Pathogenomics Project Cross-Domain Horizontal Gene Transfer Analysis

1. 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 • 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. 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?

8. 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

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. 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

11. 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

12. Development of first database: Sequence similarity-based approach • 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

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

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. 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

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

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. NCBI GI Protein description Subcellular localization in plants 4377270 Glycyl tRNA Synthetase Chloroplast 4376626 cADP/ATP Translocase Chloroplast 4376667 cGlycogen Hydrolase Chloroplast 4377189 GTP Cyclohydratase & DHBP Synthase Chloroplast 4377237 cBeta-Ketoacyl-ACP Synthase Chloroplast 4376686 cEnoy-Acyl-Carrier Reductase Chloroplast 4376591 cThioredoxin Reductase Chloroplast 4377185 Metal Transport P-type ATPase Chloroplast 4377346 Similar to NA+/H+ Antiporter Chloroplast 4376650 cPhosphate Permease Chloroplast 4376637 GcpE protein Chloroplast 4376637 Tyrosyl tRNA Synthetase Chloroplast 4377360 cMalate Dehydrogenase Chloroplast 4376763 GTP Binding protein Chloroplast 4376911 cADP/ATP Translocase Chloroplast 3329179 Phosphoglycerate Mutase Chloroplast 4377281 cGlycerol-3-Phosphate Acyltransferase Chloroplast 4376993 ABC Transporter ATPase Chloroplast 4376509 dDeoxyoctulonosic Acid Synthetase Chloroplast 4376872 eSugar Nucleotide Phosphorylase Chloroplast 4377368 cShikimate 5-Dehydrogenase Chloroplast 4377054 Geranyl Transferase Chloroplast 3328465 1-Deoxyxylulose 5-Phosphate Reductoisomerase Chloroplast “Plant-like” genes in Chlamydia

28. 6578112 rRNA Methytransferase Chloroplast 3329217 HSP60 Chloroplast 3328745 cPhosphoribosylanthranilate Isomerase Chloroplast 6578104 cAspartate Aminotransferase Chloroplastf 4377328 cPolyribonucleotide Nucleotidyltransferase Chloroplastf 4377362 Putative D-Amino Acid Dehydrogenase Chloroplastg 4377331 Cytosine Deaminase Chloroplast?h 4376915 Lipoate-Protein Ligase A Mitochondrial 4377272 Glycogen Synthase N/Ai 4377065 cDihydropteroate Synthase N/Ai 4377239 cInorganic Pyrophosphatase N/Ai 4376904 Uridine 5’-Monophosphate Synthase N/Ai 4377173 cUDP-Glucose Pyrophosphorylase N/Ai 4376815 GutQ/Kpsf Family Sugar-Phosphate Isomerase Mitochondrial?j “Plant-like” genes in Chlamydia

29. Pyrococcusfuriosus (Archaea) Thermotogamaritima Chlamydiaceae share an ancestral relationship with Cyanobacteria and Chloroplast Aquifexpyrophilus Bacillussubtilis Chlamydophila pneumoniae Chlamydiaceae 538 Chlamydophila psittaci 1000 704 Chlamydia muridarum 1000 Chlamydia trachomatis 1000 Chlamydomonasreinhardtii 530 Chloroplasts Klebsormidiumflaccidum 998 988 Zeamays 1000 Nicotianatabacum 1000 SynechococcusPCC6301 349 Cyanobacteria 1000 SynechocystisPCC6803 1000 Microcystisviridis Escherichia coli Zeamaysmitochondrion 764 Rickettsia prowazekii 986 868 Caulobactercrescentus 0.1

30. Chlamydiaceae share an ancestral relationship with Cyanobacteria and Chloroplast S10 L23 L29 S14 L22 L16 L14 L24 L18 L30 L15 S19 S17 S3 S8 S5 L3 L4 L2 L5 L6 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 Pathogenicity Islands: Uro/Entero-pathogenic E. coli Salmonella typhimurium Yersinia spp. Helicobacter pylori Vibrio cholerae Transposons: ST enterotoxin genes in E. coli Prophages: Shiga-like toxins in EHEC Diptheria toxin gene, Cholera toxin Botulinum toxins Plasmids: Shigella, Salmonella, Yersinia

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

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