Transcript No Slide Title

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
P. aeruginosa Genome Sequence Analysis:
Outer Membrane Proteins (OMPs)
Approximately 150 OMPs predicted including three large
paralogous families:
 OprM homology (3 previously known, now 18 predicted)
 OprD homology (2 previously known, now 19)
 TonB-dependent domain (8 previously known, now 34)
OprM
OpmJ
OpmA
OprM
Family
(Multidrug
Efflux?)
OprJ
OpmB
OpmG
OpmI
OpmE
OprN
OpmD
OpmQ
AprF
OpmM
Protein
Secretion?
OpmN
OpmL
OpmH
TolC
OpmK
OpmF
Gram Negative Cell Envelope
LPS
PORE
PORIN
+ +
Mg
Outer
membrane
Peptidoglycan
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
Planar Lipid Bilayer Apparatus
Voltage
Source
Current
Amplifier
Protein
Bathing
Solution
Planar
Bilayer
Membrane
The N-terminus of OprF forms channels
in a lipid bilayer membrane
40
30
25
20
15
10
5
Single channel conductance (nS)
3
2.
8
2.
6
2.
4
2.
2
2
1.
8
1.
6
1.
4
1.
2
1
0.
8
0.
6
0.
4
0
0.
2
No. of events
35
Current and Future Research
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
Pathogenomics
Opportunistic pathogen Pseudomonas aeruginosa
- Genome analysis and membrane protein
bioinformatics
UBC Pathogenomics Project
- Identifying eukaryote:pathogen gene
homologs
- 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.
Rank candidates - evolutionary analysis.
Modify screening
method /algorithm
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
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
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 – 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)
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
•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
0.74x 106
Neutropenic
mice
challenged
per group
7
0.74x 105
% Mortality
Wildtype
LemA-
100
100
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 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
–
–
–
–
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
39.95
51.96
39.13
40.00
42.86
34.74
43.96
40.83
42.34
47.99
45.32
37.14
31.67
37.57
20.38
45.69
51.35
SD
-1
-1
-1
-1
-2
-1
-1
-1
-2
-1
-2
Location
Strand Product
1834676..1835113
+
virulence associated pro. homolog
1835110..1835211
cryptic plasmid A-related
1835357..1835701
+
hypothetical
1836009..1836203
+
hypothetical
1836558..1836788
+
hypothetical
1837037..1837249
+
hypothetical
1837432..1838796
+
conserved hypothetical
1839157..1839663
+
conserved hypothetical
1839826..1841079
+
conserved hypothetical
1841404..1843191
put. hemolysin activ. HecB
1843246..1843704
put. toxin-activating
1843870..1844184
hypothetical
1844196..1844495
hypothetical
1844476..1845489
hypothetical
1845558..1845974
hypothetical
1845978..1853522
hemagglutinin/hemolysin-rel.
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