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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