Cross-Domain and Within-Domain Horizontal Gene Transfer: Implications for Bacterial Pathogenicity

1. Cross-Domain and Within-Domain Horizontal Gene Transfer: Implications for Bacterial Pathogenicity • Pathogenomics Project • Cross-Domain Horizontal Gene Transfer Analysis • 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. Approach Search pathogen genes against databases. Identify those with eukaryotic similarity. Modify screening method /algorithm Evolutionary significance. - Horizontal transfer? Similar by chance? • Prioritize for biological study. • - Previously studied in the laboratory? • Can UBC microbiologists study it? • C. elegans homolog?

9. 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

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

12. 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

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. Bacillus subtilis Escherichia coli Salmonella typhimurium Staphylococcua aureus Clostridium perfringens Clostridium difficile Trichomonas vaginalis Haemophilus influenzae Acinetobacillus actinomycetemcomitans 0.1 Pasteurella multocida First case: Bacterium Eukaryote Lateral Transfer 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) 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: ?

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. Streptomyces Histidine Kinase. The Missing Link? Pseudomonas aeruginosaPhoQ Xanthomonas campestris RpfC 100 Vibrio cholerae TorS 100 Escherichia coli TorS Escherichia coli RcsC Candida albicans CaNIK1 39 100 Neurospora crassa NIK-1 100 Fungi Fusarium solani FIK1 100 51 54 Fusarium solani FIK2 Streptomyces coelicolorSC4G10.06c 100 Streptomyces coelicolorSC7C7.03 virulence factor ? Pseudomonas aeruginosaGacS 100 100 Pseudomonas fluorescensGacS / ApdA 100 Pseudomonas tolaasii RtpA / PheN 100 Pseudomonas syringaeGacS / LemA 100 86 Pseudomonas viridiflavaRepA 100 Azotobacter vinelandii GacS Erwinia carotovora RpfA / ExpS virulence factor 100 = Escherichia coli BarA 100 Salmonella typhimurium BarA 0.1

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 106 bacteria Wildtype LD50= 10  1 bacteria gacS mutant LD50= 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. Rat 0.1 Human Escherichia coli Caenorhabditis elegans Pig roundworm Methanococcus jannaschii Methanobacterium thermoautotrophicum Bacillus subtilis Streptococcus pyogenes Aquifex aeolicus Acinetobacter calcoaceticus Haemophilus influenzae Chlorobium vibrioforme Eukaryote Bacteria Horizontal Transfer? 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 Aquifex aeolicus 96 Haemophilus influenza 100 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 Escherichia coli Anabaena 100 Synechocystis 100 Chlamydia trachomatis 63 Petunia x hybrida 64 Nicotiana tabacum 83 Brassica napus 99 Arabidopsis thaliana 0.1 52 Oryza sativa

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

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? 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