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Purification and characterization of Kanagawa haemolysin from Vibrio parahaemolyticus
J P Douet1, M Castroviejo, A Dodin
1Université de Bordeaux I, Talence, France.
Abstract:
The haemolysin of a Kanagawa-phenomenon-positive Vibrio parahaemolyticus strain was purified to apparent homogeneity by acid precipitation, DEAE-Trisacryl, hydroxyapatite and FPLC (Mono-Q) columns: 1.4 micrograms of protein gave a single band on conventional SDS-PAGE with silver staining. The haemolysin was not inactivated by heating for 10 min at 100 degrees C. It was a monomeric protein with a molecular weight estimated to be 29 kDa by PAGE under denaturing and non-denaturing conditions. The haemolysin caused fluid accumulation in the ligated mouse ileum, was cytolytic against cultured mammalian cells and also lysed erythrocytes of various animal species (equine erythrocytes being the most resistant).
Insights
The purified Kanagawa-phenomenon-positive Vibrio parahaemolyticus haemolysin is a heat-stable 29 kDa protein. This potent toxin causes fluid accumulation in mice and cell lysis in vitro.
Area of Science:
- Microbiology
- Protein Biochemistry
- Toxicology
Background:
- Vibrio parahaemolyticus is a significant foodborne pathogen.
- The Kanagawa phenomenon is associated with virulence in V. parahaemolyticus strains.
- Haemolysins are key virulence factors in many bacterial pathogens.
Purpose of the Study:
- To purify and characterize the haemolysin from a Kanagawa-phenomenon-positive V. parahaemolyticus strain.
- To investigate the biological activities of the purified haemolysin.
Main Methods:
- Haemolysin purification using sequential chromatography (DEAE-Trisacryl, hydroxyapatite, FPLC Mono-Q).
- Protein homogeneity assessed by SDS-PAGE and silver staining.
- Molecular weight determination by PAGE.
- Heat stability assay.
- In vivo fluid accumulation assay in ligated mouse ileum.
- In vitro cytolytic assay against mammalian cells.
- Erythrocyte lysis assay.
Main Results:
- The haemolysin was purified to apparent homogeneity.
- The purified haemolysin is a heat-stable monomeric protein with an estimated molecular weight of 29 kDa.
- The haemolysin induced fluid accumulation in mouse ileum, exhibited cytolytic activity against mammalian cells, and lysed erythrocytes from various animal species.
Conclusions:
- The study successfully isolated and characterized a heat-stable haemolysin from V. parahaemolyticus.
- The purified haemolysin possesses significant cytotoxic and enterotoxic activities, contributing to the pathogenicity of the bacterium.