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Pathogenomics Using bioinformatics to focus studies of bacterial pathogenicity

Pathogenomics Using bioinformatics to focus studies of bacterial pathogenicity. Explosion of data 23 of the 34 publicly available microbial genome sequences are for bacterial pathogens Approximately 21,000 pathogen genes with no known function!

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Pathogenomics Using bioinformatics to focus studies of bacterial pathogenicity

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  1. Pathogenomics Using bioinformatics to focus studies of bacterialpathogenicity

  2. Explosion of data • 23 of the 34 publicly available microbial genome sequences are for bacterial pathogens • Approximately 21,000 pathogen genes with no known function! • >95 bacterial pathogen genome projects in progress …

  3. Pathogenomics Opportunistic pathogen Pseudomonas aeruginosa • Genome analysis and membrane protein bioinformatics UBC Pathogenomics Project • Identifying eukaryote:pathogen gene homologs • Detecting pathogenicity islands

  4. Pseudomonas aeruginosa • Found in soil, water, plants, animals • Common cause of hospital acquired infection: ICU patients, Burn victims, cancer patients • Almost all cystic fibrosis (CF) patients infected by age 10 • Intrinsically resistant to many antibiotics • No vaccine

  5. P. aeruginosa Genome Sequence Analysis: Outer Membrane Proteins (OMPs) Approximately 150 OMPs predicted including three large paralogous families: • OprM homology(3 previously known, now 18 predicted) • OprD homology(2 previously known, now 19) • TonB-dependent domain(8 previously known, now 34)

  6. OprJ OprM OpmJ OpmB OpmA OprM Family (Multidrug Efflux?) OpmG OpmE OpmI OprN OpmD OpmQ AprF OpmM Protein Secretion? OpmN OpmH TolC OpmK OpmL OpmF

  7. Gram Negative Cell Envelope PORE LPS PORIN + + Mg Outer membrane Peptidoglycan Periplasm Cytoplasmic membrane

  8. Outer membrane Periplasm P. aeruginosa OprM structural model based on E. coli TolC

  9. Residues implicated in blocking channel formation in OmpA are not conserved in OprF

  10. Planar Lipid Bilayer Apparatus Voltage Current Source Amplifier Protein Planar Bathing Bilayer Solution Membrane

  11. The N-terminus of OprF forms channels in a lipid bilayer membrane

  12. Current and Future Research Improve computational prediction of… • membrane and secreted proteins • surface exposed regions of membrane proteins

  13. Current and Future Research Omp85 membrane protein family studies • Antigenic, conserved, vaccine candidate • Two copies in most pathogenic bacteria genomes – why? • Structure unknown, may have conformational epitopes

  14. Pathogenomics Opportunistic pathogen Pseudomonas aeruginosa • Genome analysis and membrane protein bioinformatics UBC Pathogenomics Project • Identifying eukaryote:pathogen gene homologs • Detecting pathogenicity islands

  15. Genome data for… Anthrax Necrotizing fasciitis Cat scratch disease Paratyphoid/enteric fever Chancroid Peptic ulcers and gastritis Chlamydia Periodontal disease Cholera Plague Dental caries Pneumonia Diarrhea (E. coli etc.) Salmonellosis Diphtheria Scarlet fever Epidemic typhus Shigellosis Mediterranean fever Strep throat Gastroenteritis Syphilis Gonorrhea Toxic shock syndrome Legionnaires' disease Tuberculosis Leprosy Tularemia Leptospirosis Typhoid fever Listeriosis Urethritis Lyme disease Urinary Tract Infections Meliodosis Whooping cough Meningitis +Hospital-acquired infections

  16. Bacterial Pathogenicity • Processes of microbial pathogenicity at the molecular level are still minimally understood • Pathogen proteins identified that manipulate host cells by interacting with, or mimicking, host proteins

  17. Yersinia Type III secretion system

  18. Approach Idea: Could we identify novel virulence factors by identifying pathogen genes more similar to host genes than you would expect based on phylogeny?

  19. Approach Search pathogen genes against databases. Identify those with eukaryotic similarity. Modify screening method /algorithm Rank candidates - evolutionary analysis. Prioritize for biological study Collaborations with others Study function in model host (C. elegans) Study function in bacterium Infection of mutant in model host DATABASE World Research Community C. elegans

  20. Interdisciplinary group • Informatics/Bioinformatics • BC Genome Sequence Centre • Centre for Molecular Medicine and Therapeutics • Evolutionary Theory • Dept of Zoology • Dept of Botany • Canadian Institute for Advanced Research Coordinator • Pathogen Functions • Dept. Microbiology • Biotechnology Laboratory • Dept. Medicine • BC Centre for Disease Control • Host Functions • Dept. Medical Genetics • C. elegans Reverse Genetics Facility • Dept. Biological Sciences SFU

  21. Bacillus subtilis Escherichia coli Salmonella typhimurium Staphylococcua aureus Clostridium perfringens Clostridium difficile Trichomonas vaginalis Haemophilus influenzae Acinetobacillus actinomycetemcomitans 0.1 Pasteurella multocida Bacterium Eukaryote Horizontal Transfer N-acetylneuraminate lyase (NanA) of the protozoan Trichomonas vaginalis is 92-95% similar to NanA of Pasteurellaceae bacteria.

  22. N-acetylneuraminate lyase – role in pathogenicity? • Pasteurellaceae • Mucosal pathogens of the respiratory tract • T. vaginalis • Mucosal pathogen, causative agent of the STD Trichomonas

  23. N-acetylneuraminate lyase (sialic acid lyase, NanA) Hydrolysis of glycosidic linkages of terminal sialic residues in glycoproteins, glycolipids Sialidase Free sialic acid Transporter Free sialic acid NanA N-acetyl-D-mannosamine + pyruvate Involved in sialic acid metabolism Role in Bacteria: Proposed to parasitize the mucous membranes of animals for nutritional purposes Role in Trichomonas: ?

  24. Sensor Histidine Kinase for 2-component Regulation System Signal Transduction Histidine kinases common in bacteria Ser/Thr/Tyr kinases common in eukaryotes However, a histidine kinase was recently identified in fungi, including pathogens Fusarium solani and Candida albicans How did it get there? Candida

  25. A Histidine Kinase in Streptomyces.The Missing Link? Neurospora crassa NIK-1 Streptomyces coelicolor SC7C7 Fusarium solani FIK Candida albicans CHIK1 Erwinia carotovora EXPS Escherichia coli BARA Pseudomonas aeruginosa LEMA Pseudomonas syringae LEMA Pseudomonas viridiflava LEMA Pseudomonas tolaasii RTPA 0.1

  26. Universal role of this Histidine Kinase in pathogenicity? • Pathogenic Fungi • Senses change in osmolarity of the environment • Proposed role in pathogenicity • Pseudomonas species plant pathogens • Role in excretion of secondary metabolites that are virulence factors or antimicrobials • Virulence factor for human opportunistic pathogen Pseudomonas aeruginosa?

  27. Cells challenged per mouse Neutropenic mice challenged per group % Mortality Wildtype LemA- 0.74x 106 7 100 100 0.74x 105 7 100 85.7 0.74x 104 7 100 50 0.74x 103 8 75 50 0.74x 102 8 62.5 50 0.74x 101 8 37.5 25 Reduced virulence of a Pseudomonas aeruginosa transposon mutant disrupted in the histidine kinase lemA

  28. Trends in the Current Analysis • Identifies the strongest cases of lateral gene transfer between bacteria and eukaryotes • Most common “cross-kingdom” horizontal transfers: • Bacteria Unicellular Eukaryote • A control: Method identifies all previously reported Chlamydia trachomatis eukaryotic-like genes.

  29. Horizontal Gene Transfer and Bacterial Pathogenicity Transposons: ST enterotoxin genes in E. coli Prophages: Shiga-like toxins in EHEC Diptheria toxin gene, Cholera toxin Botulinum toxins Plasmids: Shigella, Salmonella, Yersinia

  30. Horizontal Gene Transfer and Bacterial Pathogenicity Pathogenicity Islands: Uropathogenic and Enteropathogenic E. coli Salmonella typhimurium Yersinia spp. Helicobacter pylori Vibrio cholerae

  31. Pathogenicity Islands Associated with • Atypical %G+C • tRNA sequences • Transposases, Integrases and other mobility genes • Flanking repeats

  32. IslandPath: Identifying Pathogenicity Islands Yellow circle = high %G+C Pink circle = low %G+C tRNA gene lies between the two dots rRNA gene lies between the two dots Both tRNA and rRNA lie between the two dots Dot is named a transposase Dot is named an integrase

  33. Neisseria meningitidis serogroup B strain MC58 Mean %G+C: 51.37 STD DEV: 7.57 %G+C SD Location Strand Product 39.95 -1 1834676..1835113 + virulence associated pro. homolog 51.96 1835110..1835211 - cryptic plasmid A-related 39.13 -1 1835357..1835701 + hypothetical 40.00 -1 1836009..1836203 + hypothetical 42.86 -1 1836558..1836788 + hypothetical 34.74 -2 1837037..1837249 + hypothetical 43.96 1837432..1838796 + conserved hypothetical 40.83 -1 1839157..1839663 + conserved hypothetical 42.34 -1 1839826..1841079 + conserved hypothetical 47.99 1841404..1843191 - put. hemolysin activ. HecB 45.32 1843246..1843704 - put. toxin-activating 37.14 -1 1843870..1844184 - hypothetical 31.67 -2 1844196..1844495 - hypothetical 37.57 -1 1844476..1845489 - hypothetical 20.38 -2 1845558..1845974 - hypothetical 45.69 1845978..1853522 - hemagglutinin/hemolysin-rel. 51.35 1854101..1855066 + transposase, IS30 family

  34. Variance of the Mean %G+C for all Genes in a Genome: Correlation with bacteria’s clonal nature

  35. Variance of the Mean %G+C for all Genes in a Genome • Is this a measure of clonality of a bacterium? • Are intracellular bacteria more clonal because they are ecologically isolated from other bacteria?

  36. Pathogenomics Project: Future Developments • Identify eukaryotic motifs and domains in pathogen genes • Identify further motifs associated with • Pathogenicity islands • Virulence determinants • Functional tests for new predicted virulence factors

  37. Acknowledgements • Pseudomonas Genome Project: PathoGenesis Corp. (Ken Stover) and University of Washington (Maynard Olsen) • Membrane proteins: Manjeet Bains, Kendy Wong, Canadian Cystic Fibrosis Foundation • Animal infection studies: Hong Yan

  38. Pathogenomics group • Ann M. Rose, Yossef Av-Gay, David L. Baillie, Fiona S. L. Brinkman, Robert Brunham, Stefanie Butland, Rachel C. Fernandez, B. Brett Finlay, Hans Greberg, Robert E.W. Hancock, Steven J. Jones,Patrick Keeling, Audrey de Koning, Don G. Moerman, Sarah P. Otto, B. Francis Ouellette, Ivan Wan. Peter Wall Foundation www.pathogenomics.bc.ca

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