of 3D structure of proteins and 3D docking between proteins and biochemicals for rational drug design, difference analysis between pathogenic and non-pathogenic strains to identify candidate genes for vaccines and anti-microbial agents, and the whole genome comparison to understand the microbial evolution. The development of bioinformatics techniques has enhanced the pace of biological discovery by automated analysis of large number of microbial genomes. We are on the verge of using all this knowledge to understand cellular mechanisms at the systemic level. The developed bioinformatics techniques have potential to facilitate (i) the discovery of causes of diseases, (ii) vaccine and rational drug design, and (iii) improved cost effective agents for bioremediation by pruning out the dead ends. Despite the fast paced global effort, the current analysis is limited by the lack of available gene-functionality from the wet-lab data, the lack of computer algorithms to explore vast amount of data with unknown functionality, limited availability of protein-protein and protein-DNA interactions, and the lack of knowledge of temporal and transient behavior of genes and pathways. [Abstract/Link to Full Text]

Rogé J, Betton JM
Use of pIVEX plasmids for protein overproduction in Escherichia coli.
Microb Cell Fact. 2005 Jun 2;418.
BACKGROUND: The pIVEX plasmids are vectors optimized for expression in the Rapid Translation System (RTS) cell-free system under control of bacteriophage T7 transcription elements. Even if these plasmids are intended for use in vitro, it is usually worthwhile to compare both cell-free and bacterial expression from the same genetic construct. However, some RTS users encountered problems when they introcuded these plasmids into Escherichia coli host strains producing the T7 RNA polymerase. RESULTS: We verified that difficulties in transforming the commonly used BL21(lambdaDE3) strain with pIVEX arose from the presence of a strong T7 promoter combined with a high-copy number plasmid, independent of gene expression. When these vectors were introduced into this strain harboring a compatible plasmid carrying the lactose repressor (lacI), we improved the transformation efficiency by 4 orders of magnitude. Moreover, we designed a transformation protocol that allows, after induction, the overproduction of pIVEX-encoded proteins in the BL21(lambdaDE3) strain. CONCLUSION: Using the correct plasmid/host combination and transformation-expression protocol, we could directly compare overproduction of the same pIVEX-encoded proteins from both in vivo and in vitro expression systems. [Abstract/Link to Full Text]

Wojtowicz A, Mazurkiewicz-Pisarek A, Plucienniczak G, Mikiewicz-Sygula D, Chojnacka L, Lukasiewicz N, Plucienniczak A
Expression of yeast deubiquitination enzyme UBP1 analogues in E. coli.
Microb Cell Fact. 2005 May 30;4(1):17.
BACKGROUND: It has been shown that proteins fused to ubiquitin undergo greater expression in E. coli and are easier to purify and renaturate than nonhybrid foreign proteins. However, there is no commercial source of large quantities of specific deubiquitinating proteases. This is the reason why hybrid proteins containing ubiquitin at their N-end cannot be used in large scale biotechnological processes. RESULTS AND CONCLUSION: We have described the synthesis of the yeast deubiquination enzyme UBP1 muteins in E. coli. We have shown that an efficient overproduction of the enzyme in E. coli may be achieved after the introduction of several changes in the nucleotide sequence encoding UBP1. One of the conditions of an effective synthesis of the UBP1 muteins is the removal of the 5'-end sequence encoding the transmembrane region of the enzyme. The obtained variants of the enzyme may be successfully used for processing large amounts of hybrid proteins comprising ubiquitin or tagged ubiquitin at their N-ends. [Abstract/Link to Full Text]

Mierau I, Olieman K, Mond J, Smid EJ
Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications.
Microb Cell Fact. 2005 May 30;4(1):16.
BACKGROUND: The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein. RESULTS: In a series of pH-controlled fermentations the following parameters were optimized: pH of the culture, use of NaOH or NH4OH as neutralizing agent, the addition of zinc and phosphate, the fermentation temperature, the time point of induction (cell density of the culture), the amount of nisin added for induction and the amount of three basic medium components, i.e. yeast extract, peptone and lactose. For each culture growth and lysostaphin production was followed. Lysostaphin production yields depended on all parameters that were varied. In the course of the optimization a three-fold increase in lysostaphin yield was achieved from 100 mg/l to 300 mg/l. CONCLUSION: Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction. Careful optimization of key parameters lead to a significant increase in the yield of the target protein. [Abstract/Link to Full Text]

Mierau I, Leij P, van Swam I, Blommestein B, Floris E, Mond J, Smid EJ
Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin.
Microb Cell Fact. 2005 May 27;415.
BACKGROUND: The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation. RESULTS: Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials. CONCLUSION: This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme. [Abstract/Link to Full Text]

Gosset G
Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system.
Microb Cell Fact. 2005 May 16;4(1):14.
The application of metabolic engineering in Escherichia coli has resulted in the generation of strains with the capacity to produce metabolites of commercial interest. Biotechnological processes with these engineered strains frequently employ culture media containing glucose as the carbon and energy source. In E. coli, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports glucose when this sugar is present at concentrations like those used in production fermentations. This protein system is involved in phosphoenolpyruvate-dependent sugar transport, therefore, its activity has an important impact on carbon flux distribution in the phosphoenolpyruvate and pyruvate nodes. Furthermore, PTS has a very important role in carbon catabolite repression. The properties of PTS impose metabolic and regulatory constraints that can hinder strain productivity. For this reason, PTS has been a target for modification with the purpose of strain improvement. In this review, PTS characteristics most relevant to strain performance and the different strategies of PTS modification for strain improvement are discussed. Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities has resulted in significant improvements in product yield from glucose and productivity for several classes of metabolites. In addition, inactivation of PTS components has been applied successfully as a strategy to abolish carbon catabolite repression, resulting in E. coli strains that use more efficiently sugar mixtures, such as those obtained from lignocellulosic hydrolysates. [Abstract/Link to Full Text]

Rawlings DE
Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates.
Microb Cell Fact. 2005 May 6;4(1):13.
Microorganisms are used in large-scale heap or tank aeration processes for the commercial extraction of a variety of metals from their ores or concentrates. These include copper, cobalt, gold and, in the past, uranium. The metal solubilization processes are considered to be largely chemical with the microorganisms providing the chemicals and the space (exopolysaccharide layer) where the mineral dissolution reactions occur. Temperatures at which these processes are carried out can vary from ambient to 80 degrees C and the types of organisms present depends to a large extent on the process temperature used. Irrespective of the operation temperature, biomining microbes have several characteristics in common. One shared characteristic is their ability to produce the ferric iron and sulfuric acid required to degrade the mineral and facilitate metal recovery. Other characteristics are their ability to grow autotrophically, their acid-tolerance and their inherent metal resistance or ability to acquire metal resistance. Although the microorganisms that drive the process have the above properties in common, biomining microbes usually occur in consortia in which cross-feeding may occur such that a combination of microbes including some with heterotrophic tendencies may contribute to the efficiency of the process. The remarkable adaptability of these organisms is assisted by several of the processes being continuous-flow systems that enable the continual selection of microorganisms that are more efficient at mineral degradation. Adaptability is also assisted by the processes being open and non-sterile thereby permitting new organisms to enter. This openness allows for the possibility of new genes that improve cell fitness to be selected from the horizontal gene pool. Characteristics that biomining microorganisms have in common and examples of their remarkable adaptability are described. [Abstract/Link to Full Text]

Su WW
Fluorescent proteins as tools to aid protein production.
Microb Cell Fact. 2005 Apr 25;4(1):12.
Fluorescent proteins are genetically encoded, highly versatile reporters useful for monitoring various aspects of recombinant protein production. In addition to the widely popular green fluorescent protein (GFP) from Aequorea victoria, a variety of other fluorescent proteins have been discovered that display a wide range of spectral properties. Synthetic variants have also been developed to overcome limitations associated with their wild-type counterparts. Having a large repertoire of fluorescent proteins with diverse traits opens new opportunities for rapid monitoring and optimization of recombinant protein production. [Abstract/Link to Full Text]

Ventura S
Sequence determinants of protein aggregation: tools to increase protein solubility.
Microb Cell Fact. 2005 Apr 22;4(1):11.
Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, very often the target protein accumulates into insoluble aggregates in a misfolded and biologically inactive form. Bacterial inclusion bodies are major bottlenecks in protein production and are hampering the development of top priority research areas such structural genomics. Inclusion body formation was formerly considered to occur via non-specific association of hydrophobic surfaces in folding intermediates. Increasing evidence, however, indicates that protein aggregation in bacteria resembles to the well-studied process of amyloid fibril formation. Both processes appear to rely on the formation of specific, sequence-dependent, intermolecular interactions driving the formation of structured protein aggregates. This similarity in the mechanisms of aggregation will probably allow applying anti-aggregational strategies already tested in the amyloid context to the less explored area of protein aggregation inside bacteria. Specifically, new sequence-based approaches appear as promising tools to tune protein aggregation in biotechnological processes. [Abstract/Link to Full Text]

Villaverde A
Focusing in bioproduction science.
Microb Cell Fact. 2005 Apr 6;4(1):10.
As in other Biotechnological fields, the microbial production of recombinant proteins and other biomolecules can be approached from multiple angles through the help of diverse technologies of increasing complexity. To better reach all the specialized niches in bioproduction, Microbial Cell Factories is now inviting authors to prepare concise Reviews (eventually miniReviews), covering relevant areas that deserve specific and highly focused attention. By the publication of such contributions, the journal will promote the revision of new insights around the Cell Factory concept in a highly comprehensive way, in molecular, cellular and environmental contexts. [Abstract/Link to Full Text]

Soini J, Falschlehner C, Mayer C, Böhm D, Weinel S, Panula J, Vasala A, Neubauer P
Transient increase of ATP as a response to temperature up-shift in Escherichia coli.
Microb Cell Fact. 2005 Apr 1;4(1):9.
SUMMARY: BACKGROUND: Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift. RESULTS: The current study shows that a temperature up-shift is followed by a very fast significant transient increase of the cellular ATP concentration within the first minutes. This increase is connected to a longer lasting elevation of the cellular respiration and glucose uptake. Also the mRNA level of typical heat shock genes increases within only one minute after the heat-shock. CONCLUSION: The presented data prove the very fast response of E. coli to a heat-shock and that the initial response includes the increase of the ATP pool which is important to fulfil the need of the cell for new syntheses, as well as for the function of chaperones and proteases. [Abstract/Link to Full Text]

Yun J, Ryu S
Screening for novel enzymes from metagenome and SIGEX, as a way to improve it.
Microb Cell Fact. 2005 Mar 25;4(1):8.
Metagenomics has been successfully applied to isolate novel biocatalysts from the uncultured microbiota in the environment. Two types of screening have been used to identify clones carrying desired traits from metagenomic libraries: function-based screening, and sequence-based screening. Both function- and sequence- based screening have individual advantages and disadvantages, and they have been applied successfully to discover biocatalysts from metagenome. However, both strategies are laborious and tedious because of the low frequency of screening hits. A recent paper introduced a high throughput screening strategy, termed substrate-induced gene-expression screening (SIGEX). SIGEX is designed to select the clones harboring catabolic genes induced by various substrates in concert with fluorescence activated cell sorting (FACS). This method was applied successfully to isolate aromatic hydrocarbon-induced genes from a metagenomic library. Although SIGEX has many limitations, it is expected to provide economic advantages, especially to industry. [Abstract/Link to Full Text]

Nakashima N, Mitani Y, Tamura T
Actinomycetes as host cells for production of recombinant proteins.
Microb Cell Fact. 2005 Mar 23;4(1):7.
Actinomycetes (Actinobacteria) are highly attractive as cell factories or bioreactors for applications in industrial, agricultural, environmental, and pharmaceutical fields. Genome sequencing of several species of actinomycetes has paved the way for biochemical and structural analysis of important proteins and the production of such proteins as recombinants on a commercial scale. In this regard, there is a need for improved expression vectors that will be applicable to actinomycetes. Recent advancements in gene expression systems, knowledge regarding the intracellular environment, and identification and characterization of plasmids has made it possible to develop practicable recombinant expression systems in actinomycetes as described in this review. [Abstract/Link to Full Text]

Lethanh H, Neubauer P, Hoffmann F
The small heat-shock proteins IbpA and IbpB reduce the stress load of recombinant Escherichia coli and delay degradation of inclusion bodies.
Microb Cell Fact. 2005 Feb 11;4(1):6.
BACKGROUND: The permanently impaired protein folding during recombinant protein production resembles the stress encountered at extreme temperatures, under which condition the putative holding chaperones, IbpA/IbpB, play an important role. We evaluated the impact of ibpAB deletion or overexpression on stress responses and the inclusion body metabolism during production of yeast alpha-glucosidase in Escherichia coli. RESULTS: Deletion of ibpAB, which is innocuous under physiological conditions, impaired culture growth during alpha-glucosidase production. At higher temperatures, accumulation of stress proteins including disaggregation chaperones (DnaK and ClpB) and components of the RNA degradosome, enolase and PNP, was intensified. Overexpression of ibpAB, conversely, suppressed the heat-shock response under these conditions. Inclusion bodies of alpha-glucosidase started to disaggregate after arrest of protein synthesis in a ClpB and DnaK dependent manner, followed by degradation or reactivation. IbpA/IbpB decelerated disaggregation and degradation at higher temperatures, but did hardly influence the disaggregation kinetics at 15 degrees C. Overexpression of ibpAB concomitant to production at 42 degrees C increased the yield of alpha-glucosidase activity during reactivation. CONCLUSIONS: IbpA/IbpB attenuate the accumulation of stress proteins, and - at high temperatures - save disaggregated proteins from degradation, at the cost, however, of delayed removal of aggregates. Without ibpAB, inclusion body removal is faster, but cells encounter more intense stress and growth impairment. IbpA/IbpB thus exert a major function in cell protection during stressful situations. [Abstract/Link to Full Text]

De D, Dutta D, Kundu M, Mahato S, Schiavone MT, Chaudhuri S, Giri A, Gupta V, Bhattacharya SK
Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase) as sugar binders for ribulose-1,5-bisphosphate regeneration reactors.
Microb Cell Fact. 2005 Feb 2;4(1):5.
BACKGROUND: Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose1,5-bisphosphate (RuBP) for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. RESULTS: We demonstrate specific binding of 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd) for 3-phosphoglycerate (655-796 muM) compared to 3-phosphoglyceraldehyde (822-966 muM) indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. CONCLUSIONS: The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors. [Abstract/Link to Full Text]

Di Lorenzo A, Varcamonti M, Parascandola P, Vignola R, Bernardi A, Sacceddu P, Sisto R, de Alteriis E
Characterization and performance of a toluene-degrading biofilm developed on pumice stones.
Microb Cell Fact. 2005 Jan 17;4(1):4.
BACKGROUND: Hydrocarbon-degrading biofilms in the treatment of contaminated groundwaters have received increasing attention due to the role played in the so-called "biobarriers". These are bioremediation systems in which a microbial consortium adherent to a solid support is placed across the flow of a contaminated plume, thus promoting biodegradation of the pollutant. RESULTS: A microbial consortium adherent to pumice granules (biofilm) developed from a toluene-enriched microflora in a mini-scale system, following continuous supply of a mineral medium containing toluene, over a 12-month period. Observation by scanning electron microscopy, together with quantification of the biomass attached to pumice, evidenced the presence of abundant exopolymeric material surrounding the cells in the biofilm. Toluene removal monitored during 12-month operation, reached 99%. Identification of the species, based on comparative 16S ribosomal DNA (rDNA) sequence analysis, revealed that Rhodococcus erythropolis and Pseudomonas marginalis were the predominant bacterial species in the microbial consortium. CONCLUSION: A structurally complex toluene-degrading biofilm, mainly formed by Rhodococcus erythropolis and Pseudomonas marginalis, developed on pumice granules, in a mini-scale apparatus continuously fed with toluene. [Abstract/Link to Full Text]

Vethanayagam JG, Flower AM
Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase.
Microb Cell Fact. 2005 Jan 7;4(1):3.
BACKGROUND: Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure. RESULTS: We demonstrated that decreases in protein overproduction levels are not due to significant plasmid loss nor to mutations arising on the plasmid, but instead largely are attributable to chromosomal mutations that diminish the level of functional T7 RNA polymerase, resulting in decreased expression from the plasmid. Isolation of plasmid DNA from non-expressing strains and reintroduction of the plasmid into a T7 RNA polymerase-producing strain such as BL21(lambdaDE3) reproducibly restored high level protein production. CONCLUSIONS: Our results suggest that a major contributing factor to decreased expression levels in T7 based systems is chromosomal mutation resulting in loss of functional T7 RNA polymerase. Consistent with this hypothesis, we found that optimal protein overproduction was obtained reproducibly from T7 promoters using freshly transformed cells that had not been subjected to outgrowth during which mutations could accumulate. [Abstract/Link to Full Text]

Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, Bermúdez-Humarán LG, Nouaille S, Ribeiro LA, Leclercq S, Gabriel JE, Guimaraes VD, Oliveira MN, Charlier C, Gautier M, Langella P
Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production.
Microb Cell Fact. 2005 Jan 4;4(1):2.
Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species. [Abstract/Link to Full Text]

Sřrensen HP, Mortensen KK
Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli.
Microb Cell Fact. 2005 Jan 4;4(1):1.
Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags. [Abstract/Link to Full Text]

Sauer M, Branduardi P, Gasser B, Valli M, Maurer M, Porro D, Mattanovich D
Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation.
Microb Cell Fact. 2004 Dec 20;3(1):17.
BACKGROUND: Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. RESULTS: We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified. CONCLUSIONS: We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase. [Abstract/Link to Full Text]

Philibert P, Martineau P
Directed evolution of single-chain Fv for cytoplasmic expression using the beta-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation.
Microb Cell Fact. 2004 Dec 17;3(1):16.
BACKGROUND: Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli. RESULTS: We used the beta-galactosidase alpha-complementation system to monitor and evolve two antibody fragments for high expression levels in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>107 clones), clones exhibiting high levels of beta-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the beta-galactosidase activity present in the cells. CONCLUSION: This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free alpha-peptide released from the protein fusion by the host proteases. This means that the alpha-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released alpha-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein. [Abstract/Link to Full Text]

Vadeboncoeur C, Moineau S
The relevance of genetic analysis to dairy bacteria: building upon our heritage.
Microb Cell Fact. 2004 Dec 10;3(1):15.
Lactic acid bacteria (LAB) are essential for the manufacture of fermented dairy products. Studies on the physiology, biochemistry and genetics of these microorganisms over the last century have contributed considerably to the improvement of fermentation processes and have resulted in better and safer products. Nevertheless, the potential of LAB is far from being maximized. The sophistication of biotechnologies and the availability of complete genome sequences have opened the door to the metabolic engineering of LAB. In this regard, the recent publication of the complete genome sequences of two Streptococcus thermophilus strains will provide a key tool to facilitate the genetic manipulation of this important dairy species. [Abstract/Link to Full Text]

Scheibel T
Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins.
Microb Cell Fact. 2004 11 16;3(1):14.
Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins. [Abstract/Link to Full Text]

---

Recent Articles in Antimicrobial Agents and Chemotherapy

---

Shi M, Wang X, De Clercq E, Takao S, Baba M
Selective inhibition of porcine endogenous retrovirus replication in human cells by acyclic nucleoside phosphonates.
Antimicrob Agents Chemother. 2007 Jul;51(7):2600-4.
Several anti-human immunodeficiency virus type 1 reverse transcriptase inhibitors were evaluated for their antiviral activities against porcine endogenous retrovirus in human cells. Among the test compounds, zidovudine was found to be the most active. The order of potency was zidovudine > phosphonylmethoxyethoxydiaminopyrimidine = phosphonylmethoxypropyldiaminopurine > tenofovir > or = adefovir > stavudine. [Abstract/Link to Full Text]

Stickney DR, Noveljic Z, Garsd A, Destiche DA, Frincke JM
Safety and activity of the immune modulator HE2000 on the incidence of tuberculosis and other opportunistic infections in AIDS patients.
Antimicrob Agents Chemother. 2007 Jul;51(7):2639-41.
Twenty-five AIDS patients were treated with HE2000, a synthetic adrenal hormone. The drug was well tolerated and safe and reduced both the incidence of tuberculosis coinfection by 42.2% (P < 0.05) and the cumulative incidence of opportunistic infections (P < 0.05). These results warrant further clinical investigation of HE2000. [Abstract/Link to Full Text]

White LK, Yoon JJ, Lee JK, Sun A, Du Y, Fu H, Snyder JP, Plemper RK
Nonnucleoside inhibitor of measles virus RNA-dependent RNA polymerase complex activity.
Antimicrob Agents Chemother. 2007 Jul;51(7):2293-303.
Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe measles and silence viral outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhibitors. The potential for preexisting or emerging resistance in the field constitutes the rationale for the identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and implementation of a cell-based assay for high-throughput screening of MV antivirals, which has yielded several hit candidates. Following confirmation by secondary assays and chemical synthesis, the most potent hit was found to act as a target-specific inhibitor of MV replication with desirable drug-like properties. The compound proved highly active against multiple primary isolates of diverse MV genotypes currently circulating worldwide, showing active concentrations of 35 to 145 nM. Significantly, it does not interfere with viral entry and lacks cross-resistance with the MV fusion inhibitor class. Mechanistic characterization on a subinfection level revealed that the compound represents a first-in-class nonnucleoside inhibitor of MV RNA-dependent RNA polymerase complex activity. Singly or in combination with the fusion inhibitors, this novel compound class has high developmental potential as a potent therapeutic against MV and will likely further the mechanistic characterization of the viral polymerase complex. [Abstract/Link to Full Text]

Lund KC, Peterson LL, Wallace KB
Absence of a universal mechanism of mitochondrial toxicity by nucleoside analogs.
Antimicrob Agents Chemother. 2007 Jul;51(7):2531-9.
Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 microM; 2.7 and 13.4 microg/ml), didanosine (ddI) (10 and 50 microM; 2.4 and 11.8 microg/ml), and zalcitabine (ddC) (1 and 5 microM; 0.21 and 1.1 microg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 microg/ml; 1.3 microM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 microM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard. [Abstract/Link to Full Text]

Borkow G, Sidwell RW, Smee DF, Barnard DL, Morrey JD, Lara-Villegas HH, Shemer-Avni Y, Gabbay J
Neutralizing viruses in suspensions by copper oxide-based filters.
Antimicrob Agents Chemother. 2007 Jul;51(7):2605-7.
We report the capacity of copper oxide-containing filters to reduce infectious titers of a panel of viruses spiked into culture media. Enveloped, nonenveloped, RNA, and DNA viruses were affected, suggesting the possibility of using copper oxide-containing devices to deactivate a wide spectrum of infectious viruses found in filterable suspensions. [Abstract/Link to Full Text]

García-Cobos S, Campos J, Lázaro E, Román F, Cercenado E, García-Rey C, Pérez-Vázquez M, Oteo J, de Abajo F
Ampicillin-resistant non-beta-lactamase-producing Haemophilus influenzae in Spain: recent emergence of clonal isolates with increased resistance to cefotaxime and cefixime.
Antimicrob Agents Chemother. 2007 Jul;51(7):2564-73.
The sequence of the ftsI gene encoding the transpeptidase domain of penicillin-binding protein 3 (PBP 3) was determined for 354 nonconsecutive Haemophilus influenzae isolates from Spain; 17.8% of them were ampicillin susceptible, 56% were beta-lactamase nonproducing ampicillin resistant (BLNAR), 15.8% were beta-lactamase producers and ampicillin resistant, and 10.4% displayed both resistance mechanisms. The ftsI gene sequences had 28 different mutation patterns and amino acid substitutions at 23 positions. Some 93.2% of the BLNAR strains had amino acid substitutions at the Lys-Thr-Gly (KTG) motif, the two most common being Asn526 to Lys (83.9%) and Arg517 to His (9.3%). Amino acid substitutions at positions 377, 385, and 389, which conferred cefotaxime and cefixime MICs 10 to 60 times higher than those of susceptible strains, were found for the first time in Europe. In 72 isolates for which the repressor acrR gene of the AcrAB efflux pump was sequenced, numerous amino acid substitutions were found. Eight isolates with ampicillin MICs of 0.25 to 2 microg/ml showed changes that predicted the early termination of the acrR reading frame. Pulsed-field gel electrophoresis analysis demonstrated that most BLNAR strains were genetically diverse, although clonal dissemination was detected in a group of isolates presenting with increased resistance to cefotaxime and cefixime. Background antibiotic use at the community level revealed a marked trend toward increased amoxicillin-clavulanic acid consumption. BLNAR H. influenzae strains have arisen by vertical and horizontal spread and have evolved to adapt rapidly to the increased selective pressures posed by the use of oral penicillins and cephalosporins. [Abstract/Link to Full Text]

Perilli M, Caporale B, Celenza G, Pellegrini C, Docquier JD, Mezzatesta M, Rossolini GM, Stefani S, Amicosante G
Identification and characterization of a new metallo-beta-lactamase, IND-5, from a clinical isolate of Chryseobacterium indologenes.
Antimicrob Agents Chemother. 2007 Aug;51(8):2988-90.
A new natural IND-type metallo-beta-lactamase variant, IND-5, was identified in a clinical isolate of Chryseobacterium indologenes. IND-5 shared 92.8% and 92.4% amino acid homology with IND-1 and IND-3, respectively. Purified enzyme (pI = 8.8, M(r) = 25,000) was able to hydrolyze penicillins, some narrow- and expanded-spectrum cephalosporins, and carbapenems but not monobactams. [Abstract/Link to Full Text]

Lartigue MF, Zinsius C, Wenger A, Bille J, Poirel L, Nordmann P
Extended-spectrum beta-lactamases of the CTX-M type now in Switzerland.
Antimicrob Agents Chemother. 2007 Aug;51(8):2855-60.
The epidemiology of clavulanic acid-inhibited extended-spectrum beta-lactamases (ESBLs) was investigated among infection-associated enterobacterial isolates at the University Hospital in Lausanne, Switzerland, from January 2004 to June 2005. Out of 57 nonrepetitive ESBL producers (prevalence rate of 0.7%), 45 produced CTX-M-like ESBLs. CTX-M enzymes were mostly from clonally nonrelated Escherichia coli isolates, from urinary infections and community-acquired infections. Pediatric patients (20 out of 57) accounted for a large number of CTX-M producers. CTX-M-15 was the most frequent CTX-M-type enzyme. The plasmid-located bla(CTX-M) genes were associated with either ISEcp1 or ISCR1 insertion sequences. This study is the first published report of CTX-M-type beta-lactamases in Switzerland. [Abstract/Link to Full Text]

Griffith ME, Moon JE, Johnson EN, Clark KP, Hawley JS, Hospenthal DR, Murray CK
Efficacy of fluoroquinolones against Leptospira interrogans in a hamster model.
Antimicrob Agents Chemother. 2007 Jul;51(7):2615-7.
Ciprofloxacin, gatifloxacin, and levofloxacin were evaluated for their abilities to prevent mortality in hamsters infected with a lethal inoculum of Leptospira interrogans serovar Portlandvere. Each agent produced a statistically significant survival advantage compared to no treatment and demonstrated survival similar to that seen with doxycycline therapy. [Abstract/Link to Full Text]

Bugatti A, Urbinati C, Ravelli C, De Clercq E, Liekens S, Rusnati M
Heparin-mimicking sulfonic acid polymers as multitarget inhibitors of human immunodeficiency virus type 1 Tat and gp120 proteins.
Antimicrob Agents Chemother. 2007 Jul;51(7):2337-45.
Human immunodeficiency virus (HIV) Tat and gp120 intriguingly share the feature of being basic peptides that, once released by HIV(+) cells, bind to polyanionic heparan sulfate proteoglycans (HSPGs) on target uninfected cells, contributing to the onset of AIDS-associated pathologies. To identify multitarget anti-HIV prodrugs, we investigated the gp120 and Tat antagonist potentials of a series of polyanionic synthetic sulfonic acid polymers (SSAPs). Surface plasmon resonance revealed that SSAPs inhibit with a competitive mechanism of action the binding of Tat and gp120 to surface-immobilized heparin, an experimental condition that resembles binding to cellular HSPGs. Accordingly, SSAPs inhibited HSPG-dependent cell internalization and the transactivating activity of Tat. Little is known about the binding of free gp120 to target cells. Here, we identified two classes of gp120 receptors expressed on endothelial cells, one of which was consistent with an HSPG-binding, low-affinity/high-capacity receptor that is inhibited by free heparin. SSAPs inhibited the binding of free gp120 to endothelial cells, as well as its capacity to induce apoptosis in the same cells. In all the assays, poly(4-styrenesulfonic acid) (PSS) proved to be the most potent antagonist of Tat and gp120. Accordingly, PSS bound both proteins with high affinity. In conclusion, SSAPs represent an interesting class of compounds that bind both gp120 and Tat and inhibit their HSPG-dependent cell surface binding and pathological effects. As these activities contribute to both AIDS progression and associated pathologies, SSAPs can be considered prototypic molecules for the development of multitarget drugs for the treatment of HIV infection and AIDS-associated pathologies. [Abstract/Link to Full Text]

Stone ND, Dunaway SB, Flexner C, Tierney C, Calandra GB, Becker S, Cao YJ, Wiggins IP, Conley J, MacFarland RT, Park JG, Lalama C, Snyder S, Kallungal B, Klingman KL, Hendrix CW
Multiple-dose escalation study of the safety, pharmacokinetics, and biologic activity of oral AMD070, a selective CXCR4 receptor inhibitor, in human subjects.
Antimicrob Agents Chemother. 2007 Jul;51(7):2351-8.
AMD070 is an oral CXCR4 antagonist with in vitro activity against X4-tropic human immunodeficiency virus type 1. Thirty fasting healthy male volunteers received oral doses of AMD070 ranging from a single 50-mg dose to seven 400-mg doses given every 12 h (q12h). Nine subjects received a 200-mg dose during fasting and prior to a meal. Subjects were monitored for safety and pharmacokinetics. AMD070 was well tolerated, without serious adverse events. Transient headaches (13 subjects) and neurocognitive (8 subjects) and gastrointestinal (7 subjects) symptoms were the most common complaints. Seven subjects had sinus tachycardia, and two were symptomatic. AMD070 plasma concentrations peaked 1 to 2 h after patient dosing. The estimated terminal half-life ranged from 11.2 to 15.9 h among cohorts. Dose proportionality was not demonstrated. Less than 1% of the drug appeared unchanged in the urine. Food reduced the maximum concentration of drug in serum and the area under the concentration-time curve from 0 to 24 h by 70% and 56%, respectively (P < or = 0.01). A dose-dependent elevation of white blood cells (WBC) demonstrated a maximum twofold increase over baseline (95% confidence interval, 2.0- to 2.1-fold) in an E(max) model. In healthy volunteers, AMD070 was well tolerated and demonstrated mixed-order pharmacokinetics, and food reduced drug exposure. AMD070 induced a dose-related elevation of WBC which was attributed to CXCR4 blockade. Using leukocytosis as a surrogate marker for CXCR4 inhibition, this dose-response relationship suggests that the doses used in this study were active in vivo, though not maximal, throughout the dosing interval. Trough concentrations with the 400-mg dose q12h exceeded the antiviral in vitro 90% effective concentration of AMD070. [Abstract/Link to Full Text]

Moise PA, Sakoulas G, Forrest A, Schentag JJ
Vancomycin in vitro bactericidal activity and its relationship to efficacy in clearance of methicillin-resistant Staphylococcus aureus bacteremia.
Antimicrob Agents Chemother. 2007 Jul;51(7):2582-6.
We examined the relationship between the time to clearance of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia while patients were receiving vancomycin therapy and the in vitro bactericidal activity of vancomycin. Vancomycin killing assays were performed with 34 MRSA bloodstream isolates (17 accessory gene regulator group II [agr-II] and 17 non-agr-II isolates) from 34 different patients with MRSA bacteremia for whom clinical and microbiological outcomes data were available. Vancomycin doses were prospectively adjusted to achieve peak plasma concentrations of 28 to 32 mug/ml and trough concentrations of 8 to 12 microg/ml. Bactericidal assays were performed over 24 h with approximately 10(7) to 10(8) CFU/ml in broth containing 16 microg/ml vancomycin. The median time to clearance of bacteremia was 6.5 days for patients with MRSA isolates demonstrating > or =2.5 reductions in log(10) CFU/ml at 24 h and >10.5 days for patients with MRSA isolates demonstrating <2.5 log(10) CFU/ml by 24 h (P = 0.025). The median time to clearance was significantly longer with MRSA isolates with vancomycin MICs of 2.0 microg/ml compared to that with MRSA isolates with MICs of < or =1.0 microg/ml (P = 0.019). The bacteremia caused by MRSA isolates with absent or severely reduced delta-hemolysin expression was of a longer duration of bacteremia (10 days and 6.5 days, respectively; P = 0.27) and had a decreased probability of eradication (44% and 78%, respectively; P = 0.086). We conclude that strain-specific microbiological features of MRSA, such as increased vancomycin MICs and decreased killing by vancomycin, appear to be predictive of prolonged MRSA bacteremia while patients are receiving vancomycin therapy. Prolonged bacteremia and decreased delta-hemolysin expression may also be related. Evaluation of these properties may be useful in the consideration of antimicrobial therapies that can be used as alternatives to vancomycin for the treatment of MRSA bacteremia. [Abstract/Link to Full Text]

Serena C, Rodríguez MM, Mariné M, Pastor FJ, Guarro J
Combined therapies in a murine model of blastoschizomycosis.
Antimicrob Agents Chemother. 2007 Jul;51(7):2608-10.
In a murine model of blastoschizomycosis, amphotericin B combined with micafungin, flucytosine or voriconazole did not improve the efficacy of fluconazole. However, such combinations can constitute therapeutic options for those cases where fluconazole fails. [Abstract/Link to Full Text]

Ruslami R, Nijland HM, Alisjahbana B, Parwati I, van Crevel R, Aarnoutse RE
Pharmacokinetics and tolerability of a higher rifampin dose versus the standard dose in pulmonary tuberculosis patients.
Antimicrob Agents Chemother. 2007 Jul;51(7):2546-51.
Rifampin is a key drug for tuberculosis (TB) treatment. The available data suggest that the currently applied 10-mg/kg of body weight dose of rifampin may be too low and that increasing the dose may shorten the treatment duration. A double-blind randomized phase II clinical trial was performed to investigate the effect of a higher dose of rifampin in terms of pharmacokinetics and tolerability. Fifty newly diagnosed adult Indonesian TB patients were randomized to receive a standard (450-mg, i.e., 10-mg/kg in Indonesian patients) or higher (600-mg) dose of rifampin in addition to other TB drugs. A full pharmacokinetic curve for rifampin, pyrazinamide, and ethambutol was recorded after 6 weeks of daily TB treatment. Tolerability was assessed during the 6-month treatment period. The geometric means of exposure to rifampin (area under the concentration-time curve from 0 to 24 h [AUC(0-24)]) were increased by 65% (P < 0.001) in the higher-dose group (79.7 mg.h/liter) compared to the standard-dose group (48.5 mg.h/liter). Maximum rifampin concentrations (C(max)) were 15.6 mg/liter versus 10.5 mg/liter (49% increase; P < 0.001). The percentage of patients for whom the rifampin C(max) was > or =8 mg/liter was 96% versus 79% (P = 0.094). The pharmacokinetics of pyrazinamide and ethambutol were similar in both groups. Mild (grade 1 or 2) hepatotoxicity was more common in the higher-dose group (46 versus 20%; P = 0.054), but no patient developed severe hepatotoxicity. Increasing the rifampin dose was associated with a more than dose-proportional increase in the mean AUC(0-24) and C(max) of rifampin without affecting the incidence of serious adverse effects. Follow-up studies are warranted to assess whether high-dose rifampin may enable shortening of TB treatment. [Abstract/Link to Full Text]

Morrey JD, Siddharthan V, Olsen AL, Wang H, Julander JG, Hall JO, Li H, Nordstrom JL, Koenig S, Johnson S, Diamond MS
Defining limits of treatment with humanized neutralizing monoclonal antibody for West Nile virus neurological infection in a hamster model.
Antimicrob Agents Chemother. 2007 Jul;51(7):2396-402.
A potent anti-West Nile virus (anti-WNV)-neutralizing humanized monoclonal antibody, hE16, was previously shown to improve the survival of WNV-infected hamsters when it was administered intraperitoneally (i.p.), even after the virus had infected neurons in the brain. In this study, we evaluated the therapeutic limit of hE16 for the treatment of WNV infection in hamsters by comparing single-dose peripheral (i.p.) therapy with direct administration into the pons through a convection-enhanced delivery (CED) system. At day 5 after infection, treatments with hE16 by the peripheral and the CED routes were equally effective at reducing morbidity and mortality. In contrast, at day 6 only the treatment by the CED route protected the hamsters from lethal infection. These experiments suggest that hE16 can directly control WNV infection in the central nervous system. In support of this, hE16 administered i.p. was detected in a time-dependent manner in the serum, cerebrospinal fluid (CSF), cerebral cortex, brain stem, and spinal cord in CSF. A linear relationship between the hE16 dose and the concentration in serum was observed, and maximal therapeutic activity occurred at doses of 0.32 mg/kg of body weight or higher, which produced serum hE16 concentrations of 1.3 microg/ml or higher. Overall, these data suggest that in hamsters hE16 can ameliorate neurological disease after significant viral replication has occurred, although there is a time window that limits therapeutic efficacy. [Abstract/Link to Full Text]

Cai L, Gochin M
A novel fluorescence intensity screening assay identifies new low-molecular-weight inhibitors of the gp41 coiled-coil domain of human immunodeficiency virus type 1.
Antimicrob Agents Chemother. 2007 Jul;51(7):2388-95.
A metallopeptide-based fluorescence assay has been designed for the detection of small-molecule inhibitors of human immunodeficiency virus type 1 gp41, the viral protein involved in membrane fusion. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer C-terminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The two peptides span a hydrophobic pocket previously defined in the literature. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorophore-labeled HR2 peptide to be followed by fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and we show that they can be detected in the assay from an increase in the fluorescence that is correlated with the potency of the compound. Assay optimization and validation have resulted in a simple quantitative competitive inhibition assay for fusion inhibitors that bind in the hydrophobic pocket. The assay has an assay quality factor (Z') of 0.88 and can rank order inhibitors at 10 microM concentration with K(i)s in the range of 0.2 microM to 30 microM, an ideal range for drug discovery. Screening of a small peptidomimetic library has yielded three new low-molecular-weight gp41 inhibitors. In vitro syncytium inhibition assays confirmed that the compounds inhibited cell-cell fusion in the low micromolar range. These lead compounds provide a new molecular scaffold for the development of fusion inhibitors. [Abstract/Link to Full Text]

Barklis E, Still A, Sabri MI, Hirsch AJ, Nikolich-Zugich J, Brien J, Dhenub TC, Scholz I, Alfadhli A
Sultam thiourea inhibition of West Nile virus.
Antimicrob Agents Chemother. 2007 Jul;51(7):2642-5.
We have identified sultam thioureas as novel inhibitors of West Nile virus (WNV) replication. One such compound inhibited WNV, with a 50% effective concentration of 0.7 microM, and reduced reporter expression from cells that harbored a WNV-based replicon. Our results demonstrate that sultam thioureas can block a postentry, preassembly step of WNV replication. [Abstract/Link to Full Text]

Cattoir V, Poirel L, Mazel D, Soussy CJ, Nordmann P
Vibrio splendidus as the source of plasmid-mediated QnrS-like quinolone resistance determinants.
Antimicrob Agents Chemother. 2007 Jul;51(7):2650-1. [Abstract/Link to Full Text]

Almyroudis NG, Sutton DA, Fothergill AW, Rinaldi MG, Kusne S
In vitro susceptibilities of 217 clinical isolates of zygomycetes to conventional and new antifungal agents.
Antimicrob Agents Chemother. 2007 Jul;51(7):2587-90.
We evaluated the in vitro susceptibilities of 217 zygomycetes to amphotericin B, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, caspofungin, and flucytosine. The significant in vitro activity of posaconazole against several species appears to support its reported clinical efficacy. Decreased susceptibility to amphotericin B was noted with Cunninghamella bertholletiae. [Abstract/Link to Full Text]

Coelho AC, Messier N, Ouellette M, Cotrim PC
Role of the ABC transporter PRP1 (ABCC7) in pentamidine resistance in Leishmania amastigotes.
Antimicrob Agents Chemother. 2007 Aug;51(8):3030-2.
Pentamidine is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance mechanism are not well understood. In this work, we show that the intracellular ABC protein PRP1 (pentamidine resistance protein 1) (ABCC7) can confer resistance to pentamidine in Leishmania sp. parasites in the intracellular stage. [Abstract/Link to Full Text]

Daikos GL, Kosmidis C, Tassios PT, Petrikkos G, Vasilakopoulou A, Psychogiou M, Stefanou I, Avlami A, Katsilambros N
Enterobacteriaceae bloodstream infections: presence of integrons, risk factors, and outcome.
Antimicrob Agents Chemother. 2007 Jul;51(7):2366-72.
A prospective observational study was conducted to identify factors associated with bloodstream infections (BSIs) caused by integron-carrying Enterobacteriaceae and to evaluate the clinical significance of integron carriage. Consecutive patients with Enterobacteriaceae BSIs were identified and followed up until discharge or death. Identification of blood isolates and susceptibility testing were performed by the Wider I automated system. int-1-specific PCR, conserved-segment PCR, and DNA sequencing were used to determine the presence, length, and content of integrons. The relatedness among the isolates was examined by pulsed-field gel electrophoresis. Two hundred fifty episodes of Enterobacteriaceae BSI occurred in 233 patients; 109 (43.6%) were nosocomial, 82 (32.8%) were community acquired, and 59 (23.6%) were health care associated. Integrons were detected in 11 (13.4%) community-acquired, 24 (40.7%) health care-associated, and 46 (42.2%) nosocomial isolates. Integron-carrying organisms were more likely to exhibit resistance to three or more classes of antimicrobials (odds ratio [OR], 9.84; 95% confidence interval [95% CI], 5.31 to 18.23; P < 0.001) or to produce extended-spectrum beta-lactamases (OR, 5.75; 95% CI, 2.38 to 13.89; P < 0.001) or a VIM-type metallo-beta-lactamase (P, 0.003). Inter- or intraspecies integron transfer and cross-transmission of integron-carrying clones were observed. Use of cotrimoxazole (OR, 4.77; 95% CI, 1.81 to 12.54; P < 0.001) and a nosocomial or other health care setting (OR, 3.07; 95% CI, 1.30 to 7.22; P, 0.01) were independently associated with BSIs caused by integron-carrying Enterobacteriaceae. Patients with a nonurinary source of bacteremia (OR, 9.46; 95% CI, 2.77 to 32.32; P < 0.001) and a Pitt bacteremia score of > or =4 (OR, 23.36; 95% CI, 7.97 to 68.44; P < 0.001) had a significantly higher 14-day mortality rate, whereas integron carriage did not affect clinical outcomes. These findings may have implications affecting antibiotic policies and infection control measures. [Abstract/Link to Full Text]

Azpiroz MF, Lavińa M
Modular structure of microcin H47 and colicin V.
Antimicrob Agents Chemother. 2007 Jul;51(7):2412-9.
Microcins are gene-encoded peptide antibiotics produced by enterobacteria that act on strains of gram-negative bacteria. In this work, we concentrated on higher-molecular-mass microcins, i.e., those possessing 60 or more amino acids. They can be subdivided into unmodified and posttranslationally modified peptides. In both cases, they exhibit conserved C-terminal sequences that appear to be characteristic of each subgroup. In the hypothesis that these sequences could correspond to domains, gene fusions between the activity genes for the unmodified microcin colicin V and the modified microcin H47 were constructed. These two microcins differ in their mode of synthesis, uptake, target, and specific immunity. Through this experimental approach, chimeric peptides with exchanged C-terminal sequences were encoded. Cells carrying the fusions in different genetic contexts were then assayed for antibiotic production. Many of them produced antibiotic activities with recombinant properties: the toxicity of one microcin and the mode of uptake of the other. The results led to the identification of a modular structure of colicin V and microcin H47, with the recognition of two domains in their peptide chains: a toxic N-terminal domain and an uptake C-terminal domain. This modular design would be shared by other microcins from each subgroup. [Abstract/Link to Full Text]

Serrano-Gómez D, Martínez-Nuńez RT, Sierra-Filardi E, Izquierdo N, Colmenares M, Pla J, Rivas L, Martinez-Picado J, Jimenez-Barbero J, Alonso-Lebrero JL, González S, Corbí AL
AM3 modulates dendritic cell pathogen recognition capabilities by targeting DC-SIGN.
Antimicrob Agents Chemother. 2007 Jul;51(7):2313-23.
AM3 (Inmunoferon) is an orally effective immunomodulator that influences the regulatory and effector functions of the immune system whose molecular mechanisms of action are mostly unknown. We hypothesized that the polysaccharide moiety of AM3 (IF-S) might affect immune responses by modulating the lectin-dependent pathogen recognition abilities of human dendritic cells. IF-S inhibited binding of viral, fungal, and parasite pathogens by human monocyte-derived dendritic cells in a dose-dependent manner. IF-S specifically impaired the pathogen recognition capabilities of DC-SIGN, as it reduced the attachment of Candida, Aspergillus, and Leishmania to DC-SIGN transfectants. IF-S also inhibited the interaction of DC-SIGN with both its cellular counterreceptor (intercellular adhesion molecule 3) and the human immunodeficiency virus (HIV) type 1 gp120 protein and blocked the DC-SIGN-dependent capture of HIV virions and the HIV trans-infection capability of DC-SIGN transfectants. IF-S promoted DC-SIGN internalization in DCs without affecting mannose receptor expression, and (1)D saturation transfer difference nuclear magnetic resonance demonstrated that IF-S directly interacts with DC-SIGN on the cell surface. Therefore, the polysaccharide moiety of AM3 directly influences pathogen recognition by dendritic cells by interacting with DC-SIGN. Our results indicate that DC-SIGN is the target for an immunomodulator and imply that the adjuvant and immunomodulatory actions of AM3 are mediated, at least in part, by alteration of the DC-SIGN functional activities. [Abstract/Link to Full Text]

Legrand E, Demar M, Volney B, Ekala MT, Quinternet M, Bouchier C, Fandeur T, Rogier C, Carme B, Puijalon OM, Esterre P
First case of emergence of atovaquone resistance in Plasmodium falciparum during second-line atovaquone-proguanil treatment in South America.
Antimicrob Agents Chemother. 2007 Jun;51(6):2280-1. [Abstract/Link to Full Text]

Focher F, Lossani A, Verri A, Spadari S, Maioli A, Gambino JJ, Wright GE, Eberle R, Black DH, Medveczky P, Medveczky M, Shugar D
Sensitivity of monkey B virus (Cercopithecine herpesvirus 1) to antiviral drugs: role of thymidine kinase in antiviral activities of substrate analogs and acyclonucleosides.
Antimicrob Agents Chemother. 2007 Jun;51(6):2028-34.
Herpes B virus (B virus [BV]) is a macaque herpesvirus that is occasionally transmitted to humans where it can cause rapidly ascending encephalitis that is often fatal. To understand the low susceptibility of BV to the acyclonucleosides, we have cloned, expressed, and characterized the BV thymidine kinase (TK), an enzyme that is expected to "activate" nucleoside analogs. This enzyme is similar in sequence and properties to the TK of herpes simplex virus (HSV), i.e., it has a broad substrate range and low enantioselectivity and is sensitive to inhibitors of HSV TKs. The BV enzyme phosphorylates some modified nucleosides and acyclonucleosides and l enantiomers of thymidine and related antiherpetic analogs. However, the potent anti-HSV drugs acyclovir (ACV), ganciclovir (GCV), and 5-bromovinyldeoxyuridine were poorly or not phosphorylated by the BV enzyme under the experimental conditions. The antiviral activities of a number of marketed antiherpes drugs and experimental compounds were compared against BV strains and, for comparison, HSV type 1 (HSV-1) in Vero cell cultures. For most compounds tested, BV was found to be about as sensitive as HSV-1 was. However, BV was less sensitive to ACV and GCV than HSV-1 was. The abilities of thymidine analogs and acyclonucleosides to inhibit replication of BV in Vero cell culture were not always proportional to their substrate properties for BV TK. Our studies characterize BV TK for the first time and suggest new lead compounds, e.g., 5-ethyldeoxyuridine and pencyclovir, which may be superior to ACV or GCV as treatment for this emerging infectious disease. [Abstract/Link to Full Text]

Chamilos G, Lewis RE, Albert N, Kontoyiannis DP
Paradoxical effect of Echinocandins across Candida species in vitro: evidence for echinocandin-specific and candida species-related differences.
Antimicrob Agents Chemother. 2007 Jun;51(6):2257-9.
Paradoxical growth of some Candida isolates occurs at concentrations above the MIC for echinocandins. In 60 Candida bloodstream isolates from cancer patients (20 C. albicans isolates and 10 isolates each of C. parapsilosis, C. tropicalis, C. krusei, and C. glabrata), paradoxical growth was more frequent with caspofungin than micafungin or anidulafungin, was unrelated to MIC, and was strikingly absent in C. glabrata isolates. [Abstract/Link to Full Text]

Müller J, Wastling J, Sanderson S, Müller N, Hemphill A
A novel Giardia lamblia nitroreductase, GlNR1, interacts with nitazoxanide and other thiazolides.
Antimicrob Agents Chemother. 2007 Jun;51(6):1979-86.
The nitrothiazole analogue nitazoxanide [NTZ; 2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] represents the parent compound of a class of drugs referred to as thiazolides and exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. NTZ and other thiazolides are active against a wide range of other intracellular and extracellular protozoan parasites in vitro and in vivo, but their mode of action and respective subcellular target(s) have only recently been investigated. In order to identify potential targets of NTZ and other thiazolides in Giardia lamblia trophozoites, we have developed an affinity chromatography system using the deacetylated derivative of NTZ, tizoxanide (TIZ), as a ligand. Affinity chromatography on TIZ-agarose using cell extracts of G. lamblia trophozoites resulted in the isolation of an approximately 35-kDa polypeptide, which was identified by mass spectrometry as a nitroreductase (NR) homologue (EAA43030.1). NR was overexpressed as a six-histidine-tagged recombinant protein in Escherichia coli, purified, and then characterized using an assay for oxygen-insensitive NRs with dinitrotoluene as a substrate. This demonstrated that the NR was functionally active, and the protein was designated GlNR1. In this assay system, NR activity was severely inhibited by NTZ and other thiazolides, demonstrating that the antigiardial activity of these drugs could be, at least partially, mediated through inhibition of GlNR1. [Abstract/Link to Full Text]

Doh-Ura K, Kuge T, Uomoto M, Nishizawa K, Kawasaki Y, Iha M
Prophylactic effect of dietary seaweed Fucoidan against enteral prion infection.
Antimicrob Agents Chemother. 2007 Jun;51(6):2274-7.
Dietary seaweed fucoidan delays the onset of disease of enterally infected mice with scrapie when given orally for 6 days after infection, but not when given before the infection. This effect was not modified at a tested fucoidan dose range and appeared to reach the maximum level at a concentration of 2.5% or less in feed. Daily uptake of fucoidan might be prophylactic against prion diseases caused by ingestion of prion-contaminated materials, although further evaluation of its pharmacology remains to be done. [Abstract/Link to Full Text]

Arias CA, Singh KV, Panesso D, Murray BE
Time-kill and synergism studies of ceftobiprole against Enterococcus faecalis, including beta-lactamase-producing and vancomycin-resistant isolates.
Antimicrob Agents Chemother. 2007 Jun;51(6):2043-7.
Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of Enterococcus faecalis (including 16 beta-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10(7) CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two beta-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 microg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of </=1 and </=4 microg/ml, respectively, at the standard inoculum concentration. Ceftobiprole MICs at a high inoculum concentration for a subset of five Bla(+) E. faecalis isolates were </=1 microg/ml. Bactericidal activity was observed against four isolates tested at concentrations as low as 1 microg/ml regardless of the production of beta-lactamase or vancomycin resistance. A combination of ceftobiprole (0.5 microg/ml) and streptomycin (25 microg/ml) was synergistic against Bla(+) TX0630 and TX5070. Ceftobiprole (0.5 microg/ml) plus gentamicin (10 microg/ml) was synergistic against VanB isolate TX2484 and showed enhanced killing, but not synergism, against TX2784 (VanA), despite the absence of high-level resistance to gentamicin. In conclusion, ceftobiprole exhibited good in vitro activity against E. faecalis, including Bla(+) and vancomycin-resistant strains, and exhibited synergism with aminoglycosides against selected isolates. [Abstract/Link to Full Text]

Kwon DH, Lu CD
Polyamine effects on antibiotic susceptibility in bacteria.
Antimicrob Agents Chemother. 2007 Jun;51(6):2070-7.
Biogenic polyamines (e.g., spermidine and spermine) are a group of essential polycationic compounds found in all living cells. The effects of spermine and spermidine on antibiotic susceptibility were examined with gram-negative Escherichia coli and Salmonella enterica serovar Typhimurium bacteria and clinical isolates of Pseudomonas aeruginosa and with gram-positive Staphylococcus aureus bacteria, including methicillin-resistant S. aureus (MRSA). Exogenous spermine exerted a dose-dependent inhibition effect on the growth of E. coli, S. enterica serovar Typhimurium, and S. aureus but not P. aeruginosa, as depicted by MIC and growth curve measurements. While the MICs of polymyxin and ciprofloxacin were in general increased by exogenous spermine and spermidine in P. aeruginosa, this adverse effect was not observed in enteric bacteria and S. aureus. It was found that spermine and spermidine can decrease the MICs of beta-lactam antibiotics in all strains as well as other types of antibiotics in a strain-dependent manner. Significantly, the MICs of oxacillin for MRSA Mu50 and N315 were decreased more than 200-fold in the presence of spermine, and this effect of spermine was retained when assessed in the presence of divalent ions (magnesium or calcium; 3 mM) or sodium chloride (150 mM). The effect of spermine on the sensitization of P. aeruginosa and MRSA to antibiotics was further demonstrated by population analysis and time-killing assays. The results of checkerboard assays with E. coli and S. aureus indicated a strong synergistic effect of spermine in combination with beta-lactams and chloramphenicol. The decreased MICs of beta-lactams implied that the possible blockage of outer membrane porins by exogenous spermine or spermidine did not play a crucial role in most cases. In contrast, only the MIC of imipenem against P. aeruginosa was increased by exogenous spermine and spermidine, and this resistance effect was abolished in a mutant strain devoid of the outer membrane porin OprD. In E. coli, the MICs of carbenicillin, chloramphenicol, and tetracycline were decreased in two acrA mutants devoid of a major efflux pump, AcrAB. However, retention of the spermine effect on antibiotic susceptibility in two acrA mutants of E. coli suggested that the AcrAB efflux pump was not the target for a synergistic effect by spermine and antibiotics and ruled out the hypothesis of spermine serving as an efflux pump inhibitor in this organism. In summary, this interesting finding of the effect of spermine on antibiotic susceptibility provides the basis for a new potential approach against drug-resistant pathogens by use of existing beta-lactam antibiotics. [Abstract/Link to Full Text]

San Millan A, Escudero JA, Catalan A, Nieto S, Farelo F, Gibert M, Moreno MA, Dominguez L, Gonzalez-Zorn B
Beta-lactam resistance in Haemophilus parasuis Is mediated by plasmid pB1000 bearing blaROB-1.
Antimicrob Agents Chemother. 2007 Jun;51(6):2260-4.
beta-Lactam resistance in Haemophilus parasuis is an emerging phenomenon that has not yet been characterized from a molecular perspective. Clinical high-level beta-lactam-resistant isolates from Spain bore a novel plasmid, pB1000, expressing a functionally active ROB-1 beta-lactamase. Pulsed-field gel electrophoresis was applied for the first time to H. parasuis and showed that beta-lactam resistance is due to clonal spread of a resistant strain, BB1018, bearing pB1000. [Abstract/Link to Full Text]

---

Recent Articles in Malaria Journal

---

Skorokhod OA, Schwarzer E, Ceretto M, Arese P
Malarial pigment haemozoin, IFN-gamma, TNF-alpha, IL-1beta and LPS do not stimulate expression of inducible nitric oxide synthase and production of nitric oxide in immuno-purified human monocytes.
Malar J. 2007;673.
BACKGROUND: Enhanced production of nitric oxide (NO) following upmodulation of the inducible isoform of NO synthase (iNOS) by haemozoin (HZ), inflammatory cytokines and LPS may provide protection against Plasmodium falciparum malaria by killing hepatic and blood forms of parasites and inhibiting the cytoadherence of parasitized erythrocytes (RBC) to endothelial cells. Monocytes and macrophages are considered to contribute importantly to protective upregulation of iNOS and production of NO. Data obtained with murine phagocytes fed with human HZ and synthetic HZ (sHZ) indicate that supplemental treatment of those cells with IFN-gamma elicited significant increases in protein and mRNA expression of iNOS and NO production, providing a potential mechanism linking HZ phagocytosis and increased production of NO. Purpose of this study was to analyse the effect of P. falciparum HZ and sHZ supplemental to treatment with IFN-gamma and/or a stimulatory cytokine-LPS mix on iNOS protein and mRNA expression in immuno-purified human monocytes. METHODS: Adherent immunopurified human monocytes (purity >85%), and murine phagocytic cell lines RAW 264.7, N11 and ANA1 were fed or not with P. falciparum HZ or sHZ and treated or not with IFN-gamma or a stimulatory cytokine-LPS mix. Production of NO was quantified in supernatants, iNOS protein and mRNA expression were measured after immunoprecipitation and Western blotting and quantitative RT-PCT, respectively. RESULTS: Phagocytosis of HZ/sHZ by human monocytes did not increase iNOS protein and mRNA expression and NO production either after stimulation by IFN-gamma or the cytokine-LPS mix. By contrast, in HZ/sHZ-laden murine macrophages, identical treatment with IFN-gamma and the cytokine-LPS mix elicited significant increases in protein and mRNA expression of iNOS and NOS metabolites production, in agreement with literature data. CONCLUSION: Results indicate that human monocytes fed or not with HZ/sHZ were constantly unable to express iNOS and generate NOS metabolites even after stimulation with IFN-gamma or a cytokine-LSP mix that were very active on HZ-fed murine phagocytic lines. Present data do not support the hypothesis that monocytes are mediators of anti-parasitic defence in clinical malaria via activation of iNOS and production of NO, and suggest caution in extrapolating data obtained with murine or hybrid systems to human malaria. [Abstract/Link to Full Text]

Amin AA, Zurovac D, Kangwana BB, Greenfield J, Otieno DN, Akhwale WS, Snow RW
The challenges of changing national malaria drug policy to artemisinin-based combinations in Kenya.
Malar J. 2007;672.
BACKGROUND: Sulphadoxine/sulphalene-pyrimethamine (SP) was adopted in Kenya as first line therapeutic for uncomplicated malaria in 1998. By the second half of 2003, there was convincing evidence that SP was failing and had to be replaced. Despite several descriptive investigations of policy change and implementation when countries moved from chloroquine to SP, the different constraints of moving to artemisinin-based combination therapy (ACT) in Africa are less well documented. METHODS: A narrative description of the process of anti-malarial drug policy change, financing and implementation in Kenya is assembled from discussions with stakeholders, reports, newspaper articles, minutes of meetings and email correspondence between actors in the policy change process. The narrative has been structured to capture the timing of events, the difficulties and hurdles faced and the resolutions reached to the final implementation of a new treatment policy. RESULTS: Following a recognition that SP was failing there was a rapid technical appraisal of available data and replacement options resulting in a decision to adopt artemether-lumefantrine (AL) as the recommended first-line therapy in Kenya, announced in April 2004. Funding requirements were approved by the Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) and over 60 million US$ were agreed in principle in July 2004 to procure AL and implement the policy change. AL arrived in Kenya in May 2006, distribution to health facilities began in July 2006 coincidental with cascade in-service training in the revised national guidelines. Both training and drug distribution were almost complete by the end of 2006. The article examines why it took over 32 months from announcing a drug policy change to completing early implementation. Reasons included: lack of clarity on sustainable financing of an expensive therapeutic for a common disease, a delay in release of funding, a lack of comparative efficacy data between AL and amodiaquine-based alternatives, a poor dialogue with pharmaceutical companies with a national interest in antimalarial drug supply versus the single sourcing of AL and complex drug ordering, tendering and procurement procedures. CONCLUSION: Decisions to abandon failing monotherapy in favour of ACT for the treatment of malaria can be achieved relatively quickly. Future policy changes in Africa should be carefully prepared for a myriad of financial, political and legislative issues that might limit the rapid translation of drug policy change into action. [Abstract/Link to Full Text]

Yé Y, Kimani-Murage E, Kebaso J, Mugisha F
Assessing the risk of self-diagnosed malaria in urban informal settlements of Nairobi using self-reported morbidity survey.
Malar J. 2007;671.
BACKGROUND: Because of the belief that Nairobi is a low risk zone for malaria, little empirical data exists on malaria risk in the area. The aim of this study was to explore the risk of perceived malaria and some associated factors in Nairobi informal settlements using self-reported morbidity survey. METHODS: The survey was conducted from May to August 2004 on 7,288 individuals in two informal settlements of Nairobi. Participants were asked to report illnesses they experienced in the past 14 days. Logistic regression was used to estimate the odds of perceived-malaria. The model included variables such as site of residence, age, ethnicity and number of reported symptoms. RESULTS: Participants reported 165 illnesses among which malaria was the leading cause (28.1%). The risk of perceived-malaria was significantly higher in Viwandani compared to Korogocho (OR 1.61, 95%CI: 1.10-2.26). Participants in age group 25-39 years had significantly higher odds of perceived-malaria compared to those under-five years (OR 2.07, 95%CI: 1.43-2.98). The Kikuyu had reduced odds of perceived-malaria compared to other ethnic groups. Individuals with five and more symptoms had higher odds compared to those with no symptoms (OR 23.69, 95%CI: 12.98-43.23). CONCLUSION: Malaria was the leading cause of illness as perceived by the residents in the two informal settlements. This was rational as the number of reported symptoms was highly associated with the risk of reporting the illness. These results highlight the need for a more comprehensive assessment of malaria epidemiology in Nairobi to be able to offer evidence-based guidance to policy on malaria in Kenya and particularly in Nairobi. [Abstract/Link to Full Text]

Na-Bangchang K, Ruengweerayut R, Karbwang J, Chauemung A, Hutchinson D
Pharmacokinetics and pharmacodynamics of fosmidomycin monotherapy and combination therapy with clindamycin in the treatment of multidrug resistant falciparum malaria.
Malar J. 2007;670.
BACKGROUND: The study investigated the pharmacokinetics of fosmidomycin when given alone and in combination with clindamycin in patients with acute uncomplicated falciparum malaria. METHODS: A total of 15 and 18 patients with acute uncomplicated Plasmodium falciparum malaria who fulfilled the enrollment criteria were recruited from out-patient department of Mae Sot Hospital, Tak Province, Thailand. Patients were treated with monotherapy with fosmidomycin at the dose of 1,200 mg every 8 hours for 7 days (n = 15) or combination therapy with fosmidomycin (900 mg every 12 hours for 7 days) and clindamycin (600 mg every 12 hours for 7 days) (n = 18). Blood samples were taken for pharmacokinetic investigations of clindamycin and/or fosmidomycin and 24-hour urine samples were collected during dosing period. Efficacy assessments included clinical and parasitological evaluation. Safety and tolerability were assessed based on clinical and laboratory investigations. RESULTS: Both mono- and combination therapy regimens of fosmidomycin were well tolerated with no serious adverse events. Combination therapy with fosmidomycin and clindamycin was proven highly effective with 100% cure rate, whereas cure rate of monotherapy was 22% (28-day follow up). Pharmacokientics of fosmidomycin following mono- and combination therapy were similar except Vz/F and CL/F, which were significantly smaller in the combination regimen. Plasma concentration-time profiles of both fosmidomycin and clindamycin were best fit with a one-compartment open model with first-order absorption and elimination and with absorption lag time. Steady-state plasma concentrations of fosmidomycin and clindamycin were attained at about the second or third dose. There was no evidence of dose accumulation during multiple dosing. Urinary recovery of fosmidomycin was 18.7 and 20% following mono- and combination therapy, respectively. CONCLUSION: Pharmacokinetic dose optimization of fosmidomycin-clindamycin combination therapy with the course of treatment of not longer than three days is required to obtain a regimen which is safe and produced 100% cure for multidrug-resistant P. falciparum. [Abstract/Link to Full Text]

Futagbi G, Welbeck JE, Tetteh JK, Hviid L, Akanmori BD
Selective activation of TCR-gammadelta+ cells in endemic Burkitt's lymphoma.
Malar J. 2007;669.
BACKGROUND: The overlap in geographical distribution of Plasmodium falciparum malaria and endemic Burkitt's lymphoma (eBL)--an aggressive Epstein-Barr virus (EBV)-associated B-cell tumour occurring almost exclusively in the tropics--strongly suggests a link between the two diseases. It is suspected that the polyclonal B-cell activation in P. falciparum malaria may precipitate a breakdown in homeostatic T-cell control of EBV-immortalized B-cell proliferation. Previous studies have suggested that a particular T-cell subset, characterized by expression of Vdelta1+ gammadelta T-cell receptors, is important for maintaining B-cell homeostasis, both in P. falciparum- exposed populations and in individuals subject to polyclonal B-cell activation of other aetiology. The objective of the present study was, therefore, to characterize lymphocyte phenotypes and to investigate possible differences in T-cell subset composition and activation status in P. falciparum-exposed Ghanaian children with and without eBL. METHODS: Venous blood samples in heparin from 21 eBL patients (mean age: 7.0 years; range: 3-11 years), referred to the Burkitt's Tumour Centre at Korle-Bu Teaching Hospital, Accra and 15 healthy, age and sex matched children, were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, R-phycoerythrin (RPE)- and RPE-Cy5-conjugated antibodies (CD3, CD4, CD8, CD25, CD69, CD95, HLA-DR, TCR-gammadelta, Vdelta1, Vdelta3, Vgamma9 and B-cells) and acquired on a flow cytometer. RESULTS: A reduction in the proportion of CD3+ cells in eBL patients, due mainly to perturbations among TCR-gammadelta+ cells was observed. In contrast, the proportions of CD4+ or CD8+ cells were relatively unaffected, as were the mean numbers of peripheral blood mononuclear cells. CONCLUSION: Selective changes in numbers and activation status of TCR-gammadelta+ cells occurs in Ghanaian children with eBL, a pattern which is similar to P. falciparum-induced changes. The data supports the hypothesis of a regulatory role for Vdelta1+ TcR-gammadelta T-cells in maintaining B-cell homeostasis and provides insights into the pathogenesis of eBL. [Abstract/Link to Full Text]

Thera MA, Keita F, Sissoko MS, Traoré OB, Coulibaly D, Sacko M, Lameyre V, Ducret JP, Doumbo O
Acceptability and efficacy of intra-rectal quinine alkaloids as a pre-transfer treatment of non-per os malaria in peripheral health care facilities in Mopti, Mali.
Malar J. 2007;668.
BACKGROUND: The acceptability and efficacy of a new kit with a new formulation of quinine alkaloids designed for the intra-rectal administration in the treatment of non-per os malaria was assessed in the peripheral health care system of Mopti, Mali. METHODS: A single-arm trial was conducted from August 2003 to January 2004. An initial dose of diluted quinine alkaloids (20 mg/kg Quinimax) was administered by the intra-rectal route to children with presumptive non per-os malaria at six peripheral heath care centres. The children were then referred to two referral hospitals where standard inpatient care including intravenous route were routinely provided. A malaria thick smear was done at inclusion and a second malaria thick smear after arrival at the referral facility, where a more complete clinical examination and laboratory testing was done to confirm diagnosis. Confirmed cases of severe malaria or others diseases were treated according to national treatment guidelines. Cases of non per-os malaria received a second dose of intra rectal quinine alkaloids. Primary outcome was acceptability of the intra rectal route by children and their parents as well as the ease to handle the kit by health care workers. RESULTS: The study included 134 children with a median age of 33 months and 53.7% were male. Most of the children (67%) and 92% of parents or guardians readily accepted the intra-rectal route; 84% of health care workers found the kit easy to use. At the peripheral health care centres, 32% of children had a coma score < or = 3 and this was reduced to 10% at the referral hospital, following one dose of intra-rectal quinine alkaloids (IRQA). The mean time to availability of oral route treatment was 1.8 +/- 1.1 days. Overall, 73% of cases were confirmed severe malaria and for those the case fatality rate was 7.2%. CONCLUSION: IRQA was well accepted by children, their parents/guardians and by the health workers at peripheral health facilities in Mopti, Mali. There was also a quick recovery from deep coma and a reduced case fatality rate in severe malaria. [Abstract/Link to Full Text]

Carneiro IA, Drakeley CJ, Owusu-Agyei S, Mmbando B, Chandramohan D
Haemoglobin and haematocrit: is the threefold conversion valid for assessing anaemia in malaria-endemic settings?
Malar J. 2007;667.
BACKGROUND: Anaemic status is determined by haemoglobin using the HemoCue system or haematocrit measurements, and a threefold conversion is commonly used to equate the two measures (haemoglobin = haematocrit/3). The validity of this conversion in malaria endemic settings was assessed. METHODS: Concurrent measures of haemoglobin and centrifuged haematocrit in children aged 6-59 months were compared by modelling the difference between the two measures against their average. A random effects linear regression of the difference of the measures on their average was used to describe the line of best agreement and 95% limits of agreement for these two measures over a range of values after adjusting for statistically significant covariates. RESULTS: There was a consistent bias between the two measures, with haemoglobin less than haematocrit/3 in 87% (899/1,030) of observations. This difference was non-uniform, decreasing with the average measure, i.e. less difference at higher haemoglobin and haematocrit values. In these studies, use of haematocrit would have underestimated the prevalence of anaemia by misclassifying 10% (89/920) of individuals with haemoglobin < 11 g/dl, 66% (252/380) of individuals with haemoglobin < 8 g/dl and 100% (23/23) of individuals with haemoglobin < 5 g/dl. The mean difference between the measures was greater in males than females, increased with age between 6-59 months, and was greater in the wet than dry season suggesting that the relationship between haemoglobin and haematocrit may be modified by exposure to malaria. CONCLUSION: The regression model indicated that the standard threefold conversion from haematocrit to haemoglobin underestimates the prevalence of haemoglobin < 11 g/dl in children under five years of age in malaria endemic settings. This bias was more acute for more severe anaemia defined by haemoglobin < 8 g/dl and haemoglobin < 5 g/dl. This has important implications for the comparability of studies using these different measures. Direct determination of haemoglobin should be the measurement of choice for assessing anaemia outcomes in malaria intervention trials and surveys. [Abstract/Link to Full Text]

Sharling L, Sowa KM, Thompson J, Kyriacou HM, Arnot DE
Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites.
Malar J. 2007;666.
BACKGROUND: Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. METHODS: After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. RESULTS: Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in surface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. CONCLUSION: Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations. [Abstract/Link to Full Text]

Ménard D, Andrianina NN, Ramiandrasoa Z, Randriamanantena A, Rasoarilalao N, Jahevitra M, Ratsimbasoa A, Tuseo L, Raveloson A
Randomized clinical trial of artemisinin versus non-artemisinin combination therapy for uncomplicated falciparum malaria in Madagascar.
Malar J. 2007;665.
BACKGROUND: Data concerning antimalarial combination treatment for uncomplicated malaria in Madagascar are largely lacking. Randomized clinical trial was designed to assess therapeutic efficacies of chloroquine (CQ), amodiaquine (AQ), sulphadoxine-pyrimethamine (SP), amodiaquine plus sulphadoxine-pyrimethamine combination (AQ+SP) and artesunate plus amodiaquine combination (AQ+AS). METHODS: 287 children between 6 months and 15 years of age, with uncomplicated falciparum malaria, were enrolled in the study. Primary endpoints were the day-14 and day-28 risks of parasitological failure, either unadjusted or adjusted by genotyping. RESULTS: All treatment regimens, except for CQ treatment, gave clinical cure rates above 97% by day-14 and 92% by day-28 (PCR-corrected). AQ+SP was as effective as AQ+AS. The risk of new infection within the month after therapy was generally higher for AQ+AS than AQ+SP. CONCLUSION: These findings show that the inexpensive and widely available combination AQ+SP may be valuable in for the treatment of uncomplicated malaria in Madagascar and could have an important role in this country, where much of the drugs administered go to patients who do not have malaria. [Abstract/Link to Full Text]

Williams M, Louw AI, Birkholtz LM
Deletion mutagenesis of large areas in Plasmodium falciparum genes: a comparative study.
Malar J. 2007;664.
BACKGROUND: The increasing emergence of Plasmodium falciparum parasites resistant to most of the cost-effective drugs has necessitated the identification of novel leads and drug targets. Parasite-specific inserts in enzymes that are essential for the differentiation and proliferation of malarial parasites have received considerable interest since it distinguishes these proteins from their human counterparts. The functions of these inserts, which include mediations of protein activities or protein-protein interactions, are being investigated by several strategies including deletion mutagenesis. A comparative study of five widely used PCR-based mutagenesis methods identified a modified inverse PCR method as particularly suitable for the deletion of large areas (>100 bp) in malaria parasite genes. METHODS: The restriction enzyme-mediated inverse PCR method described here incorporates unique restriction enzyme sites at the 5'-ends of inverse tail-to-tail primers. The entire gene-containing vector is amplified except the desired region to be deleted and cloned using the unique restriction sites to increase ligation efficiency. This method was compared in its efficiency to delete a ~400 bp parasite-specific insert in malarial S-adenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC) to existing PCR-based site-directed deletion mutagenesis methods including the QuickChange site-directed mutagenesis, ExSite, overlapping primer and inverse PCR. In addition, the modified method was applied in the deletion of a >600 bp parasite-specific insert in another malarial gene, pyridoxal kinase (PfPdxK). RESULTS: The modified and optimized restriction enzyme-mediated inverse PCR method resulted in 80% compared to 40% deletion mutagenesis efficiency of the overlapping primer method in the deletion of a large area (411 bp) from a large malaria gene (PfAdoMetDC/ODC, gene size 4257 bp). In contrast, deletion mutagenesis methods such as the well-known QuickChange site-directed mutagenesis, ExSite and inverse PCR methods produced insignificant results. A 100% mutagenesis efficiency was obtained with the restriction enzyme-mediated inverse PCR method to delete 618 bp from a smaller gene (PfPdxK, gene size 1536 bp). CONCLUSION: An efficient method was developed for the deletion of large areas (>100 bp) in significantly sized genes such as those of the A+T-rich P. falciparum genome. [Abstract/Link to Full Text]

Okumu FO, Knols BG, Fillinger U
Larvicidal effects of a neem (Azadirachta indica) oil formulation on the malaria vector Anopheles gambiae.
Malar J. 2007;663.
BACKGROUND: Larviciding is a key strategy used in many vector control programmes around the world. Costs could be reduced if larvicides could be manufactured locally. The potential of natural products as larvicides against the main African malaria vector, Anopheles gambiae s.s was evaluated. METHODS: To assess the larvicidal efficacy of a neem (Azadirachta indica) oil formulation (azadirachtin content of 0.03% w/v) on An. gambiae s.s., larvae were exposed as third and fourth instars to a normal diet supplemented with the neem oil formulations in different concentrations. A control group of larvae was exposed to a corn oil formulation in similar concentrations. RESULTS: Neem oil had an LC50 value of 11 ppm after 8 days, which was nearly five times more toxic than the corn oil formulation. Adult emergence was inhibited by 50% at a concentration of 6 ppm. Significant reductions on growth indices and pupation, besides prolonged larval periods, were observed at neem oil concentrations above 8 ppm. The corn oil formulation, in contrast, produced no growth disruption within the tested range of concentrations. CONCLUSION: Neem oil has good larvicidal properties for An. gambiae s.s. and suppresses successful adult emergence at very low concentrations. Considering the wide distribution and availability of this tree and its products along the East African coast, this may prove a readily available and cheap alternative to conventional larvicides. [Abstract/Link to Full Text]

Karunaweera N, Wanasekara D, Chandrasekharan V, Mendis K, Carter R
Plasmodium vivax: paroxysm-associated lipids mediate leukocyte aggregation.
Malar J. 2007;662.
BACKGROUND: Paroxysms are recurrent febrile episodes, characteristic of Plasmodium vivax infections, which coincide with the rupture of schizont-infected erythrocytes in the patients' circulation. The present study describes the formation of prominent aggregates of leukocytes in vitro in the presence of parasite and host factors released during paroxysms. METHODS: Whole blood cells from uninfected malaria-naďve donors were incubated with plasma taken during a paroxysm or normal human plasma as a control and cell smears were observed under the microscope for the presence of leukocyte aggregates. Plasma factors involved in mediating the leukocyte aggregation were identified using immune depletion and reconstitution experiments. Furthermore, biochemical characterization was carried out to determine the chemical nature of the active moieties in plasma present during paroxysms. RESULTS: Leukocyte aggregates were seen exclusively when cells were incubated in plasma collected during a paroxysm. Immune depletion and reconstitution experiments revealed that the host cytokines TNF-alpha, GM-CSF, IL-6 and IL-10 and two lipid fractions of paroxysm plasma comprise the necessary and sufficient mediators of this phenomenon. The two lipid components of the paroxysm plasmas speculated to be of putative parasite origin, were a phospholipid-containing fraction and another containing cholesterol and triglycerides. The phospholipid fraction was dependent upon the presence of cytokines for its activity unlike the cholesterol/triglyceride-containing fraction which in the absence of added cytokines was much more active than the phospholipids fraction. The biological activity of the paroxysm plasmas from non-immune patients who presented with acute P. vivax infections was neutralized by immune sera raised against schizont extracts of either P. vivax or Plasmodium falciparum. However, immune sera against P. vivax were more effective than that against P. falciparum indicating that the parasite activity involved may be antigenically at least partially parasite species-specific. CONCLUSION: Leukocyte aggregation was identified as associated with paroxysms in P. vivax infections. This phenomenon is mediated by plasma factors including host-derived cytokines and lipids of putative parasite origin. The characteristics of the phospholipid fraction in paroxysm plasma are congruent with those of the parasite-derived, TNF-inducing GPI moieties described by others. The more active cholesterol/triglyceride(s), however, represent a novel malarial toxin, which is a new class of biologically active lipid associated with the paroxysm of P. vivax malaria. [Abstract/Link to Full Text]

Glushakova S, Yin D, Gartner N, Zimmerberg J
Quantification of malaria parasite release from infected erythrocytes: inhibition by protein-free media.
Malar J. 2007;661.
BACKGROUND: Intracellular malaria parasites leave their host erythrocytes to infect neighbouring cells after each cycle of asexual replication. No method is currently available for the direct quantification of parasite release. METHOD AND RESULTS: To quantify parasite release process, human erythrocytes infected with Plasmodium falciparum were injected into sealed chambers at optimal density, where they progressed through the end of the erythrocyte cycle. Each event of parasite release inside the chamber at the site of erythrocyte rupture leaves on the chamber wall a footprint, composed of 1) separated parasites, 2) a digestive vacuole with haemozoin, and 3) fragments of the ruptured membranes. These footprints are stable for hours, allowing precise identification using differential interference contrast (DIC) microscopy. The relative rate of parasite release is defined as the percent of such footprints out of all schizonts injected and incubated into chamber at 37 degrees C for two hours. The method is highly reproducible, easy to perform, and does not require expensive equipment. Additionally, this method allows one to analyse cell and release site morphology, which adds information about the release process and the quality of the culture. The method is used here to show that swelling of schizonts caused by protein-free media inhibits parasite release. CONCLUSION: In this study, a novel method is described to count sites of parasite release by microscopy. Besides the direct estimation of parasite release from infected erythrocytes, this method provides a morphological evaluation of normal infected cells approaching the end of the plasmodial life cycle, or pathological forms accumulated as the result of experimental intervention in the parasite release process. One may now accurately estimate the relative parasite release rate at the time of cycle transition, without any obligatory coupling to parasite invasion. [Abstract/Link to Full Text]

Joshi H, Valecha N, Verma A, Kaul A, Mallick PK, Shalini S, Prajapati SK, Sharma SK, Dev V, Biswas S, Nanda N, Malhotra MS, Subbarao SK, Dash AP
Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India.
Malar J. 2007;660.
BACKGROUND: Molecular techniques have facilitated the studies on genetic diversity of Plasmodium species particularly from field isolates collected directly from patients. The msp-1 and msp-2 are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the msp-1 gene and the central repetitive domain (block3) of the msp-2 gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among Plasmodium falciparum population of India, this report evaluates the extent of genetic diversity in the field isolates of P. falciparum in eastern and north-eastern regions of India. METHODS: A study was designed to assess the diversity of msp-1 and msp-2 among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa. RESULTS: P. falciparum isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of msp-1 and msp-2. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the P. falciparum populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of msp-1 was observed to be monomorphic with an allele size of 150/160 bp. The observed 80-90% sequence identity of Indian isolates with data of other regions suggests that Indian P. falciparum population is a mixture of different strains. CONCLUSION: The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of msp-1 (block 2) and msp-2 (central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity. [Abstract/Link to Full Text]

Carvalho LJ, Ferreira-da-Cruz MF, Daniel-Ribeiro CT, Pelajo-Machado M, Lenzi HL
Germinal center architecture disturbance during Plasmodium berghei ANKA infection in CBA mice.
Malar J. 2007;659.
BACKGROUND: Immune responses to malaria blood stage infection are in general defective, with the need for long-term exposure to the parasite to achieve immunity, and with the development of immunopathology states such as cerebral malaria in many cases. One of the potential reasons for the difficulty in developing protective immunity is the poor development of memory responses. In this paper, the potential association of cellular reactivity in lymphoid organs (spleen, lymph nodes and Peyer's patches) with immunity and pathology was evaluated during Plasmodium berghei ANKA infection in CBA mice. METHODS: CBA mice were infected with 1 x 10(6) P. berghei ANKA-parasitized erythrocytes and killed on days 3, 6-8 and 10 of infection. The spleen, lymph nodes and Peyer's patches were collected, fixed in Carson's formalin, cut in 5 mum sections, mounted in glass slides, stained with Lennert's Giemsa and haematoxylin-eosin and analysed with bright-field microscopy. RESULTS: Early (day 3) strong activation of T cells in secondary lymphoid organs was observed and, on days 6-8 of infection, there was overwhelming activation of B cells, with loss of conventional germinal center architecture, intense centroblast activation, proliferation and apoptosis but little differentiation to centrocytes. In the spleen, the marginal zone disappeared and the limits between the disorganized germinal center and the red pulp were blurred. Intense plasmacytogenesis was observed in the T cell zone. CONCLUSION: The observed alterations, especially the germinal center architecture disturbance (GCAD) with poor centrocyte differentiation, suggest that B cell responses during P. berghei ANKA infection in mice are defective, with potential impact on B cell memory responses. [Abstract/Link to Full Text]

Swarthout TD, Counihan H, Senga RK, van den Broek I
Paracheck-Pf accuracy and recently treated Plasmodium falciparum infections: is there a risk of over-diagnosis?
Malar J. 2007;658.
BACKGROUND: An assessment of the accuracy of Paracheck Pf, a malaria rapid diagnostic test (RDT) detecting histidine rich protein 2 was undertaken amongst children aged 6-59 months in eastern Democratic Republic of Congo. METHODS: This RDT assessment occurred in conjunction with an ACT efficacy trial. Febrile children were simultaneously screened with both RDT and high quality microscopy and those meeting inclusion criteria were followed for 35 days. RESULTS: 358 febrile children were screened with 180 children recruited for five weeks follow-up. On screening, the RDT accurately diagnosed all 235 true malaria cases, indicating 100% RDT sensitivity. Of the 123 negative slides, the RDT gave 59 false-positive results, indicating 52.0% (64/123) RDT specificity. During follow-up after treatment with an artemisinin-based combination therapy, 98.2% (110/112), 94.6% (106/112), 92.0% (103/112) and 73.5% (50/68) of effectively treated children were still false-positive by RDT at days 14, 21, 28 and 35, respectively. CONCLUSION: Results show that though the use of Paracheck-Pf is as sensitive as microscopy in detecting true malaria cases, a low specificity did present a high frequency of false-positive RDT results. What's more, a duration of RDT false-positivity was found that significantly surpassed the 'fortnight' after effective treatment reported by its manufacturer. Though further research is needed in assessing RDT accuracy, study results showing the presence of frequent false positivity should be taken into consideration to avoid clinicians inappropriately focusing on malaria, not identifying the true cause of illness, and providing unnecessary treatment. [Abstract/Link to Full Text]

Abuya TO, Mutemi W, Karisa B, Ochola SA, Fegan G, Marsh V
Use of over-the-counter malaria medicines in children and adults in three districts in Kenya: implications for private medicine retailer interventions.
Malar J. 2007;657.
BACKGROUND: Global malaria control strategies highlight the need to increase early uptake of effective antimalarials for childhood fevers in endemic settings, based on a presumptive diagnosis of malaria in this age group. Many control programmes identify private medicine sellers as important targets to promote effective early treatment, based on reported widespread inadequate childhood fever treatment practices involving the retail sector. Data on adult use of over-the-counter (OTC) medicines is limited. This study aimed to assess childhood and adult patterns of OTC medicine use to inform national medicine retailer programmes in Kenya and other similar settings. METHODS: Large-scale cluster randomized surveys of treatment seeking practices and malaria parasite prevalence were conducted for recent fevers in children under five years and recent acute illnesses in adults in three districts in Kenya with differing malaria endemicity. RESULTS: A total of 12, 445 households were visited and data collected on recent illnesses in 11, 505 children and 19, 914 adults. OTC medicines were the most popular first response to fever in children with fever (47.0%; 95% CI 45.5, 48.5) and adults with acute illnesses (56.8%; 95% CI 55.2, 58.3). 36.9% (95% CI 34.7, 39.2) adults and 22.7% (95% CI 20.9, 24.6) children using OTC medicines purchased antimalarials, with similar proportions in low and high endemicity districts. 1.9% (95% CI 0.8, 4.2) adults and 12.1% (95% CI 16.3,34.2) children used multidose antimalarials appropriately. Although the majority of children and adults sought no further treatment, self-referral to a health facility within 72 hours of illness onset was the commonest pattern amongst those seeking further help. CONCLUSION: In these surveys, OTC medicines were popular first treatments for fever in children or acute illnesses in adults. The proportions using OTC antimalarials were similar in areas of high and low malaria endemicity. In all districts, adults were more likely to self-treat with OTC antimalarial medicines than febrile children were to receive them, and less likely to use them in recommended ways. Government health centres were the most common second resort for treatment and were often used within 72 hours. In view of these practices, more research is needed to assess the impact on the popularity of private medicine sellers of strengthened public sector policies on access to malaria treatment and insecticide-treated bed nets. Improved targeting of OTC antimalarials to high risk groups, better communication strategies regarding adult as well as children's dosages, and facilitating more rapid referral to trained health workers where needed are important challenges to private medicine seller programmes. [Abstract/Link to Full Text]

Djouaka RF, Bakare AA, Bankole HS, Doannio JM, Kossou H, Akogbeto MC
Quantification of the efficiency of treatment of Anopheles gambiae breeding sites with petroleum products by local communities in areas of insecticide resistance in the Republic of Benin.
Malar J. 2007;656.
BACKGROUND: The emergence of Anopheles populations capable of withstanding lethal doses of insecticides has weakened the efficacy of most insecticide based strategies of vector control and, has highlighted the need for developing new insecticidal molecules or, improving the efficacy of existing insecticides or abandoning those to which resistance has emerged. The use of petroleum products (PP) against mosquito larvae had an immense success during early programmes of malaria control, but these compounds were abandoned and replaced in the 1950s by synthetic insecticides probably because of the high performances given by these new products. In the current context of vector resistance, it is important to elucidate the empirical use of PP by quantifying their efficiencies on resistant strains of Anopheles. METHODS: Larvae of Anopheles Ladji a local resistant strain were exposed to increasing concentrations of various PP (kerosene, petrol and engine oils) for 24 hours and the lethal activities recorded. The highest concentration (HiC) having no lethal activity (also referred as the NOEL or no effect level) and the lowest concentration (LoC100) yielding 100% mortality were rated for each PP on the Ladji strain. Prior to laboratory analysis, KAP studies were conducted in three traditional communities were insecticide resistance is clearly established to confirm the use of PP against mosquitoes. RESULTS: Laboratory analysis of petrol, kerosene and engine oils, clearly established their lethal activities on resistant strains of Anopheles larvae. Contrary to existing references, this research revealed that exposed larvae of Anopheles were mostly killed by direct contact toxicity and not by suffocation as indicated in some earlier reports. CONCLUSION: This research could serve as scientific basis to backup the empirical utilisation of PP on mosquito larvae and to envisage possibilities of using PP in some traditional settings where Anopheles have developed resistance to currently used insecticides. [Abstract/Link to Full Text]

Meremikwu M, Okomo U, Nwachukwu C, Oyo-Ita A, Eke-Njoku J, Okebe J, Oyo-Ita E, Garner P
Antimalarial drug prescribing practice in private and public health facilities in South-east Nigeria: a descriptive study.
Malar J. 2007;655.
BACKGROUND: Nigeria's national standard has recently moved to artemisinin combination treatments for malaria. As clinicians in the private sector are responsible for attending a large proportion of the population ill with malaria, this study compared prescribing in the private and public sector in one State in Nigeria prior to promoting ACTs. OBJECTIVE: To assess prescribing for uncomplicated malaria in government and private health facilities in Cross River State. METHOD: Audit of 665 patient records at six private and seven government health facilities in 2003. RESULTS: Clinicians in the private sector were less likely to record history or physical examination than those in public facilities, but otherwise practice and prescribing were similar. Overall, 45% of patients had a diagnostic blood slides; 77% were prescribed monotherapy, either chloroquine (30.2%), sulphadoxine-pyrimethamine (22.7%) or artemisinin derivatives alone (15.8%). Some 20.8% were prescribed combination therapy; the commonest was chloroquine with sulphadoxine-pyrimethamine. A few patients (3.5%) were prescribed sulphadoxine-pyrimethamine-mefloquine in the private sector, and only 3.0% patients were prescribed artemisinin combination treatments. CONCLUSION: Malaria treatments were varied, but there were not large differences between the public and private sector. Very few are following current WHO guidelines. Monotherapy with artemisinin derivatives is relatively common. [Abstract/Link to Full Text]

Bonnet M, Roper C, Félix M, Coulibaly L, Kankolongo GM, Guthmann JP
Efficacy of antimalarial treatment in Guinea: in vivo study of two artemisinin combination therapies in Dabola and molecular markers of resistance to sulphadoxine-pyrimethamine in N'Zérékoré.
Malar J. 2007;654.
BACKGROUND: In the last five years, countries have been faced with changing their malaria treatment policy to an artemisinin-based combination therapy (ACT), many with no national data on which to base their decision. This is particularly true for a number of West African countries, including Guinea, where these studies were performed. Two studies were conducted in 2004/2005 in programmes supported by Medecins Sans Frontieres, when chloroquine was still national policy, but artesunate (AS)/sulphadoxine-pyrimethamine (SP) had been used in refugee camps for two years. METHODS: In Dabola (central Guinea), 220 children aged 6-59 months with falciparum malaria were randomized to receive either AS/amodiaquine (AQ) or AS/SP. In vivo efficacy was assessed following the 2003 World Health Organization guidelines. In a refugee camp in Laine (south of Guinea), where an in vivo study was not feasible due to the unstable context, a molecular genotyping study in 160 patients assessed the prevalence of mutations in the dihydrofolate reductase (dhfr) (codons 108, 51, 59) and dihydropteroate synthase (dhps) (codons 436, 437, 540) genes of Plasmodium falciparum, which have been associated with resistance to pyrimethamine and sulphadoxine, respectively. RESULTS: In Dabola, after 28 days of follow-up, Polymerase Chain Reaction (PCR)-adjusted failure rates were 1.0% (95%CI 0-5.3) for AS/AQ and 1.0% (95%CI 0-5.5) for AS/SP. In the refugee camp in Laine, the molecular genotyping study found three dhfr mutations in 85.6% (95%CI 79.2-90.7) patients and quintuple dhfr/dhps mutations in 9.6% (95%CI 5.2-15.9). CONCLUSION: Both AS/AQ and AS/SP are highly efficacious in Dabola, whereas there is molecular evidence of established SP resistance in Laine. This supports the choice of the national programme of Guinea to adopt AS/AQ as first line antimalarial treatment. The results highlight the difficulties faced by control programmes, which have gone through the upheaval of implementing ACTs, but cannot predict how long their therapeutic life will be, especially in countries which have chosen drugs also available as monotherapies. [Abstract/Link to Full Text]

Smith TA
Measures of clinical malaria in field trials of interventions against Plasmodium falciparum.
Malar J. 2007;653.
BACKGROUND: Standard methods for defining clinical malaria in intervention trials in endemic areas do not guarantee that efficacy estimates will be unbiased, and do not indicate whether the intervention has its effect by modifying the force of infection, the parasite density, or the risk of pathology at given parasite density. METHODS: Three different sets, each of 500 Phase IIb or III malaria vaccine trials were simulated corresponding to each of a pre-erythrocytic, blood stage, and anti-disease vaccine, each in a population with 80% prevalence of patent malaria infection. Simulations considered only the primary effects of vaccination in a homogeneous trial population. The relationships between morbidity and parasite density and the performance of different case definitions for clinical malaria were analysed using conventional likelihood ratio tests to compare incidence of episodes defined using parasite density cut-offs. Bayesian latent class models were used to compare the overall frequencies of clinical malaria episodes in analyses that did not use diagnostic cut-offs. RESULTS: The different simulated interventions led to different relationships between clinical symptoms and parasite densities. Consequently, the operating characteristics of parasitaemia cut-offs in general differ between vaccine and placebo arms of the simulated trials, leading to different patterns of bias in efficacy estimates depending on the type of intervention effect. Efficacy was underestimated when low parasitaemia cut-offs were used but the efficacy of an asexual blood stage vaccine was overestimated when a high parasitaemia cut-off was used. The power of a trial may be maximal using case definitions that are associated with substantial bias in efficacy. CONCLUSION: Secondary analyses of the data of malaria intervention trials should consider the relationship between clinical symptoms and parasite density, and attempt to estimate overall numbers of clinical episodes and the degree of bias of the primary efficacy measure. Such analyses would help to clarify whether the effect of an intervention corresponds to that anticipated on the basis of the parasite stage that is targeted, and would highlight whether the primary measure of efficacy results from unexpected behaviour in the parasitological and clinical data used to estimate it. [Abstract/Link to Full Text]

Sharp BL, Ridl FC, Govender D, Kuklinski J, Kleinschmidt I
Malaria vector control by indoor residual insecticide spraying on the tropical island of Bioko, Equatorial Guinea.
Malar J. 2007;652.
BACKGROUND: A comprehensive malaria control intervention was initiated in February 2004 on Bioko Island, Equatorial Guinea. This manuscript reports on the continuous entomological monitoring of the indoor residual spray (IRS) programme during the first two years of its implementation. METHODS: Mosquitoes were captured daily using window traps at 16 sentinel sites and analysed for species identification, sporozoite rates and knockdown resistance (kdr) using polymerase chain reaction (PCR) to assess the efficacy of the vector control initiative from December 2003 to December 2005. RESULTS: A total of 2,807 and 10,293 Anopheles funestus and Anopheles gambiae s.l. respectively were captured throughout the study period. Both M and S molecular forms of An. gambiae s.s. and Anopheles melas were identified. Prior to the first round of IRS, sporozoite rates were 6.0, 8.3 and 4.0 for An. gambiae s.s., An. melas and An. funestus respectively showing An. melas to be an important vector in areas in which it occurred. After the third spray round, no infective mosquitoes were identified. After the first spray round using a pyrethroid spray the number of An. gambiae s.s. were not reduced due to the presence of the kdr gene but An funestus and An. melas populations declined from 23.5 to 3.1 and 5.3 to 0.8 per trap per 100 nights respectively. After the introduction of a carbamate insecticide in the second round, An. gambiae s.s. reduced from 25.5 to 1.9 per trap per 100 nights and An. funestus and An. melas remained at very low levels. Kdr was found only in the M-form of An. gambiae s.s. with the highest frequency at Punta Europa (85%). CONCLUSION: All three vectors that were responsible for malaria transmission before the start of the intervention were successfully controlled once an effective insecticide was used.Continuous entomological surveillance including resistance monitoring is of critical importance in any IRS based malaria vector control programme. This paper demonstrates that sufficient resources for such monitoring should be included in any proposal in order to avoid programme failures. [Abstract/Link to Full Text]

Gérardin P, Rogier C, Ka AS, Jouvencel P, Diatta B, Imbert P
Outcome of life-threatening malaria in African children requiring endotracheal intubation.
Malar J. 2007;651.
BACKGROUND: Little is known about children undergoing critical care for malaria. The purpose of this survey was to evaluate the outcome in African children requiring endotracheal intubation for life-threatening malaria. METHODS: All children with a primary diagnosis of severe malaria (2000 WHO definition) requiring endotracheal intubation, hospitalised over a five-year period, within a tertiary-care hospital in Dakar, Senegal, were enrolled in a retrospective cohort study. RESULTS: 83 consecutive patients were included (median PRISM h24 score: 14; IQR: 10-19, multiple organ dysfunctions: 91.5%). The median duration of ventilation was 36 hrs (IQR: 4-72). Indications for intubation were deep coma (Glasgow score </=7, n = 16), overt cortical or diencephalic injury, i.e, status epilepticus/decorticate posturing (n = 20), severe brainstem involvement, i.e., decerebrate posturing/opisthotonus (n = 15), shock (n = 15), cardiac arrest (n = 13) or acute lung injury (ALI) (PaO2/FiO2 <300 Torr, n = 4). Death occurred in 50 cases (case fatality rate (CFR), 60%) and was associated with multiple organ dysfunctions (median PELODh24 scores: 12.5 among non-survivors versus 11 among survivors, p = 0.02). Median PRISMh24 score was significantly lower when testing deep coma against other indications (10 vs 15, p < 0.001), ditto for PELODh24 score (2.5 vs 13, p = 0.02). Multivariate analysis identified deep coma as having a better outcome than other indications (CFR, 12.5% vs 40.0 to 93.3%, p < 0.0001). Decerebrate posturing/opisthotonus (CFR 73.3%, adjusted relative risk (aRR) 10.7, 95% CI 2.3-49.5) were associated with a far worse prognosis than status epilepticus/decorticate posturing (CFR 40.0%, aRR 5.7, 95% CI 1.2-27.1). Thrombocytopaenia (platelet counts <100,000/mm3) was associated with death (aRR 2.6, 95% CI 1.2-5.8) and second-line anticonvulsant use (clonazepam or thiopental) with survival (aRR 0.4, 95% CI 0.2-0.9). Complications, mostly nosocomial infections (n = 20), ALI/ARDS (n = 9) or sub-glottic stenosis (n = 3), had no significant prognostic value. CONCLUSION: In this study, the outcome of children requiring intubation for malaria depends more on clinical presentation and progression towards organ failures than on critical care complications per se. In sub-Saharan Africa, mechanical ventilation for life-threatening childhood malaria is feasible, but seems unlikely to dramatically improve the prognosis. [Abstract/Link to Full Text]

Okech BA, Gouagna LC, Yan G, Githure JI, Beier JC
Larval habitats of Anopheles gambiae s.s. (Diptera: Culicidae) influences vector competence to Plasmodium falciparum parasites.
Malar J. 2007;650.
BACKGROUND: The origin of highly competent malaria vectors has been linked to productive larval habitats in the field, but there isn't solid quantitative or qualitative data to support it. To test this, the effect of larval habitat soil substrates on larval development time, pupation rates and vector competence of Anopheles gambiae to Plasmodium falciparum were examined. METHODS: Soils were collected from active larval habitats with sandy and clay substrates from field sites and their total organic matter estimated. An. gambiae larvae were reared on these soil substrates and the larval development time and pupation rates monitored. The emerging adult mosquitoes were then artificially fed blood with infectious P. falciparum gametocytes from human volunteers and their midguts examined for oocyst infection after seven days. The wing sizes of the mosquitoes were also measured. The effect of autoclaving the soil substrates was also evaluated. RESULTS: The total organic matter was significantly different between clay and sandy soils after autoclaving (P = 0.022). A generalized liner model (GLM) analysis identified habitat type (clay soil, sandy soil, or lake water) and autoclaving (that reduces presence of microbes) as significant factors affecting larval development time and oocyst infection intensities in adults. Autoclaving the soils resulted in the production of significantly smaller sized mosquitoes (P = 0.008). Autoclaving clay soils resulted in a significant reduction in Plasmodium falciparum oocyst intensities (P = 0.041) in clay soils (unautoclaved clay soils (4.28 +/- 0.18 oocysts/midgut; autoclaved clay soils = 1.17 +/- 0.55 oocysts/midgut) although no difference (P = 0.480) in infection rates was observed between clay soils (10.4%), sandy soils (5.3%) or lake water (7.9%). CONCLUSION: This study suggests an important nutritional role for organic matter and microbial fauna on mosquito fitness and vector competence. It shows that the quality of natural aquatic habitats of mosquito larvae may influence malaria parasite transmission potential by An. gambiae. This information can be important in targeting larval habitats for malaria control. [Abstract/Link to Full Text]

Deressa W
Treatment-seeking behaviour for febrile illness in an area of seasonal malaria transmission in rural Ethiopia.
Malar J. 2007;649.
BACKGROUND: Very little is known about the management of malaria and treatment-seeking patterns among children and adults in areas of seasonal malaria transmission particularly in east Africa. OBJECTIVES: The aim of this study was to assess treatment-seeking behaviour for reported malaria among all age groups in an area of seasonal transmission. METHODS: A community-based cross-sectional study was carried out among 2,253 households in 12 randomly selected rural kebeles in Adami Tulu district in south-central Ethiopia, during October-November 2003, using a pre-tested interviewer-administered structured questionnaire. RESULTS: Reported malaria was 14% among 12,225 people assessed during the last 14 days. Family/self-diagnosis was most common and the main first responses included visiting village-based community health workers (CHWs) (33%), public health facility (23%) and private clinic (17%). Home treatment was the least reported first response (3%). Only 13% had sought treatment within the first 24 hours of symptom onset. Early treatment-seeking pattern was reported among those who visited CHWs and practiced home treatment, with more delays among public facility users. Treatment-seeking behaviour was similar in all age groups. CONCLUSION: A considerable proportion of visits were made to CHWs and private providers, necessitating the importance of strengthening both community-based interventions and peripheral public and private facilities. Finally, the community should be informed and educated about the importance of early diagnosis and prompt treatment with effective antimalarials. [Abstract/Link to Full Text]

Opiyo P, Mukabana WR, Kiche I, Mathenge E, Killeen GF, Fillinger U
An exploratory study of community factors relevant for participatory malaria control on Rusinga Island, western Kenya.
Malar J. 2007;648.
BACKGROUND: Capacity strengthening of rural communities, and the various actors that support them, is needed to enable them to lead their own malaria control programmes. Here the existing capacity of a rural community in western Kenya was evaluated in preparation for a larger intervention. METHODS: Focus group discussions and semi-structured individual interviews were carried out in 1,451 households to determine (1) demographics of respondent and household; (2) socio-economic status of the household; (3) knowledge and beliefs about malaria (symptoms, prevention methods, mosquito life cycle); (4) typical practices used for malaria prevention; (5) the treatment-seeking behaviour and household expenditure for malaria treatment; and (6) the willingness to prepare and implement community-based vector control. RESULTS: Malaria was considered a major threat to life but relevant knowledge was a chimera of scientific knowledge and traditional beliefs, which combined with socio-economic circumstances, leads to ineffective malaria prevention. The actual malaria prevention behaviour practiced by community members differed significantly from methods known to the respondents. Beside bednet use, the major interventions implemented were bush clearing and various hygienic measures, even though these are ineffective for malaria prevention. Encouragingly, most respondents believed malaria could be controlled and were willing to contribute to a community-based malaria control program but felt they needed outside assistance. CONCLUSION: Culturally sensitive but evidence-based education interventions, utilizing participatory tools, are urgently required which consider traditional beliefs and enable understanding of causal connections between mosquito ecology, parasite transmission and the diagnosis, treatment and prevention of disease. Community-based organizations and schools need to be equipped with knowledge through partnerships with national and international research and tertiary education institutions so that evidence-based research can be applied at the grassroots level. [Abstract/Link to Full Text]

Zuluaga L, Pabón A, López C, Ochoa A, Blair S
Amodiaquine failure associated with erythrocytic glutathione in Plasmodium falciparum malaria.
Malar J. 2007;647.
OBJECTIVE: To establish the relationship between production of glutathione and the therapeutic response to amodiaquine (AQ) monotherapy in Plasmodium falciparum non-complicated malaria patients. METHODOLOGY: Therapeutic response to AQ was evaluated in 32 patients with falciparum malaria in two townships of Antioquia, Colombia, and followed-up for 28 days. For every patient, total glutathione and enzymatic activity (glutathione reductase, GR, and gamma-glutamylcysteine synthetase, gamma-GCS) were determined in parasitized erythrocytes, non-infected erythrocytes and free parasites, on the starting day (day zero, before ingestion of AQ) and on the day of failure (in case of occurrence). RESULTS: There was found an AQ failure of 31.25%. Independent of the therapeutic response, on the starting day and on the day of failure, lower total glutathione concentration and higher GR activities in parasitized erythrocytes were found, compared with non-infected erythrocytes (p < 0.003). In addition, only on the day of failure, gamma-GCS activity of parasitized erythrocytes was higher, compared with that of healthy erythrocytes (p = 0.01). Parasitized and non-parasitized erythrocytes in therapeutic failure patients (TF) had higher total glutathione on the starting day compared with those of adequate clinical response (ACR) (p < 0.02). Parasitized erythrocytes of TF patients showed lower total glutathione on the failure day, compared with starting day (p = 0.017). No differences was seen in the GR and gamma-GCS activities by compartment, neither between the two therapeutic response groups nor between the two treatment days. CONCLUSION: This study is a first approach to explaining P. falciparum therapeutic failure in humans through differences in glutathione metabolism in TF and ACR patients. These results suggest a role for glutathione in the therapeutic failure to antimalarials. [Abstract/Link to Full Text]

Yé Y, Kyobutungi C, Louis VR, Sauerborn R
Micro-epidemiology of Plasmodium falciparum malaria: Is there any difference in transmission risk between neighbouring villages?
Malar J. 2007;646.
BACKGROUND: Malaria control strategies are designed as a solution for either the whole region or the whole country and are assumed to suit every setting. There is a need to shift from this assumption because transmission may be different from one local setting to another. The aim of this study was to assess the risk of clinical malaria given the village of residence among under-five children in rural north-western Burkina Faso. METHODS: 867 children (6-59 months) were randomly selected from four sites. Interviewers visited the children weekly at home over a one-year period and tested them for fever. Children with fever were tested for malaria parasites. An episode of clinical malaria was defined as fever (axillary temperature > or = 37.5 degrees C) + parasites density > or = 5,000 parasites/microl. Logistic regression was used to assess the risk of clinical malaria among children at a given site of residence. RESULTS: Children accumulated 758 person years (PYs). Overall, 597 episodes of clinical malaria were observed, giving an incidence rate of 0.79 per PY. The risk of clinical malaria varied amongst the four sites. Taking one village as reference the odds ratio for the other three sites ranged from 0.66; 95% CI: 0.44-0.98 to 1.49; 95% CI: 1.10-2.01. CONCLUSION: Malaria control strategies should be designed to fit the local context. The heterogeneity of transmission should be assessed at the district level to allow cost-effective resource allocation that gives priority to locations with high risk. Functional routine health information systems could provide the necessary data for context specific risk assessment. [Abstract/Link to Full Text]

Pal-Bhowmick I, Vora HK, Jarori GK
Sub-cellular localization and post-translational modifications of the Plasmodium yoelii enolase suggest moonlighting functions.
Malar J. 2007;645.
BACKGROUND: Enolase (2-Phospho-D-glycerate hydrolase; EC 4.2.1.11) is one of the glycolytic enzymes, whose levels are highly elevated in malaria parasite infected red blood cells. In several organisms, enolases have been shown to have diverse non glycolytic (moonlighting) biological functions. As functional diversity of a protein would require diverse sub-cellular localization, the possibility of involvement of Plasmodium enolase in moonlighting functions was examined by investigating its sub-cellular distribution in the murine malarial parasite, Plasmodium yoelii. METHODS: Cellular extracts of P. yoelii were fractionated in to soluble (cytosolic) and particulate (membranes, nuclear and cytoskeletal) fractions and were analysed by one and two-dimensional gel electrophoresis. These were probed by Western blotting using antibodies raised against recombinant Plasmodium falciparum enolase. Immunofluorescence assay was used for in situ localization. Fe+3 based metal affinity chromatography was used to isolate the phospho-proteome fraction from P. yoelii extracts. RESULTS: Apart from the expected presence of enolase in cytosol, this enzyme was also found to be associated with membranes, nuclei and cytoskeletal fractions. Nuclear presence was also confirmed by in situ immunofluorescence. Five different post translationally modified isoforms of enolase could be identified, of which at least three were due to the phosphorylation of the native form. in situ phosphorylation of enolase was also evident from the presence of enolase in purified phosphor-proteome of P. yoelii. Different sub-cellular fractions showed different isoform profiles. CONCLUSION: Association of enolase with nuclei, cell membranes and cytoskeletal elements suggests non-glycolytic functions for this enzyme in P. yoelii. Sub-cellular fraction specific isoform profiles indicate the importance of post-translational modifications in diverse localization of enolase in P. yoelii. Further, it is suggested that post-translational modifications of enolase may govern the recruitment of enolase for non-glycolytic functions. [Abstract/Link to Full Text]

Briolant S, Parola P, Fusaď T, Madamet-Torrentino M, Baret E, Mosnier J, Delmont JP, Parzy D, Minodier P, Rogier C, Pradines B
Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay.
Malar J. 2007;644.
OBJECTIVE: The main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC50) for chloroquine. METHODS: The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake. RESULTS: From 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoďtes and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC50 > 100 nM) at 10% O2, 17 were sensitive to chloroquine (IC50 < 100 nM) at 21% O2. CONCLUSION: Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance. [Abstract/Link to Full Text]