The role of respiration in virulence gene expression of Vibrio cholerae HHMI 2011 Sara Fassio Dr. Claudia Häse Dr. Yusuke Minato.

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