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:
AprFOpmM
OpmH
OpmFOpmKOpmL
OpmN
OpmQ
OpmD
OprN
OpmE
OpmJOpmA
OprM OprJ
OpmB
OpmGOpmI
OprMFamily
(MultidrugEfflux?)
ProteinSecretion? TolC
POREPORIN
Peptidoglycan
LPS Mg++
Outermembrane
Cytoplasmicmembrane
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
BathingSolution
PlanarBilayer
Membrane
VoltageSource
CurrentAmplifier
Protein
Planar Lipid Bilayer Apparatus
The N-terminus of OprF forms channels in a lipid bilayer membrane
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2.8 3
Single channel conductance (nS)
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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…
Anthrax Necrotizing fasciitis Cat scratch disease Paratyphoid/enteric feverChancroid Peptic ulcers and gastritisChlamydia Periodontal diseaseCholera PlagueDental caries PneumoniaDiarrhea (E. coli etc.) SalmonellosisDiphtheria Scarlet feverEpidemic typhus ShigellosisMediterranean fever Strep throatGastroenteritis SyphilisGonorrhea Toxic shock syndromeLegionnaires' disease Tuberculosis Leprosy TularemiaLeptospirosis Typhoid feverListeriosis UrethritisLyme disease Urinary Tract InfectionsMeliodosis 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
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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 SialidaseFree 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?
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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
Wildtype LemA-
0.74x 106 7 100 100
0.74x 105 7 100 85.7
0.74x 104 7 100 50
0.74x 103 8 75 50
0.74x 102 8 62.5 50
0.74x 101 8 37.5 25
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 EHECDiptheria toxin gene, Cholera toxinBotulinum toxins
Plasmids:Shigella, Salmonella, Yersinia
Horizontal Gene Transfer and Bacterial Pathogenicity
Pathogenicity Islands:
Uropathogenic and Enteropathogenic E. coliSalmonella typhimuriumYersinia spp.Helicobacter pyloriVibrio 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: 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 - 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
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
www.pathogenomics.bc.ca