The role of respiration in virulence
gene expression of Vibrio cholerae
HHMI 2011
Sara Fassio
Dr. Claudia Häse
Dr. Yusuke Minato
Cholera
•
•
•
•
•
Infection of small intestine
Causes severe diarrhea and electrolyte loss
3-5 million cases a year
100,000- 120,000 deaths per year
Occurs in areas with poor sanitation,
contaminated water supplies
Vibrio cholerae
• Gram negative bacterium, causes cholera
• Two main virulence factors for establishing infection
Virulence Factors
• Toxin Coregulated Pilus (TCP)
- Responsible for colonization/ aggregation of V. cholerae
in small intestine
Initial
attachment in
small intestine
TCP expression
Colony formation,
cholera toxin
released
Virulence Factors
• Cholera Toxin (CT)
- Increases chloride secretion and inhibits sodium chloride
absorption
- Results in massive outpouring of fluids
Na+-transporting NADH:ubiquinone
oxidoreductase (NQR)
• Respiration-linked primary sodium pump
• Inactivation known to alter virulence gene
expression
7
Na+
Cholera Toxin levels WT vs. NQR
periplasm
CT (μg/ml/O.D. 600)
6
Q
5
NQR
4
membrane
QH
3
2
NADH
1
0
WT
nqr
NAD+
cytoplasm
mRNA levels relative to
WT strain (fold change)
Virulence gene expression in the NQR
mutant
1.2
1
WT
nqr
0.8
0.6
0.4
0.2
0
ctxB
ctxB- gene encoding cholera toxin
tcpA
tcpA- gene encoding TCP
Na+
Na+
Role of sodium
in virulence
gene expression
Na+
mrp
H+
H+
H+
Q
NQR
QH
NADH
Na+
NAD+
CT ELISA
Detection
Substrate
Secondory
antibody-HRP
conjugate
Anti-CT
CT
GM1 ganglioside
CT production in sodium pump knockouts
CT production
(% of WT)
150
100
50
0
Na+
Q
NQR
QH
NADH
NAD+
Hypothesis
• Changes in respiration status causes changes
in virulence gene expression in V. cholerae
• Small intestine transition point between
aerobic and anaerobic respiration
• Changes in respiration status throughout life
cycle could be key to inducing transcription at
infection site
Na+
Q
NQR
QH
NADH
NAD+
HHMI Summer Project
Investigate the role of respiration on virulence
gene expression via:
1. Inactivation of complex II with malonate
2. V. cholerae quinone deficient mutant strains
NQR
• Electron transport chain in V. cholerae similar to mitochondria in
eukaryotes
• NQR instead of complex I
• Malonate- inhibitor of complex II
Effects of malonate on CT production
CT production
(% of WT)
150
100
50
0
LB
LB + Malonate
Alkaline phosphatase activity
(% of LB)
Effects of malonate on virulence gene
expression
120
LB
100
80
**
LB-malonate
**
60
40
20
0
ctx::phoA
tcpA::phoA
HHMI Summer Project
Investigate the role of respiration on virulence
gene expression via:
1. Inactivation of complex II with malonate
2. V. cholerae quinone deficient mutant strains
• Further investigate the role of respiration in
virulence gene expression:
– Construct mutant V. cholerae knockout strains
lacking the ability to synthesize quinones
• ubiC gene, encoding the ubiquinone-8 precursor
synthesis enzyme
- Ubiquinone – aerobic respiration
• menB gene, encoding the menaquinone precursor
synthesis enzyme
- Menaquinone- anaerobic respiration
Predictions
Two possibilities
Mutants express
repression of ctxB and
tcpA in comparison to
wild-type
No changes are
observed
Quinones are linked to
virulence gene
expression
Future research can focus
on other respiratory
intermediates
Future Research
• Confirm hypothesis
– Continue development of quinone knockout
strains
• Investigate mechanisms of how respiration
affects virulence gene expression
Acknowledgements
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HHMI
Dr. Yusuke Minato
Dr. Claudia Häse
Dr. Kevin Ahern