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

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

Eukaryotic-like pathogen genes - YopH, a protein- tyrosine phosphatase, of Yersinia pestis - Enoyl-acyl carrier protein reductase (involved in lipid metabolism) of Chlamydia trachomatis 0.1 Aquifex aeolicus Haemophilus influenza Escherichia coli Anabaena Synechocystis Chlamydia trachomatis Petunia x hybrida Nicotiana tabacum Brassica napus Arabidopsis thaliana Oryza sativa

Prioritize for biological study. - Previously studied biologically? - Can UBC microbiologists study it? - C. elegans homolog? Screen for candidate genes. Search pathogen genes against sequence databases. Identify those with eukaryotic similarity/motifs Rank candidates. - how much like host protein? - info available about protein? Modify screening method /algorithm Approach Evolutionary significance. - Horizontal transfer? Similar by chance?

Pathogens 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

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 similar gene in model host, C. elegans. Study function of gene. Investigate role of bacterial gene in disease: Infection study in model host C. elegans DATABASE Contact other groups for possible collaborations.

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

Interdisciplinary team unique ideas and collaborations Automated approach continually updated Better understanding: pathogen gene and similar host gene Insight into horizontal gene transfer events and the evolution of pathogen-host interactions. Public database –other researchers may capitalize on the findings –promote further collaboration Power of the Approach

Database front end

PhyloBLAST – a tool for the analysis

Bacterium Eukaryote Horizontal 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.

N-acetylneuraminate lyase (sialic acid lyase, NanA) Intracellular enzyme involved in sialic acid metabolism In Bacteria: Proposed to parasitize the mucous membranes of animals for nutritional purposes 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

N-acetylneuraminate lyase – role in pathogenicity? Pasteurellaceae Mucosal pathogens of the respiratory tract Intracellular NanA enzyme with sialic acid transporter T. vaginalis Mucosal pathogen, causative agent of the STD Trichomonas Extracellular enzyme, so avoids need for transporter?

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 Guanosine monophosphate reductase of E. coli is 81% similar to the corresponding enzyme studied in humans and rats, and shares a significant phylogenetic relationship with metazoans (left). Its role in virulence has not been investigated.

Eukaryote Bacteria Horizontal Transfer? Ralstonia solanacearum cellulase ( ENDO-1,4- BETA-GLUCANASE ) is 56% similar to endoglucanase present in a number of fungi. Demonstrated virulence factor for plant bacterial wilt Hypocrea jecorina EGLII Trichoderma viride EGL2 Penicillium janthinellum EGL2 Macrophomina phaseolina EGL2 Cryptococcus flavus CMC1 Ralstonia solanacearum egl Humicola insolens CMC3 Humicola grisea CMC3 Aspergillus aculeatus CMC2 Aspergillus nidulans EGLA Macrophomina phaseolina egl1 Aspergillus aculeatus CEL1 Aspergillus niger EGLB Vibrio species manA

Trends in the Analysis Most cases of probable recent cross-domain gene transfer involve movement of a bacterial gene to a unicellular eukaryote Identifies the strongest cases of lateral gene transfer between bacteria and eukaryotes A control: The method identifies all previously reported Chlamydia trachomatis eukaryotic-like genes.

G+C Analysis: Identifying Pathogenicity Islands %G+C S.D. Location Strand Gene Product NMB0854 histidyl-tRNA synthetase NMB0855 put. bacteriocin resist NMB0856 hypothetical protein NMB0857 hypothetical protein NMB0858 hypothetical protein NMB0859 hypothetical protein NMB0860 hypothetical protein NMB0861 hypothetical protein NMB0862 hypothetical protein NMB0863 hypothetical protein NMB0864 hypothetical protein NMB0865 hypothetical protein NMB0866 hypothetical protein NMB0867 YabO/YceC/SfhB fam. prot NMB0868 conserved hypothetical NMB0869 hypothetical protein NMB methyl-2-oxobutanoate hydroxymethyltransferase

G+C of ORFs: Analysis of Variance Low G+C variance correlates with an intracellular lifestyle for the bacterium and a clonal nature (P = 0.004) This variance is similar within a given species Useful marker for investigating the clonality of bacteria? Relationship with intracellular lifestyle may reflect the ecological isolation of intracellular bacteria?

Future Developments Incorporate unfinished genomes, plasmids into analysis (including eukaryotic) Motif-based and domain-based analyses G+C analysis graphical viewer for identification of pathogenicity islands Functional tests

Peter Wall Foundation Pathogenomics group –Ann M. Rose, Yossef Av-Gay, David L. Baillie, Fiona S. L. Brinkman, Robert Brunham, Stefanie Butland, Rachel C. Fernandez, B. Brett Finlay, Hans Greberg, Robert E.W. Hancock, Christy Haywood-Farmer, Steven J. Jones, Patrick Keeling, Audrey de Koning, Don G. Moerman, Sarah P. Otto, B. Francis Ouellette, Ivan Wan.