1.Pathogenomics Project 2.Cross-Domain Horizontal Gene Transfer Analysis 3.Horizontal Gene Transfer: Identifying Pathogenicity Islands

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

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

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

Yersinia Type III secretion system

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?

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

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

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

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

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

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

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

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

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

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

Haemophilus influenzae Rd-KW20 proteins most strongly matching eukaryotic proteins

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

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.

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:

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

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

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

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 Streptomyces Histidine Kinase. The Missing Link? virulence factor = virulence factor ? Brinkman et al. (2001) Infection and Immunity. In Press.

“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)

“Plant-like” genes in Chlamydia NCBI GIProtein descriptionSubcellular localization in plants Glycyl tRNA SynthetaseChloroplast c ADP/ATP TranslocaseChloroplast c Glycogen HydrolaseChloroplast GTP Cyclohydratase & DHBP SynthaseChloroplast c Beta-Ketoacyl-ACP SynthaseChloroplast c Enoy-Acyl-Carrier ReductaseChloroplast c Thioredoxin ReductaseChloroplast Metal Transport P-type ATPaseChloroplast Similar to NA+/H+ AntiporterChloroplast c Phosphate PermeaseChloroplast GcpE proteinChloroplast Tyrosyl tRNA SynthetaseChloroplast c Malate DehydrogenaseChloroplast GTP Binding proteinChloroplast c ADP/ATP TranslocaseChloroplast Phosphoglycerate MutaseChloroplast c Glycerol-3-Phosphate AcyltransferaseChloroplast ABC Transporter ATPaseChloroplast d Deoxyoctulonosic Acid SynthetaseChloroplast e Sugar Nucleotide PhosphorylaseChloroplast c Shikimate 5-DehydrogenaseChloroplast Geranyl TransferaseChloroplast Deoxyxylulose 5-Phosphate ReductoisomeraseChloroplast

“Plant-like” genes in Chlamydia rRNA MethytransferaseChloroplast HSP60Chloroplast c Phosphoribosylanthranilate IsomeraseChloroplast c Aspartate AminotransferaseChloroplast f c Polyribonucleotide NucleotidyltransferaseChloroplast f Putative D-Amino Acid DehydrogenaseChloroplast g Cytosine DeaminaseChloroplast? h Lipoate-Protein Ligase AMitochondrial Glycogen SynthaseN/A i c Dihydropteroate SynthaseN/A i c Inorganic PyrophosphataseN/A i Uridine 5’-Monophosphate SynthaseN/A i c UDP-Glucose PyrophosphorylaseN/A i GutQ/Kpsf Family Sugar-Phosphate IsomeraseMitochondrial? j

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 Chlamydomonas reinhardtii Klebsormidium flaccidum Zea mays Nicotiana tabacum Synechococcus PCC6301 Synechocystis PCC6803 Microcystis viridis Escherichia coli Zea mays mitochondrion Rickettsia prowazekii Caulobacter crescentus Chloroplasts Cyanobacteria Chlamydiaceae

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

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

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

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

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

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

Neisseria meningitidis serogroup B strain MC58 Mean %G+C: STD DEV: 7.57 %G+C SD Location Strand Product virulence associated pro. homolog cryptic plasmid A-related hypothetical hypothetical hypothetical hypothetical conserved hypothetical conserved hypothetical conserved hypothetical put. hemolysin activ. HecB put. toxin-activating hypothetical hypothetical hypothetical hypothetical hemagglutinin/hemolysin-rel transposase, IS30 family

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

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

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

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.