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

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Genome analysis and membrane protein bioinformatics UBC Pathogenomics Project

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Identifying eukaryote:pathogen gene homologs

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Detecting pathogenicity islands

Pseudomonas aeruginosa

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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

P. aeruginosa Genome Sequence Analysis: Outer Membrane Proteins (OMPs)

Approximately 150 OMPs predicted including three large paralogous families:

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OprM homology (3 previously known, now 18 predicted)

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OprD homology (2 previously known, now 19)

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TonB-dependent domain (8 previously known, now 34)

OprM Family (Multidrug Efflux?)

OprM OpmJ OpmA OpmE OprN OpmD OpmQ OprJ OpmB OpmG OpmI

Protein Secretion?

OpmN OpmL OpmK AprF OpmM OpmH TolC OpmF

LPS Peptidoglycan

Gram Negative Cell Envelope

PORE PORIN + Mg + Outer membrane Periplasm Cytoplasmic membrane

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

Protein

Planar Lipid Bilayer Apparatus

Voltage Source Current Amplifier Bathing Solution Planar Bilayer Membrane

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

40 35 30 25 20 15 10 5 0 0.

2 0.

4 0.

6 0.

8 1 1.

2 1.

4 1.

6 1.

8 2 2.

2 2.

4

Single channel conductance (nS)

2.

6 2.

8 3

Current and Future Research

Improve computational prediction of…

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membrane and secreted proteins

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surface exposed regions of membrane proteins

Current and Future Research

Omp85 membrane protein family studies

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Antigenic, conserved, vaccine candidate

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Two copies in most pathogenic bacteria genomes – why?

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Structure unknown, may have conformational epitopes

Pathogenomics

Opportunistic pathogen Pseudomonas aeruginosa

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Genome analysis and membrane protein bioinformatics UBC Pathogenomics Project

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Identifying eukaryote:pathogen gene homologs

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Detecting pathogenicity islands

Genome data for…

Anthrax Cat scratch disease Chancroid Chlamydia Cholera Dental caries Diarrhea (E. coli etc.) Diphtheria Epidemic typhus Mediterranean fever Gastroenteritis Gonorrhea Legionnaires' disease Leprosy Leptospirosis Listeriosis Lyme disease Meliodosis Meningitis Necrotizing fasciitis Paratyphoid/enteric fever Peptic ulcers and gastritis Periodontal disease Plague Pneumonia Salmonellosis Scarlet fever Shigellosis Strep throat Syphilis Toxic shock syndrome Tuberculosis Tularemia Typhoid fever Urethritis Urinary Tract Infections Whooping cough +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?

Approach

Search pathogen genes against databases. Identify those with eukaryotic similarity.

Modify screening method /algorithm Rank candidates - evolutionary analysis .

Prioritize for biological study Collaborations with others Study function in model host

(C. elegans)

Study function in bacterium Infection of mutant in model host DATABASE World Research Community

C. elegans

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

Pathogen Functions

• Dept. Microbiology • Biotechnology Laboratory • Dept. Medicine • BC Centre for Disease Control

Coordinator Host Functions

• Dept. Medical Genetics • C. elegans Reverse Genetics Facility • Dept. Biological Sciences SFU

Bacterium Eukaryote Horizontal Transfer

0.1

Bacillus subtilis Escherichia coli Salmonella typhimurium Staphylococcua aureus Clostridium perfringens Clostridium difficile Trichomonas vaginalis

N-acetylneuraminate lyase (NanA) of the protozoan

Trichomonas vaginalis

is 92-95% similar to NanA of Pasteurellaceae bacteria.

Haemophilus influenzae Acinetobacillus actinomycetemcomitans Pasteurella multocida

N-acetylneuraminate lyase – role in pathogenicity?

Pasteurellaceae

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Mucosal pathogens of the respiratory tract

T. vaginalis

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Mucosal pathogen, causative agent of the STD Trichomonas

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: ?

Sensor Histidine Kinase for 2-component Regulation System Signal Transduction Histidine kinases common in bacteria Ser/Thr/Tyr kinases common in eukaryotes

Candida

However, a histidine kinase was recently identified in fungi, including pathogens Fusarium solani and Candida albicans How did it get there?

A Histidine Kinase in Streptomyces.

The Missing Link? 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 0.1

Universal role of this Histidine Kinase in pathogenicity?

Pathogenic Fungi

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Senses change in osmolarity of the environment

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Proposed role in pathogenicity Pseudomonas species plant pathogens

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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

% Mortality

Cells challenged per mouse 0.74x 10 6 0.74x 10 5 0.74x 10 4 0.74x 10 3 0.74x 10 2 0.74x 10 1 Neutropenic mice challenged per group 7 7 7 8 8 8

Wildtype 100 100 100 75 62.5

37.5

LemA 100 85.7

50 50 50 25

Trends in the Current Analysis

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Identifies the strongest cases of lateral gene transfer between bacteria and eukaryotes

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Most common “cross-kingdom” horizontal transfers: Bacteria Unicellular Eukaryote

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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

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Atypical %G+C

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tRNA sequences

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Transposases, Integrases and other mobility genes

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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: 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 47.99 1841404..1843191 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 put. hemolysin activ. HecB 45.32 1843246..1843704 put. toxin-activating 37.14 -1 1843870..1844184 31.67 -2 1844196..1844495 37.57 -1 1844476..1845489 20.38 -2 1845558..1845974 hypothetical hypothetical hypothetical hypothetical 45.69 1845978..1853522 hemagglutinin/hemolysin-rel.

51.35 1854101..1855066 + 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

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Identify eukaryotic motifs and domains in pathogen genes

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Identify further motifs associated with Pathogenicity islands Virulence determinants

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Functional tests for new predicted virulence factors

Acknowledgements

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Pseudomonas Genome Project: PathoGenesis Corp. (Ken Stover) and University of Washington (Maynard Olsen)

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Membrane proteins: Manjeet Bains, Kendy Wong, Canadian Cystic Fibrosis Foundation

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Animal infection studies: Hong Yan

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Pathogenomics group

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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 www.pathogenomics.bc.ca