Steps toward broad-spectrum therapeutics: discovering virulence-associated genes present in diverse human pathogens

BackgroundNew and improved antimicrobial countermeasures are urgently needed to counteract increased resistance to existing antimicrobial treatments and to combat currently untreatable or new emerging infectious diseases. We demonstrate that computational comparative genomics, together with experimental screening, can identify potential generic (i.e., conserved across multiple pathogen species) and novel virulence-associated genes that may serve as targets for broad-spectrum countermeasures.ResultsUsing phylogenetic profiles of protein clusters from completed microbial genome sequences, we identified seventeen protein candidates that are common to diverse human pathogens and absent or uncommon in non-pathogens. Mutants of 13 of these candidates were successfully generated in Yersinia pseudotuberculosis and the potential role of the proteins in virulence was assayed in an animal model. Six candidate proteins are suggested to be involved in the virulence of Y. pseudotuberculosis, none of which have previously been implicated in the virulence of Y. pseudotuberculosis and three have no record of involvement in the virulence of any bacteria.ConclusionThis work demonstrates a strategy for the identification of potential virulence factors that are conserved across a number of human pathogenic bacterial species, confirming the usefulness of this tool.

[1]  S. Levy,et al.  Targeting virulence to prevent infection: to kill or not to kill? , 2004 .

[2]  The UniProt Consortium,et al.  The Universal Protein Resource (UniProt) 2009 , 2008, Nucleic Acids Res..

[3]  H. Tokuda.  Biogenesis of Outer Membranes in Gram-Negative Bacteria , 2009, Bioscience, biotechnology, and biochemistry.

[4]  T. Fuchs,et al.  Comparative analysis of the Photorhabdus luminescens and the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity , 2008, BMC Genomics.

[5]  M. Valvano,et al.  Characterization of SodC, a Periplasmic Superoxide Dismutase from Burkholderia cenocepacia , 2007, Infection and Immunity.

[6]  Yan P. Yuan,et al.  Predicting function: from genes to genomes and back. , 1998, Journal of molecular biology.

[7]  G. Marshall,et al.  The biology and future prospects of antivirulence therapies , 2008, Nature Reviews Microbiology.

[8]  F. Fang,et al.  Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Calderwood,et al.  Virulence of Staphylococcus aureus Small Colony Variants in the Caenorhabditis elegans Infection Model , 2006, Infection and Immunity.

[10]  J. Glass,et al.  Genome Sequence of Avery's Virulent Serotype 2 Strain D39 of Streptococcus pneumoniae and Comparison with That of Unencapsulated Laboratory Strain R6 , 2006, Journal of bacteriology.

[11]  N. Saunders,et al.  Functional genomics of pathogenic bacteria. , 2002, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[12]  N. Slonim,et al.  Ab initio genotype–phenotype association reveals intrinsic modularity in genetic networks , 2006, Molecular systems biology.

[13]  Eduardo A. Groisman,et al.  The SPI-3 Pathogenicity Island ofSalmonella enterica , 1999, Journal of bacteriology.

[14]  Yan Lin,et al.  DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes , 2008, Nucleic Acids Res..

[15]  Michelle E. Maxson,et al.  Identification of Inducers of the Yersinia enterocolitica Phage Shock Protein System and Comparison to the Regulation of the RpoE and Cpx Extracytoplasmic Stress Responses , 2004, Journal of bacteriology.

[16]  MJ Mahan,et al.  Selection of bacterial virulence genes that are specifically induced in host tissues , 1993, Science.

[17]  P. Langford,et al.  Periplasmic Superoxide Dismutase in Meningococcal Pathogenicity , 1998, Infection and Immunity.

[18]  Peer Bork,et al.  Systematic Association of Genes to Phenotypes by Genome and Literature Mining , 2005, PLoS biology.

[19]  E. Winzeler,et al.  Application of High-Density Array-Based Signature-Tagged Mutagenesis To Discover Novel YersiniaVirulence-Associated Genes , 2001, Infection and Immunity.

[20]  Matteo Pellegrini,et al.  An improved method for identifying functionally linked proteins using phylogenetic profiles , 2007, BMC Bioinformatics.

[21]  R. Bourdet-Sicard,et al.  Commensal bacteria trigger a full dendritic cell maturation program that promotes the expansion of non‐Tr1 suppressor T cells , 2008, Journal of Leukocyte Biology.

[22]  E. Marcotte,et al.  Computational genetics: finding protein function by nonhomology methods. , 2000, Current opinion in structural biology.

[23]  J. Shea,et al.  Simultaneous identification of bacterial virulence genes by negative selection. , 1995, Science.

[24]  R. Brubaker.  Factors promoting acute and chronic diseases caused by yersiniae , 1991, Clinical Microbiology Reviews.

[25]  Ian T. Paulsen,et al.  Comparative Analyses of Fundamental Differences in Membrane Transport Capabilities in Prokaryotes and Eukaryotes , 2005, PLoS Comput. Biol..

[26]  B. Finlay,et al.  Crossing the Line: Selection and Evolution of Virulence Traits , 2006, PLoS pathogens.

[27]  J. Errington,et al.  Essential Bacterial Functions Encoded by Gene Pairs , 2006, Journal of bacteriology.

[28]  Warren C. Lathe,et al.  Predicting protein function by genomic context: quantitative evaluation and qualitative inferences. , 2000, Genome research.

[29]  M. Pallen,et al.  Bacterial copper‐ and zinc‐cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis , 1997, Molecular microbiology.

[30]  C. Surawicz.  Antibiotic-Associated Diarrhea and Pseudomembranous Colitis: Are They Less Common with Poorly Absorbed Antimicrobials? , 2005, Chemotherapy.

[31]  L. Reed,et al.  A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS , 1938 .

[32]  Cathy H. Wu,et al.  The Universal Protein Resource (UniProt) , 2004, Nucleic Acids Res..

[33]  E. Mardis,et al.  An obesity-associated gut microbiome with increased capacity for energy harvest , 2006, Nature.

[34]  C. Médigue,et al.  Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[35]  B. Finlay,et al.  The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen , 2000, Molecular microbiology.

[36]  M. Maguire,et al.  Emerging themes in manganese transport, biochemistry and pathogenesis in bacteria. , 2003, FEMS microbiology reviews.

[37]  D. Payne.  Desperately Seeking New Antibiotics , 2008, Science.

[38]  I. Cooper,et al.  Galleria mellonella as an alternative infection model for Yersinia pseudotuberculosis. , 2009, Microbiology.

[39]  Gary Taubes,et al.  The Bacteria Fight Back , 2008, Science.

[40]  J. Tobias,et al.  Selection for bacterial genes that are specifically induced in host tissues: the hunt for virulence factors. , 1993, Infectious agents and disease.

[41]  M. Winkler,et al.  The Brucella abortus Cu,Zn Superoxide Dismutase Is Required for Optimal Resistance to Oxidative Killing by Murine Macrophages and Wild-Type Virulence in Experimentally Infected Mice , 2005, Infection and Immunity.

[42]  G. Riley,et al.  Detection of pathogenic Yersinia enterocolitica by using congo red-magnesium oxalate agar medium. , 1989 .

[43]  M. Pop,et al.  Metagenomic Analysis of the Human Distal Gut Microbiome , 2006, Science.

[44]  Mark Pagel,et al.  Predicting Functional Gene Links from Phylogenetic-Statistical Analyses of Whole Genomes , 2005, 2005 IEEE Computational Systems Bioinformatics Conference - Workshops (CSBW'05).

[45]  Michael Y. Galperin,et al.  Who's your neighbor? New computational approaches for functional genomics , 2000, Nature Biotechnology.

[46]  R. Freter,et al.  Role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vivo studies , 1981, Infection and immunity.

[47]  D. Frishman,et al.  A domain interaction map based on phylogenetic profiling. , 2004, Journal of molecular biology.

[48]  V. L. Miller,et al.  Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[49]  P. Bork,et al.  Analysis of genomic context: prediction of functional associations from conserved bidirectionally transcribed gene pairs , 2004, Nature Biotechnology.

[50]  D. Eisenberg,et al.  Assigning protein functions by comparative genome analysis: protein phylogenetic profiles. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[51]  J. Ghigo,et al.  A rapid and simple method for inactivating chromosomal genes in Yersinia. , 2003, FEMS immunology and medical microbiology.

[52]  H. Wolf‐Watz,et al.  Identification of Novel Virulence-Associated Genes via Genome Analysis of Hypothetical Genes , 2004, Infection and Immunity.

[53]  S. Escaich.  Antivirulence as a new antibacterial approach for chemotherapy. , 2008, Current opinion in chemical biology.

[54]  Bruno Pot,et al.  Feeding Our Immune System: Impact on Metabolism , 2008, Clinical & developmental immunology.