1. 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 26 of the 36 publicly available bacterial genome sequences are for pathogens Approximately 24,000 pathogen genes with no known function! ~177 bacterial genome projects in progress … Data as of June, 2001

4. 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

5. Yersinia Type III secretion system

6. 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?

7. 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

8. 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

9. 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

10. 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

11. 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

12. For each complete bacterial and eukaryote genome: BLASTP (and MSP Crunch) of all deduced proteins against non-redundant SWALL database Overlay NCBI taxonomy information  form ACEDB database Query database for bacterial proteins who’s top scoring hit is eukaryotic (and eukaryotic proteins who’s top hit is bacterial) Perform similar query, but filtering different taxonomic groups from the analysis Development of first database: Sequence similarity-based approach

13. BAE-watch Database: Bacterial proteins with unusual similarity with Eukaryotic proteins

14. Problem: Proteins highly conserved in the three domains of life Top hit to a protein from another domain may occur by chance. “StepRatio” score helps detect these. Example: Glucose-6- Phosphate Reductase

15. Example of a case with a high StepRatio: Enoyl ACP reductase

16. BAE-watch Database: Bacterial proteins with unusual similarity with Eukaryotic proteins

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

18. PhyloBLAST – a tool for analysis Brinkman et al. (2001) Bioinformatics. 17:385-387.

20. Trends in this Sequence-based 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 “plant-like” genes.

21. 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

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) 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

24. 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

25. 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 ? Brinkman et al. (2001) Infection and Immunity. In Press.

26. “Plant-like” genes in Chlamydia Chlamydiaceae: Obligate intracellular pathogens of humans Proteins: Unusually high number most similar to plant proteins Previous proposal: Obtained genes from a plant-like amoebal host? (a relative of Chlamydiaceae infects Acanthamoeba)

27. “Plant-like” genes in Chlamydia NCBI GIProtein descriptionSubcellular localization in plants 4377270Glycyl tRNA SynthetaseChloroplast 4376626 c ADP/ATP TranslocaseChloroplast 4376667 c Glycogen HydrolaseChloroplast 4377189GTP Cyclohydratase & DHBP SynthaseChloroplast 4377237 c Beta-Ketoacyl-ACP SynthaseChloroplast 4376686 c Enoy-Acyl-Carrier ReductaseChloroplast 4376591 c Thioredoxin ReductaseChloroplast 4377185Metal Transport P-type ATPaseChloroplast 4377346Similar to NA+/H+ AntiporterChloroplast 4376650 c Phosphate PermeaseChloroplast 4376637GcpE proteinChloroplast 4376637Tyrosyl tRNA SynthetaseChloroplast 4377360 c Malate DehydrogenaseChloroplast 4376763GTP Binding proteinChloroplast 4376911 c ADP/ATP TranslocaseChloroplast 3329179Phosphoglycerate MutaseChloroplast 4377281 c Glycerol-3-Phosphate AcyltransferaseChloroplast 4376993ABC Transporter ATPaseChloroplast 4376509 d Deoxyoctulonosic Acid SynthetaseChloroplast 4376872 e Sugar Nucleotide PhosphorylaseChloroplast 4377368 c Shikimate 5-DehydrogenaseChloroplast 4377054Geranyl TransferaseChloroplast 33284651-Deoxyxylulose 5-Phosphate ReductoisomeraseChloroplast

28. “Plant-like” genes in Chlamydia 6578112rRNA MethytransferaseChloroplast 3329217HSP60Chloroplast 3328745 c Phosphoribosylanthranilate IsomeraseChloroplast 6578104 c Aspartate AminotransferaseChloroplast f 4377328 c Polyribonucleotide NucleotidyltransferaseChloroplast f 4377362Putative D-Amino Acid DehydrogenaseChloroplast g 4377331Cytosine DeaminaseChloroplast? h 4376915Lipoate-Protein Ligase AMitochondrial 4377272Glycogen SynthaseN/A i 4377065 c Dihydropteroate SynthaseN/A i 4377239 c Inorganic PyrophosphataseN/A i 4376904Uridine 5’-Monophosphate SynthaseN/A i 4377173 c UDP-Glucose PyrophosphorylaseN/A i 4376815GutQ/Kpsf Family Sugar-Phosphate IsomeraseMitochondrial? j

29. Chlamydiaceae share an ancestral relationship with Cyanobacteria and Chloroplast 0.1 Pyrococcus furiosus (Archaea) Thermotoga maritima Aquifex pyrophilus Bacillus subtilis Chlamydophila pneumoniae Chlamydophila psittaci Chlamydia muridarum Chlamydia trachomatis 1000 704 1000 Chlamydomonas reinhardtii Klebsormidium flaccidum Zea mays Nicotiana tabacum 1000 988 998 Synechococcus PCC6301 Synechocystis PCC6803 Microcystis viridis 1000 530 Escherichia coli Zea mays mitochondrion Rickettsia prowazekii Caulobacter crescentus 868 986 764 349 1000 538 Chloroplasts Cyanobacteria Chlamydiaceae

30. Chlamydiaceae share an ancestral relationship with Cyanobacteria and Chloroplast L3L4 L23 L2 S19L22 S3 L16 L29 S17L14L24 L5 S14 S8 L6 L18 S5 L30L15 S10 Escherichia Bacillus Thermatoga Synechocystis Chlamydia Unique shared-derived characters unite Chlamydiaceae and Synechocystis

31. Chlamydiaceae “plant-like” genes reflect an ancestral relationship with Cyanobacteria and Chloroplast Chlamydia do not appear to be exchanging DNA with their hosts Existing knowledge of Cyanobacteria may stimulate ideas about the function and control of pathogenic Chlamydia? Non-unique shared characters include a multistage developmental lifecycle, storage of glucose primarily as glycogen, and non-flagellar motility

32. 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-functioning” proteins in Rickettsia, Synechocystis, and Chlamydiaceae

33. 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 Pathogenicity Islands: Uro/Entero-pathogenic 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. 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

39. Jeff Blanchard (National Centre for Genome Resources, New Mexico) Olof Emanuelsson (Stockholm Bioinformatics Center) Genome Sequence Centre, BC Cancer Agency Acknowledgements

40. Pathogenomics group Ann M. Rose, Yossef Av-Gay, David L. Baillie, Fiona S. L. Brinkman, Robert Brunham, Artem Cherkasov, 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, Nancy Price, Ivan Wan. www.pathogenomics.bc.ca