Cholera Toxin Production and Localization in Vesicles of Vibrio cholerae El Tor Genovariants

Abstract

The aim was to assess the level of toxin production in Vibrio cholerae El Tor genovariants and to determine the localization of cholera toxin in vesicles.Materials and methods. The work is performed on typical strains and genovariants of V. cholerae El Tor, which were grown in AKI liquid nutrient medium and the one prepared according to J. Hyan recipe, providing for high toxin production under aeration conditions. The decontaminated supernatants of the studied strains served as a source for extraction of toxin preparations and membrane vesicles. The localization of cholera toxin inside or on the outer surface of vesicles was determined through polyacrylamide gel electrophoresis (PAGE), immunoblotting, GM1-ELISA, indirect uncompetitive ELISA, cell culture models CHO-K1, HuTu 80.Results and discussion. Vesicle preparations containing cholera toxin have been isolated from the supernatants of genovariants and typical V. cholerae El Tor with a high level of toxin production. After separation of the vesicles using PAGE, followed by immunoblot with a specific antitoxic serum, it has been found that cholera toxin retains the complete structure, including both subunits. Unlike CT secreted into the culture medium, vesicle-associated one does not bind to both the GM1 receptor of gangliosides sensitized on plates and on the surface of cell cultures, which indicates its absence on the outer surface of vesicles. The location of CT in the cavity of vesicles is also evidenced by their positive reaction with specific antitoxic antibodies after degradation of EDTA. The absence of the toxin on the outer surface of vesicles in typical strains and strains of V. cholerae El Tor genovariants excludes its binding with the GM1 receptor and suggests the possibility of their penetration into target cells via GM-independent pathways. The choice of the pathways by which the vesicle-associated toxin is transferred to host cells is probably determined by the location of the toxin, i.e. it is associated with the internal structures of vesicles or placement on their surface.