Pathogenomics

1
Pathogenomics
Goal Identify previously unrecognized mechanisms
of microbial pathogenicity using a unique
combination of informatics, evolutionary biology,
microbiology and genetics.
2
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?
3
Eukaryotic-like pathogen genes
Aquifex aeolicus
96
- YopH, a protein-tyrosine phosphatase, of
Yersinia pestis - Enoyl-acyl carrier protein
reductase (involved in lipid metabolism) of
Chlamydia trachomatis
Haemophilus influenza
100
Escherichia coli
Anabaena
100
Synechocystis
100
Chlamydia trachomatis
63
Petunia x hybrida
64
Nicotiana tabacum
83
Brassica napus
99
Arabidopsis thaliana
0.1
52
Oryza sativa
4
Approach
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
Evolutionary significance. - Horizontal
transfer? Similar by chance?

• Prioritize for biological study.
• - Previously studied biologically?
• Can UBC microbiologists study it?
• C. elegans homolog?

5
Pathogens
Anthrax Necrotizing fasciitis Cat scratch
disease Paratyphoid/enteric fever Chancroid
Peptic ulcers and gastritis Chlamydia
Periodontal disease Cholera Plague Dental
caries Pneumonia Diarrhea (E. coli
etc.) Salmonellosis Diphtheria Scarlet
fever Epidemic typhus Shigellosis Mediterranean
fever Strep throat Gastroenteritis
Syphilis Gonorrhea Toxic shock
syndrome Legionnaires' disease Tuberculosis
Leprosy Tularemia Leptospirosis Typhoid
fever Listeriosis Urethritis Lyme disease
Urinary Tract Infections Meliodosis Whooping
cough Meningitis Hospital-acquired
infections
6
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 Pierces Disease - grapevines
Bacterial wilt
7
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
Contact other groups for possible collaborations.
C. elegans
DATABASE
World Research Community
8
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

Coordinator

• 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

9
Power of the Approach

• 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

10
Database front end
11
(No Transcript)
12
PhyloBLAST a tool for the analysis
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Bacterium Eukaryote Horizontal Transfer
N-acetylneuraminate lyase (NanA) of the protozoan
Trichomonas vaginalis is 92-95 similar to NanA
of Pasteurellaceae bacteria.
14
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
Intracellular enzyme involved in sialic acid
metabolism In Bacteria Proposed to parasitize
the mucous membranes of animals for nutritional
purposes
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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?

16
Eukaryote Bacteria Horizontal Transfer?
Rat
0.1
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.
Human
Escherichia coli
Caenorhabditis elegans
Pig roundworm
Methanococcus jannaschii
Methanobacterium thermoautotrophicum
Bacillus subtilis
Streptococcus pyogenes
Aquifex aeolicus
Acinetobacter calcoaceticus
Haemophilus influenzae
Chlorobium vibrioforme
17
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
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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.

19
GC Analysis Identifying Pathogenicity Islands
GC S.D. Location Strand Gene
Product 52.24 879443..880738 -
NMB0854 histidyl-tRNA synthetase 46.42
880832..881488 - NMB0855 put. bacteriocin
resist. 26.07 -2 881770..882237 - NMB0856
hypothetical protein 37.29 -1
882294..882470 - NMB0857 hypothetical
protein 42.29 -1 882474..882674 -
NMB0858 hypothetical protein 29.37 -2
882677..883054 - NMB0859 hypothetical
protein 35.27 -2 883112..883369 -
NMB0860 hypothetical protein 47.99
883459..884004 - NMB0861 hypothetical
protein 35.00 -2 884001..884120 -
NMB0862 hypothetical protein 26.37 -2
884167..884439 - NMB0863 hypothetical
protein 33.33 -2 884705..884995 -
NMB0864 hypothetical protein 47.05
885001..885474 - NMB0865 hypothetical
protein 53.33 885517..886386 -
NMB0866 hypothetical protein 52.38
886550..887473 NMB0867 YabO/YceC/SfhB fam.
prot. 57.63 887551..888192 - NMB0868
conserved hypothetical 54.42 888247..889038
- NMB0869 hypothetical protein 55.56
889531..890322 NMB0870
3-methyl-2-oxobutanoate
hydroxymethyltransferase
20
GC of ORFs Analysis of Variance

• Low GC 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?

21
Future Developments

• Incorporate unfinished genomes, plasmids into
  analysis (including eukaryotic)
• Motif-based and domain-based analyses
• GC analysis graphical viewer for identification
  of pathogenicity islands
• Functional tests

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• 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.
• www.pathogenomics.bc.ca