Pathogenomics Using bioinformatics to focus studies of bacterial pathogenicity

Explosion of data 23 of the 34 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 …

Pathogenomics Opportunistic pathogen Pseudomonas aeruginosa -Genome analysis and membrane protein bioinformatics UBC Pathogenomics Project -Identifying eukaryote:pathogen gene homologs -Detecting pathogenicity islands

Pseudomonas aeruginosa Found in soil, water, plants, animals Common cause of hospital acquired infection: ICU patients, Burn victims, cancer patients Almost all cystic fibrosis (CF) patients infected by age 10 Intrinsically resistant to many antibiotics No vaccine

 OprM homology (3 previously known, now 18 predicted)  OprD homology (2 previously known, now 19)  TonB-dependent domain (8 previously known, now 34) P. aeruginosa Genome Sequence Analysis: Outer Membrane Proteins (OMPs) Approximately 150 OMPs predicted including three large paralogous families:

AprF OpmM OpmH OpmF OpmK OpmL OpmN OpmQ OpmD OprN OpmE OpmJ OpmA OprM OprJ OpmB OpmG OpmI OprM Family (Multidrug Efflux?) Protein Secretion? TolC

PORE PORIN Peptidoglycan LPS Mg ++ Outer membrane Cytoplasmic membrane Gram Negative Cell Envelope Periplasm

P. aeruginosa OprM structural model based on E. coli TolC Outer membrane Periplasm

Residues implicated in blocking channel formation in OmpA are not conserved in OprF

Bathing Solution Planar Bilayer Membrane Voltage Source Current Amplifier Protein Planar Lipid Bilayer Apparatus

The N-terminus of OprF forms channels in a lipid bilayer membrane

Improve computational prediction of… -membrane and secreted proteins -surface exposed regions of membrane proteins Current and Future Research

Omp85 membrane protein family studies -Antigenic, conserved, vaccine candidate -Two copies in most pathogenic bacteria genomes – why? -Structure unknown, may have conformational epitopes Current and Future Research

Opportunistic pathogen Pseudomonas aeruginosa -Genome analysis and membrane protein bioinformatics UBC Pathogenomics Project -Identifying eukaryote:pathogen gene homologs -Detecting pathogenicity islands Pathogenomics

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 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 pathogen genes more similar to host genes than you would expect based on phylogeny?

Prioritize for biological study Search pathogen genes against databases. Identify those with eukaryotic similarity. Modify screening method /algorithm Approach World Research Community Study function in model host (C. elegans) Study function in bacterium Infection of mutant in model host Collaborations with others DATABASE Rank candidates - evolutionary analysis. C. elegans

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

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 – 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

Sensor Histidine Kinase for 2-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

A Histidine Kinase in Streptomyces. The Missing Link? 0.1 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

Universal role of this Histidine Kinase in pathogenicity? Pathogenic Fungi Senses change in osmolarity of the environment Proposed role in 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?

Reduced virulence of a Pseudomonas aeruginosa transposon mutant disrupted in the histidine kinase lemA Cells challenged per mouse Neutropenic mice challenged per group % Mortality WildtypeLemA x x x x x x

Trends in the Current Analysis Identifies the strongest cases of lateral gene transfer between bacteria and eukaryotes Most common “cross-kingdom” horizontal transfers: Bacteria Unicellular Eukaryote A control: Method identifies all previously reported Chlamydia trachomatis eukaryotic-like genes.

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

Horizontal Gene Transfer and Bacterial Pathogenicity Pathogenicity Islands: Uropathogenic and Enteropathogenic 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

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?

Pathogenomics Project: Future Developments Identify eukaryotic motifs and domains in pathogen genes Identify further motifs associated with Pathogenicity islands Virulence determinants Functional tests for new predicted virulence factors

Acknowledgements Pseudomonas Genome Project: PathoGenesis Corp. (Ken Stover) and University of Washington (Maynard Olsen) Membrane proteins: Manjeet Bains, Kendy Wong, Canadian Cystic Fibrosis Foundation Animal infection studies: Hong Yan

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, Steven J. Jones, Patrick Keeling, Audrey de Koning, Don G. Moerman, Sarah P. Otto, B. Francis Ouellette, Ivan Wan. Peter Wall Foundation