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The role of respiration in virulence gene expression of Vibrio cholerae

The role of respiration in virulence gene expression of Vibrio cholerae . HHMI 2011 Sara Fassio Dr. Claudia H ä se 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

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The role of respiration in virulence gene expression of Vibrio cholerae

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

  2. 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

  3. Vibrio cholerae • Two main virulence factors for establishing infection • Gram negative bacterium, causes cholera

  4. Virulence Factors • Toxin CoregulatedPilus (TCP) • Responsible for colonization/ aggregation of V. cholerae in small intestine Initial attachment in small intestine TCP expression Colony formation, cholera toxin released

  5. Virulence Factors • Cholera Toxin (CT) • Increases chloride secretion and inhibits sodium chloride absorption • Results in massive outpouring of fluids

  6. Na+-transporting NADH:ubiquinoneoxidoreductase (NQR) • Respiration-linked primary sodium pump • Inactivation known to alter virulence gene expression periplasm Na+ membrane Q NQR cytoplasm QH NADH NAD+

  7. Virulence gene expression in the NQR mutant mRNA levels relative to WT strain (fold change) ctxB tcpA ctxB- gene encoding cholera toxin tcpA- gene encoding TCP

  8. Role of sodium in virulence gene expression Na+ Na+ Na+ nhaA nhaD mrp H+ H+ H+ Q NQR QH Na+ NADH NAD+

  9. CT ELISA Detection Substrate Secondory antibody-HRP conjugate Anti-CT CT GM1ganglioside

  10. CT production in sodium pump knockouts CT production (% of WT) Na+ Q NQR QH NADH NAD+

  11. 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+

  12. 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

  13. NQR • Electron transport chain in V. cholerae similar to mitochondria in eukaryotes • NQR instead of complex I • Malonate- inhibitor of complex II

  14. Effects of malonate on CT production CT production (% of WT)

  15. Effects of malonate on virulence gene expression ** Alkaline phosphatase activity (% of LB) ** ctx::phoA tcpA::phoA

  16. 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

  17. Further investigate the role of respiration in virulence gene expression: • Construct mutant V. cholerae knockoutstrains 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

  18. Predictions Two possibilities

  19. Future Research • Confirm hypothesis • Continue development of quinone knockout strains • Investigate mechanisms of how respiration affects virulence gene expression

  20. Acknowledgements • HHMI • Dr. Yusuke Minato • Dr. Claudia Häse • Dr. Kevin Ahern

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