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Role of Vibrio mimicus protease in enterotoxigenicity
M A Chowdhury1, S Miyoshi, S Shinoda
1Department of Environmental Hygiene, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Abstract:
Recently, we have purified and characterised a metalloprotease produced by Vibrio mimicus. The role of V. mimicus protease (VMP) in fluid accumulation (FA) in rabbit ileal loops (RIL) was investigated. Although live cells or crude VMP prepared from cell-free culture supernatant showed activity in RIL, the purified VMP failed to induce FA in the RIL. When VMP in crude preparation was inactivated by preparing the fraction in the presence of EGTA (Ethyleneglycol-bis [beta-aminoethylether]-N, N, N1, N1,-tetraacetic acid), a chelating agent for Ca2+ (EGTA-crude), or treatment with phosphoramidon (a metalloprotease inhibitor), the preparation became negative in RIL assay. Again, the EGTA-crude induced FA in RIL when retreated with 2mM CaCl2 after elimination of EGTA. The results show evidences of attribution of VMP in RIL activity. These observations, therefore, suggest that the protease produced by V. mimicus may play a potential role in enterotoxigenicity.
Insights
Vibrio mimicus protease (VMP) is linked to fluid accumulation (FA) in rabbit ileal loops (RIL). Calcium-dependent activity of VMP suggests a role in enterotoxigenicity.
Area of Science:
- Microbiology
- Gastroenterology
- Biochemistry
Background:
- Vibrio mimicus produces a metalloprotease (VMP).
- The role of VMP in fluid accumulation (FA) is not fully understood.
Purpose of the Study:
- To investigate the role of V. mimicus protease (VMP) in fluid accumulation (FA) using rabbit ileal loops (RIL).
Main Methods:
- Purification and characterization of VMP.
- Assay of VMP activity in RIL.
- Inactivation of VMP using EGTA (a chelating agent for Ca2+) and phosphoramidon (a metalloprotease inhibitor).
- Reactivation of VMP activity with CaCl2.
Main Results:
- Live V. mimicus cells and crude VMP induced FA in RIL.
- Purified VMP did not induce FA.
- Inactivation of crude VMP with EGTA or phosphoramidon abolished FA induction.
- EGTA-inactivated VMP regained FA-inducing activity upon addition of CaCl2.
Conclusions:
- The metalloprotease produced by V. mimicus plays a role in fluid accumulation in rabbit ileal loops.
- Calcium is essential for VMP activity in inducing FA.
- VMP may contribute to the enterotoxigenicity of V. mimicus.