The role of respiration in virulence gene expression of Vibrio cholerae HHMI 2011 Sara Fassio Dr. Claudia Häse Dr. Yusuke Minato.
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The role of respiration in virulence gene expression of Vibrio cholerae
HHMI 2011Sara Fassio
Dr. Claudia HäseDr. 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
• Two main virulence factors for establishing infection
• Gram negative bacterium, causes cholera
Virulence Factors
• Toxin Coregulated Pilus (TCP)
Initial attachment in
small intestine
TCP expression
Colony formation, cholera toxin released
- Responsible for colonization/ aggregation of V. cholerae in small intestine
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
WT nqr0
1
2
3
4
5
6
7 Cholera Toxin levels WT vs. NQR
CT (μ
g/m
l/O
.D. 6
00)
cytoplasm
membrane
Na+
NADH NAD+
NQR
Q
QH
periplasm
Series10
0.2
0.4
0.6
0.8
1
1.2
WT nqr
ctxB tcpA
mR
NA
lev
els
rela
tive
to
W
T s
trai
n (
fold
ch
ang
e)Virulence gene expression in the NQR
mutant
ctxB- gene encoding cholera toxin tcpA- gene encoding TCP
Na+
H+H+
Na+
H+
Na+
nhaA nhaD
Na+NADH NAD+
NQR
Q
QH
Role of sodium in virulence
gene expression
mrp
GM1 ganglioside
CT
Anti-CT
Secondory antibody-HRPconjugate
Detection
Substrate
CT ELISA
WT
nqr
nhaAnhaD m
rp
WT +
monen
sin
0
50
100
150
CT
pro
du
ctio
n(%
of
WT
)CT production in sodium pump knockouts
Na+
NADH NAD+
NQR
Q
QH
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+
NADH NAD+
NQR
Q
QH
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
• Electron transport chain in V. cholerae similar to mitochondria in eukaryotes
NQR
• NQR instead of complex I • Malonate- inhibitor of complex II
LB LB + Malonate0
50
100
150
CT
pro
du
ctio
n(%
of
WT
)Effects of malonate on CT production
Series10
20
40
60
80
100
120
LB LB-malonate
**
Alka
line
phos
phat
ase
activ
ity
(% o
f LB) **
ctx::phoA tcpA::phoA
Effects of malonate on virulence gene expression
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
Mutants express repression of ctxB and tcpA in comparison to
wild-type
Quinones are linked to virulence gene
expression
No changes are observed
Future research can focus on other respiratory
intermediates
Two possibilities
Future Research
• Confirm hypothesis– Continue development of quinone knockout
strains• Investigate mechanisms of how respiration
affects virulence gene expression
Acknowledgements
• HHMI• Dr. Yusuke Minato• Dr. Claudia Häse• Dr. Kevin Ahern
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