1. Cross-Domain and Within-Domain Horizontal Gene Transfer: Implications for Bacterial Pathogenicity 1.Pathogenomics Project 2.Cross-Domain Horizontal Gene Transfer Analysis 3.Horizontal Gene Transfer: Identifying Pathogenicity Islands

2. Pathogenomics Goal: Identify previously unrecognized mechanisms of microbial pathogenicity using a combination of informatics, evolutionary biology, microbiology and genetics.

3. Explosion of data 23 of the 37 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 …

4. The need for new tools Prioritize new genes for further laboratory study Capitalize on the existing genomic data

5. 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

6. Yersinia Type III secretion system

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

8. Prioritize for biological study. - Previously studied in the laboratory? - Can UBC microbiologists study it? - C. elegans homolog? Search pathogen genes against databases. Identify those with eukaryotic similarity. Evolutionary significance. - Horizontal transfer? Similar by chance? Modify screening method /algorithm Approach

9. Genome data for… AnthraxNecrotizing fasciitis Cat scratch diseaseParatyphoid/enteric fever Chancroid Peptic ulcers and gastritis Chlamydia Periodontal disease CholeraPlague Dental cariesPneumonia Diarrhea (E. coli etc.)Salmonellosis DiphtheriaScarlet fever Epidemic typhusShigellosis Mediterranean feverStrep throat Gastroenteritis Syphilis GonorrheaToxic shock syndrome Legionnaires' disease Tuberculosis LeprosyTularemia Leptospirosis Typhoid fever Listeriosis Urethritis Lyme disease Urinary Tract Infections Meliodosis Whooping cough Meningitis +Hospital-acquired infections

10. Bacterial Pathogens Chlamydophila psittaci Respiratory disease, primarily in birds Mycoplasma mycoides Contagious bovine pleuropneumonia Mycoplasma hyopneumoniae Pneumonia in pigs Pasteurella haemolytica Cattle shipping fever Pasteurella multicoda Cattle septicemia, pig rhinitis Ralstonia solanacearum Plant bacterial wilt Xanthomonas citri Citrus canker Xylella fastidiosa Pierce’s Disease - grapevines Bacterial wilt

11. World Research Community Approach Prioritized candidates Study function of homolog in model host (C. elegans) Study function of gene in bacterium. Infection of mutant in model host C. elegans DATABASE Collaborations with others

12. 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 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 Interdisciplinary group Coordinator

13. Pathogenomics Database: Bacterial proteins with unusual similarity with Eukaryotic proteins

14. Haemophilus influenzae Rd-KW20 proteins most strongly matching eukaryotic proteins

16. PhyloBLAST – a tool for analysis Brinkman et al. (2001) Bioinformatics. In Press.

18. Trends in the Initial Analysis Identifies the strongest cases of lateral gene transfer between bacteria and eukaryotes Most common “cross-domain” horizontal transfers: Bacteria Unicellular Eukaryote Identifies nuclear genes with potential organelle origins A control: Method identifies all previously reported Chlamydia trachomatis “eukaryote-like” genes.

19. First case: Bacterium Eukaryote Lateral Transfer 0.1 Bacillus subtilis Escherichia coli Salmonella typhimurium Staphylococcua aureus Clostridium perfringens Clostridium difficile Trichomonas vaginalis Haemophilus influenzae Acinetobacillus actinomycetemcomitans Pasteurella multocida N-acetylneuraminate lyase (NanA) of the protozoan Trichomonas vaginalis is 92-95% similar to NanA of Pasteurellaceae bacteria. de Koning et al. (2000) Mol. Biol. Evol. 17:1769-1773

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

21. N-acetylneuraminate lyase (sialic acid lyase, NanA) Involved in sialic acid metabolism Role in Bacteria: Proposed to parasitize the mucous membranes of animals for nutritional purposes Role in Trichomonas: ? 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

22. Another case: A Sensor Histidine Kinase for a Two-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

23. Neurospora crassa NIK-1 Fusarium solani FIK2 Streptomyces coelicolor SC4G10.06c Candida albicans CaNIK1 Escherichia coli RcsC Erwinia carotovora RpfA / ExpS Escherichia coli BarA Salmonella typhimurium BarA Pseudomonas aeruginosa GacS Pseudomonas fluorescens GacS / ApdA Pseudomonas tolaasii RtpA / PheN Pseudomonas syringae GacS / LemA Pseudomonas viridiflava RepA Azotobacter vinelandii GacS 0.1 Streptomyces coelicolor SC7C7.03 Xanthomonas campestris RpfC Vibrio cholerae TorS Escherichia coli TorS Fusarium solani FIK1 Fungi Pseudomonas aeruginosa PhoQ 100 51 100 86 54 39 100 Streptomyces Histidine Kinase. The Missing Link? virulence factor = virulence factor ?

24. Reduced virulence of a Pseudomonas aeruginosa transposon mutant disrupted in the histidine kinase gene gacS Groups of 7-8 neutropenic mice challenged on two separate occasions with doses ranging from 8 to 8 x 10 6 bacteria Wildtype LD 50 = 10  1 bacteria gacS mutant LD 50 = 7,500  100 bacteria 750-fold increase

25. Recent report: P. aeruginosa eukaryote-type Phospholipase plays a role in infection Wilderman et al. 2001. Mol Microbiol 39:291-304 Phospholipase D (PLDs) virtually ubiquitous in eukaryotes (relatively uncommon in prokaryotes) P. aeruginosa expresses PLD with significant (1e-38 BLAST Expect) similarity to eukaryotic PLDs Part of a mobile 7 kb genetic element Role in P. aeruginosa persistence in a chronic pulmonary infection model

26. Eukaryote Bacteria Horizontal Transfer? 0.1 Rat Human Escherichia coli Caenorhabditis elegans Pig roundworm Methanococcus jannaschii Methanobacterium thermoautotrophicum Bacillus subtilis Streptococcus pyogenes Aquifex aeolicus Acinetobacter calcoaceticus Haemophilus influenzae Chlorobium vibrioforme E. coli Guanosine monophosphate reductase 81% similar to corresponding enzyme in humans and rats Role in virulence not yet investigated.

27. Expanding the Cross-Domain Analysis Identify cross-domain lateral gene transfer between bacteria, archaea and eukaryotes No obvious correlation seen with protein functional classification Most cases: no obvious correlation seen between “organisms involved” in potential lateral transfer Exceptions: –Unicellular eukaryotes –“Organelle-like” proteins in Rickettsia and Synechocystis –“Plant-like(?)” genes in the obligate intracellular bacteria Chlamydia

28. “Plant-like” genes in Chlamydia Enoyl-acyl carrier protein reductase (involved in lipid metabolism) of Chlamydia trachomatis is similar to those of Plants Organelle relationship? Notably more similar to plants than Synechocystis 0.1 Aquifex aeolicus Haemophilus influenza Escherichia coli Anabaena Synechocystis Chlamydia trachomatis Petunia x hybrida Nicotiana tabacum Brassica napus Arabidopsis thaliana Oryza sativa 100 96 63 64 52 83 99

29. Synechocystis

30. Rickettsia and Chlamydia

31. Proteins Homologous to Eukaryote Proteins (according to BLAST Exp=1)

32. 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

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

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

35. 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

36. 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

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

38. 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?

39. Pathogenomics Project: Future Developments Identify eukaryotic motifs and domains in pathogen genes Threader: Detect proteins with similar tertiary structure Identify more motifs associated with Pathogenicity islands Virulence determinants Functional tests for new predicted virulence factors Expand analysis to include viral genomes

40. Fundamental research Interdisciplinary Lack of fit with alternative funding sources Peter Wall Major Thematic Grant

41. Pathogenomics group Ann M. Rose, Yossef Av-Gay, David L. Baillie, Fiona S. L. Brinkman, Robert Brunham, 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. www.pathogenomics.bc.ca

42. Universal role of this Histidine Kinase in pathogenicity? Pathogenic Fungi Senses change in osmolarity of the environment Role in hyphal formation 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?

43. A Histidine Kinase in Streptomyces. The Missing Link? 0.1 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