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Pathogenomics
Goal:
Identify previously unrecognized
mechanisms of microbial
pathogenicity using a unique
combination of informatics,
evolutionary biology, microbiology
and genetics.
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?
Eukaryotic-like pathogen genes
Aquifex aeolicus
96
Haemophilus influenza
100
Escherichia coli
Anabaena
- YopH, a proteintyrosine phosphatase, of
Yersinia pestis
100
Synechocystis
100
63
Chlamydia trachomatis
64 Petunia x hybrida
83
0.1
Nicotiana tabacum
Brassica napus
99
Arabidopsis thaliana
52
Oryza sativa
- Enoyl-acyl carrier
protein reductase
(involved in lipid
metabolism) of
Chlamydia trachomatis
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?
Evolutionary significance.
- Horizontal transfer? Similar by
chance?
Prioritize for biological study.
- Previously studied biologically?
- Can UBC microbiologists study it?
- C. elegans homolog?
Modify
screening
method
/algorithm
Pathogens
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
Pathogens
Chlamydophila psittaci
Mycoplasma mycoides
Mycoplasma hyopneumoniae
Pasteurella haemolytica
Pasteurella multicoda
Ralstonia solanacearum
Xanthomonas citri
Xylella fastidiosa
Respiratory disease, primarily in birds
Contagious bovine pleuropneumonia
Pneumonia in pigs
Cattle shipping fever
Cattle septicemia, pig rhinitis
Plant bacterial wilt
Citrus canker
Pierce’s Disease - grapevines
Bacterial wilt
Approach
Prioritized candidates
Study function
of gene.
Investigate role
of bacterial
gene in disease:
Infection study
in model host
Study function
of similar gene
in model host,
C. elegans.
Contact other
groups for
possible
collaborations.
C. elegans
DATABASE
World Research
Community
Interdisciplinary group
Informatics/Bioinformatics
Evolutionary Theory
• BC Genome Sequence Centre
• Centre for Molecular
Medicine and Therapeutics
• Dept of Zoology
• Dept of Botany
• Canadian Institute for
Advanced Research
Coordinator
Pathogen Functions
Host Functions
•
•
•
•
• Dept. Medical Genetics
• C. elegans Reverse Genetics
Facility
• Dept. Biological Sciences SFU
Dept. Microbiology
Biotechnology Laboratory
Dept. Medicine
BC Centre for Disease Control
Power of the Approach
• Interdisciplinary team
• Automated approach
unique ideas and collaborations
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
Database front end
PhyloBLAST – a tool for the analysis
Bacterium Eukaryote Horizontal Transfer
Bacillus subtilis
Escherichia coli
Salmonella typhimurium
Staphylococcua aureus
Clostridium perfringens
Clostridium difficile
Trichomonas vaginalis
Haemophilus influenzae
N-acetylneuraminate
lyase (NanA) of the
protozoan
Trichomonas vaginalis
is 92-95% similar to
NanA of
Pasteurellaceae
bacteria.
Acinetobacillus actinomycetemcomitans
0.1
Pasteurella multocida
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
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?
Eukaryote Bacteria Horizontal Transfer?
Rat
0.1
Human
Escherichia coli
Caenorhabditis elegans
Pig roundworm
Methanococcus jannaschii
Methanobacterium thermoautotrophicum
Bacillus subtilis
Streptococcus pyogenes
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).
Aquifex aeolicus
Acinetobacter calcoaceticus
Haemophilus influenzae
Chlorobium vibrioforme
Its role in virulence has
not been investigated.
Eukaryote Bacteria Horizontal Transfer?
Hypocrea jecorina EGLII
Trichoderma viride EGL2
Penicillium janthinellum EGL2
Macrophomina phaseolina EGL2
Cryptococcus flavus CMC1
Ralstonia solanacearum egl
Humicola insolens CMC3
Humicola grisea CMC3
Ralstonia
solanacearum
cellulase (ENDO-1,4BETA-GLUCANASE) is
56% similar to
endoglucanase
present in a number
of fungi.
Aspergillus aculeatus CMC2
Aspergillus nidulans EGLA
Macrophomina phaseolina egl1
Aspergillus aculeatus CEL1
Demonstrated
virulence factor for
plant bacterial wilt
Aspergillus niger EGLB
Vibrio species manA
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.
G+C Analysis: Identifying Pathogenicity Islands
%G+C
52.24
46.42
26.07
37.29
42.29
29.37
35.27
47.99
35.00
26.37
33.33
47.05
53.33
52.38
57.63
54.42
55.56
S.D.
-2
-1
-1
-2
-2
-2
-2
-2
Location
879443..880738
880832..881488
881770..882237
882294..882470
882474..882674
882677..883054
883112..883369
883459..884004
884001..884120
884167..884439
884705..884995
885001..885474
885517..886386
886550..887473
887551..888192
888247..889038
889531..890322
Strand
Gene
+
+
NMB0854
NMB0855
NMB0856
NMB0857
NMB0858
NMB0859
NMB0860
NMB0861
NMB0862
NMB0863
NMB0864
NMB0865
NMB0866
NMB0867
NMB0868
NMB0869
NMB0870
Product
histidyl-tRNA synthetase
put. bacteriocin resist.
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
YabO/YceC/SfhB fam. prot.
conserved hypothetical
hypothetical protein
3-methyl-2-oxobutanoate
hydroxymethyltransferase
G+C of ORFs: Analysis of Variance
• Low G+C 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?
Future Developments
• Incorporate unfinished genomes, plasmids into
analysis (including eukaryotic)
• Motif-based and domain-based analyses
• G+C analysis graphical viewer for identification of
pathogenicity islands
• Functional tests
• 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