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

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

5. 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

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

7. Na + -transporting NADH:ubiquinone oxidoreductase (NQR) Respiration-linked primary sodium pump Inactivation known to alter virulence gene expression cytoplasm membrane Na + NADHNAD + NQR Q QH periplasm

8. ctxBtcpA mRNA levels relative to WT strain (fold change) Virulence gene expression in the NQR mutant ctxB- gene encoding cholera toxintcpA- gene encoding TCP

9. Na + H+H+ H+H+ H+H+ nhaA nhaD Na + NADHNAD + NQR Q QH Role of sodium in virulence gene expression mrp

10. GM 1 ganglioside CT Anti-CT Secondory antibody-HRP conjugate Detection Substrate CT ELISA

11. CT production (% of WT) CT production in sodium pump knockouts Na + NADHNAD + NQR Q QH

12. 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 + NADHNAD + NQR Q QH

13. 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

14. Aerobic respiration chain in V. cholerae SDH sdhA-D VC2088-91 NQR nqrA-F VC2290-95 NDH-2 ndh VC1890 Q QH 2 Bd cyt oxidase-1 cydA-B-1 VC1843-44 Fe/S bc1 complex petA-C VC0573-74 VC0577 Bd cyt oxidase-2 cydA-B-2 VCA0872-73

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

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

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

18. 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

19. 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

20. 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

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

22. Acknowledgements HHMI Dr. Yusuke Minato Dr. Claudia Häse Dr. Kevin Ahern