1. Using informatics to focus bacterial pathogenicity studies

2. Goal: Use informatic analyses to generate new testable hypotheses about pathogen protein function and pathogenicity mechanisms Test the hypotheses in the laboratory Using informatics to focus bacterial pathogenicity studies

3. Gramicidine S (Consden et al., 1947), partial insulin sequence (Sanger and Tuppy, 1951) First codon assignment UUU/phe (Nirenberg and Matthaei, 1961) 3.5 kb RNA bacteriophage MS2 (Fiers et al., 1976) 5.4 kb bacteriophage  X174 (Sanger et al., 1977) Early databases: Dayhoff, 1972; Erdmann, 1978 Need for informatics in biology: origins

4. (from the National Centre for Biotechnology Information)

5. Explosion of data 22 of the 33 publicly available microbial genome sequences are for bacterial pathogens Approximately 18,000 pathogen genes with no known function! >95 bacterial pathogen genome projects in progress…

6. -Pseudomonas aeruginosa -Three dimensional comparative protein modeling -Phylogenetic analysis of gene families -Other analyses: Regulatory network complexity -Pathogenomics Project -Detecting eukaryote:pathogen homologs -Detecting pathogenicity islands Pathogen Informatics

7. 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

8. Outer membrane protein OprF Nonspecific porin Required for –Maintenance of cell shape –Growth in low-osmolarity environments OprF - clinical mutant with multiple antimicrobial resistance being characterized Adhesin in plant colonizing Pseudomonas species Proposed vaccine component

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

10. Structure of the outer membrane protein A transmembrane domain Pautsch and Schulz (1998). Nature Structural Biology 5:1013-1017 No channel formation detected

11. OprF 1 -QGQNSVEIEAFGKRYFTDSVRNMKN-------ADLYGGSIGYFLTDDVELALSYGEYH OmpA 1 APKDNTWYTGAKLGWSQYHDTGLINNNGPTHENKLGAGAFGGYQVNPYVGFEMGYDWLG * * * * ** * * OprF 52 DVRGTYETGNKKVHGNLTSLDAIYHFGTPGVGLRPYVSAGLA-HQNITNINSDSQGRQQ OmpA 60 RMPYKGSVENGAYKAQGVQLTAKLGYPIT-DDLDIYTRLGGMVWRADTYSNVYGKNHDT * * * * * * * * OprF 110 MTMANIGAGLKYYFTENFFAKASLDGQYGLEKRDNGHQG--EWMAGLGVGFNFG OmpA 118 GVSPVFAGGVEYAITPEIATRLEYQWTNNIGDAHTIGTRPDNGMLSLGVSYRFG * * * * *** ** OprF and OmpA share only 15% identity

12. Model of the N-terminus of OprF based on OmpA Brinkman, Bains and Hancock (2000). Journal of Bacteriology 182:5251-5255

13. OprF model (yellow and green) aligned with the crystal structure of OmpA (blue) Many residues are in the same three dimensional environment, though on different strands

14. OprF 1 -QGQNSVEIEAFGKRYFTDSVRNMKN-------ADLYGGSIGYFLTDDVELALSYGEYH OmpA 1 APKDNTWYTGAKLGWSQYHDTGLINNNGPTHENKLGAGAFGGYQVNPYVGFEMGYDWLG * * * * ** * * OprF 52 DVRGTYETGNKKVHGNLTSLDAIYHFGTPGVGLRPYVSAGLA-HQNITNINSDSQGRQQ OmpA 60 RMPYKGSVENGAYKAQGVQLTAKLGYPIT-DDLDIYTRLGGMVWRADTYSNVYGKNHDT * * * * * * * * OprF 110 MTMANIGAGLKYYFTENFFAKASLDGQYGLEKRDNGHQG--EWMAGLGVGFNFG OmpA 118 GVSPVFAGGVEYAITPEIATRLEYQWTNNIGDAHTIGTRPDNGMLSLGVSYRFG * * * * *** ** OprF and OmpA similarity

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

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

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

18. Upstream of OprF is a probable sigma factor gene, sigX sigXoprF Promoter Transcription terminator

19. Disruption of sigX reduces expression of OprF 1.Marker 2.Wildtype 3.sigX - mutant 4.oprF - mutant P. aeruginosa P. fluorescens

20. oprF No SigX expression: SigX expression: sigX

21. 18 ECF sigma factors in the P. aeruginosa genome

23. 01000200030004000500060007000 Number of Genes 0 2 4 6 8 10 Regulators (%) Percent Regulators as a Function of Genome Size 1 2 3 4 5 6767 8 9 10 11 12 13 Specialized environments Free-living Genomes represented: 1, Mycoplasma genitalium; 2, Chlamydia trachomatis; 3, Treponema pallidum; 4, Borrelia burgdorferi; 5, Chlamydia pneumoniae; 6, Helicobacter pylori ---; 7, Helicobacter pylori---; 8, Haemophilus influenzae; 9, Neisseria meningitidis; 10, Mycobacterium tuberculosis; 11, Bacillus subtilis; 12, Escherichia coli; 13, Pseudomonas aeruginosa.

24.  OprM Family of putative Efflux and Type I secretion proteins (18 members)  OprD Family of putative Amino acid, Peptide and Aromatic compound transporters (19 members)  TonB Family of putative iron-siderophore receptors (34 members) P. aeruginosa Genome Sequence Analysis: Outer Membrane Proteins (OMPs) Approximately 150 OMPs predicted including three large paralogous families:

25. 0.1 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

26. OprM structural model based on TolC

29. Future Developments Modeling of other outer membrane proteins in Neisseria species. Developing a better algorithms for secondary structure prediction

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

31. 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?

32. 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 100 96 63 64 52 83 99

33. 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

34. 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 Citrus variegated chlorosis Bacterial wilt

35. 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

36. 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?

37. 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.

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

39. 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

40. 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 GMP reductase of E. coli is 81% similar to the corresponding enzyme studied in humans and rats Role in virulence not yet investigated

41. 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

42. World Research Community Functional studies 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.

43. Pathogenicity Islands Virulence genes commonly in clusters Associated with –tRNA sequences –Transposases, Integrases and other mobility genes –Flanked by repeats

44. G+C Analysis: 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

45. Neisseria meningitidis serogroup B strain MC58 Mean %G+C: 51.37 STD DEV: 7.57 %G+C SD Location Strand Product 37.22 -1 1831577..1832527 + pilin gene inverting 39.95 -1 1834676..1835113 + VapD-related 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

46. %G+C of ORFs: Analysis of Variance %G+C variance is similar within a given species Low %G+C variance correlates with an intracellular lifestyle for the bacterium and a clonal nature (P = 0.004) Neisseria meningitidis +/- 7% Chlamydia species+/- 2% Intracellular bacteria ecologically isolated?

47. Future Developments Identify eukaryotic motifs and domains in pathogen genes Identify further motifs associated with Pathogenicity islands Virulence determinants Functional tests for new potential virulence factors www.pathogenomics.bc.ca

48. Informatics as a focus Outer membrane protein modeling: Focus mutational studies and studies of surface exposed sequences Phylogenetic analyses: Focus study of gene mutants under certain environmental conditions Other analyses - Regulatory network complexity: Change focus of regulation studies Eukaryote:pathogen homologs: Focus identification of “mimics” Pathogenicity islands: Focus identification of recently obtained virulence determinants

49. Acknowledgements Pathogenomics group: Ann Rose, Steven Jones, Ivan Wan, Hans Greberg, Yossef Av- Gay, David Baillie, Bob Brunham, Stefanie Butland, Rachel Fernandez, Brett Finlay, Patrick Keeling, Audrey de Koning, Sarah Otto, Francis Ouellette, Peter Wall Institute Pseudomonas Genome Project: PathoGenesis Corp. (Ken Stover) and University of Washington (Maynard Olsen) Outer membrane proteins: Manjeet Bains, Kendy Wong, Canadian Cystic Fibrosis Foundation Bob Hancock