Transcript Powerpoint File - Centre for Microbial Diseases and Immunity

Cross-Domain and Within-Domain
Horizontal Gene Transfer: Implications
for Bacterial Pathogenicity
1. Pathogenomics Project
2. Cross-Domain Horizontal Gene
Transfer Analysis
3. Horizontal Gene Transfer:
Identifying Pathogenicity Islands
Pathogenomics
Goal:
Identify previously unrecognized
mechanisms of microbial pathogenicity
using a combination of informatics,
evolutionary biology, microbiology and
genetics.
Explosion of data
23 of the 37 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 …
The need for new tools
Prioritize new genes for further laboratory
study
Capitalize on the existing genomic data
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
bacterial 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.
Evolutionary significance.
- Horizontal transfer? Similar by chance?
Prioritize for biological study.
- Previously studied in the laboratory?
- Can UBC microbiologists study it?
- C. elegans homolog?
Modify
screening
method
/algorithm
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 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 in
bacterium.
Study function
of homolog in
model host
(C. elegans)
Collaborations
with others
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
Pathogenomics Database: Bacterial proteins with unusual
similarity with Eukaryotic proteins
Haemophilus influenzae Rd-KW20 proteins most strongly
matching eukaryotic proteins
PhyloBLAST – a tool for analysis
Brinkman et al. (2001) Bioinformatics. In Press.
Trends in the Initial Analysis
• Identifies the strongest cases of lateral gene transfer
between bacteria and eukaryotes
• Most common “cross-domain” horizontal transfers:
Bacteria
Unicellular Eukaryote
• Identifies nuclear genes with potential organelle origins
• A control: Method identifies all previously reported
Chlamydia trachomatis “eukaryote-like” genes.
First case: Bacterium Eukaryote Lateral 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
de Koning et al. (2000) Mol. Biol. Evol. 17:1769-1773
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: ?
Another case: A Sensor Histidine Kinase for a
Two-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?
Streptomyces Histidine Kinase. The Missing Link?
Pseudomonas aeruginosa PhoQ
100
100
Xanthomonas campestris RpfC
Vibrio cholerae TorS
Escherichia coli TorS
Escherichia coli RcsC
39
100
100
54
100
Candida albicans CaNIK1
100 Neurospora crassa NIK-1
51Fusarium solani FIK1
Fusarium solani FIK2
Streptomyces coelicolor SC4G10.06c
100
Streptomyces coelicolor SC7C7.03
Pseudomonas aeruginosa GacS
100
100Pseudomonas fluorescens GacS / ApdA
Fungi
virulence factor ?
Pseudomonas tolaasii RtpA / PheN
86
100
0.1
100
100Pseudomonas syringae GacS / LemA
Pseudomonas viridiflava RepA
Azotobacter vinelandii GacS
Erwinia carotovora RpfA / ExpS
100
100 Escherichia coli BarA
Salmonella typhimurium BarA
virulence
= factor
Reduced virulence of a Pseudomonas aeruginosa
transposon mutant disrupted in the
histidine kinase gene gacS
Groups of 7-8 neutropenic mice challenged on two separate
occasions with doses ranging from 8 to 8 x 106 bacteria
Wildtype LD50 = 10  1 bacteria
gacS mutant LD50 = 7,500  100 bacteria
750-fold increase
Recent report: P. aeruginosa eukaryote-type
Phospholipase plays a role in infection
Wilderman et al. 2001. Mol Microbiol 39:291-304
• Phospholipase D (PLDs) virtually ubiquitous in eukaryotes
(relatively uncommon in prokaryotes)
• P. aeruginosa expresses PLD with significant (1e-38
BLAST Expect) similarity to eukaryotic PLDs
• Part of a mobile 7 kb genetic element
• Role in P. aeruginosa persistence in a chronic pulmonary
infection model
Eukaryote Bacteria Horizontal Transfer?
Rat
0.1
Human
Escherichia coli
Caenorhabditis elegans
Pig roundworm
Methanococcus jannaschii
Methanobacterium thermoautotrophicum
E. coli Guanosine
monophosphate
reductase 81% similar
to corresponding
enzyme in humans
and rats
Bacillus subtilis
Streptococcus pyogenes
Aquifex aeolicus
Acinetobacter calcoaceticus
Haemophilus influenzae
Chlorobium vibrioforme
Role in virulence not
yet investigated.
Expanding the Cross-Domain Analysis
•
Identify cross-domain lateral gene transfer between bacteria,
archaea and eukaryotes
•
No obvious correlation seen with protein functional classification
•
Most cases: no obvious correlation seen between “organisms
involved” in potential lateral transfer
Exceptions:
–
Unicellular eukaryotes
– “Organelle-like” proteins in Rickettsia and Synechocystis
–
“Plant-like(?)” genes in the obligate intracellular bacteria
Chlamydia
“Plant-like” genes in Chlamydia
Aquifex aeolicus
96
Haemophilus influenza
100
Escherichia coli
Anabaena
100
Synechocystis
100
63
64
83
0.1
Chlamydia trachomatis
Enoyl-acyl carrier protein
reductase (involved in lipid
metabolism) of Chlamydia
trachomatis is similar to
those of Plants
Petunia x hybrida
Nicotiana tabacum
Brassica napus
99
Arabidopsis thaliana
52
Oryza sativa
Organelle relationship?
Notably more similar to
plants than Synechocystis
Eukaryote Top Hits in Bacterial Genomes
(after excluding relatives of the same Family)
No. of Eukaryotic Top Hits
300
Synechocystis
250
200
150
100
50
0
0
1000
2000
3000
4000
No. of Proteins in each Bacterial Genome
5000
6000
Eukaryote Top Hits in Bacterial Genomes
(excluding "Family" and Synechocystis )
No. of Eukaryotic Top Hits
80
70
60
50
40
30
Rickettsia and Chlamydia
20
10
0
0
1000
2000
3000
No. of Proteins
4000
5000
6000
Proteins Homologous to Eukaryote Proteins
(according to BLAST Exp=1)
No of Proteins with Eukaryotic
Homology
1800
1600
1400
1200
1000
800
600
400
200
0
0
1000
2000
3000
No. of Proteins
4000
5000
6000
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
non-clonal
clonal
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
• Threader: Detect proteins with similar tertiary structure
• Identify more motifs associated with
• Pathogenicity islands
• Virulence determinants
• Functional tests for new predicted virulence factors
• Expand analysis to include viral genomes
Peter Wall Major Thematic Grant
• Fundamental research
• Interdisciplinary
• Lack of fit with
alternative funding
sources
Pathogenomics group
Ann M. Rose, Yossef Av-Gay, David L. Baillie, Fiona S. L.
Brinkman, Robert Brunham, 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.
www.pathogenomics.bc.ca
Universal role of this Histidine Kinase in
pathogenicity?
Pathogenic Fungi
•Senses change in osmolarity of the environment
•Role in hyphal formation 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?
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
Euykaryotic top hits in bacterial genomes
(after excluding "tertiary" relatives)
350
No. of Eukaryote Hits
Synechocystis
300
250
200
150
100
Rikettsia
50
0
0
1000
2000
3000
No. of Proteins
4000
5000
6000