Dissertation
Regulation of T3SS1 genes in Vibrio parahaemolyticus and its involvement in pathogenesis
Washington State University
Doctor of Philosophy (PhD), Washington State University
12/2008
DOI:
https://doi.org/10.7273/000006178
Abstract
Vibrio parahaemolyticus is a Gram-negative bacterium belonging to the family that causes cholera. Genome annotation showed that V. parahaemolyticus harbors two chromosomes and each chromosome encodes a type III secretion system (T3SS1 and T3SS2).
T3SS1 induces cytotoxicity in mammalian epithelial cells. We showed that V. parahaemolyticus strain NY-4 was able to induce cell death in both HeLa and U937 cell lines via a T3SS1-dependent mechanism. The mechanism of cell death involves formation of a pore structure in the surface of infected HeLa and U937 cells as demonstrated by cellular swelling, uptake of cell membrane-impermeable dye, and protection of cytotoxicity by osmoprotectant (PEG3350). Poly ADP ribose polymerase (PARP) was not cleaved. T3SS1 induced-cytotoxicity was not inhibited by addition of the pan-caspase inhibitor zVAD or caspase-1 inhibitor Ac-YVAD-CHO. DNA fragmentation was not evident following infection and autophagic vacuoles were not observed by monodansylcadaverine staining. We conclude that T3SS1 of V. parahaemolyticus strain NY-4 induced a mode of cell death consistent with oncosis. v Genes encoding T3SSs are normally activated when bacteria contact host cells or are grown under certain conditions. We found that a subset of ten T3SS1 genes are transcribed when V. parahaemolyticus is grown in tissue culture medium (DMEM), while transcription of these genes (except exsD) is minimal upon growth in Luria-Bertani-Salt (LB-S). Transcription of T3SS1 genes and cytotoxicity towards HeLa cells was prevented by deletion of exsA while complementation with exsA restored these traits. Over-expression of ExsA in the wild-type strain, NY-4, activated the transcription of T3SS1 genes when bacteria were grown in LB-S. Deletion of the exsD permitted the transcription of T3SS1 genes when bacteria were grown in the LB-S medium and complementation with the wild-type exsD gene blocked transcription of T3SS1 genes. Over-expression of ExsD in NY-4 prevented the transcription of T3SS1 gene when bacteria were grown in DMEM. Purified ExsA protein binds a novel motif in the upstream region of vp1668 and vp1687 indicating that ExsA interacts directly with the promoter sequences of T3SS1 genes. ExsA positively regulates the expression and secretion of Vp1656 while ExsD negatively regulates the expression and secretion of Vp1656.
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Details
- Title
- Regulation of T3SS1 genes in Vibrio parahaemolyticus and its involvement in pathogenesis
- Creators
- Xiaohui Zhou
- Contributors
- Douglas Ruben Call (Advisor) - Washington State University, Paul G. Allen School for Global Animal HealthMichael Edward Konkel (Committee Member) - Washington State University, School of Molecular BiosciencesThomas Eugene Besser (Committee Member) - Washington State University, Department of Veterinary Microbiology and PathologyTerry F McElwain (Committee Member) - Washington State University, Paul G. Allen School for Global Animal HealthSubramaniam Srikumaran (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- College of Veterinary Medicine
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 114
- Identifiers
- 99901055123101842
- Language
- English
- Resource Type
- Dissertation