MECHANISM OF ACTION OF PORE FORMING TOXINS

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Bacteria of genus Vibrio are normal habitant in natural environmental water. Because of universal consumption of sea foods in Japan, gastroenteric infections due to pathogenic vibrios break out frequently, therefore it is an important problem to clarify the mechanism of infection of the vibrios. Many pathogenic vibrios produce hemolysin as an important pathogenic factor. It is expected that elucidation of action mechanism of the hemolysins leads to development of methods for prevention and therapy of the vibrio infections. We selected two vibrio hemolysins, Vibrio vulnificus hemolysin (VVH) and V.mimicus hemolysin (VMH), and carried out the comparative study of action mechanism in vitro and in vivo. The project started in 1994. Differences of properties of the hemolysins produced by different strains and a role of VMH as a diarrhenogenic factor were demonstrated in 1994 and 1995. In 1996, protein-chemical and immuno-chemical studies of VMH,especially structure and function, were carried out. VMH was purified with ethylene glycol as the solvent. Monoclonal antibodies (MAb) against VMH were prepared but only IgM MAbs which had binding activity to VMH but not neutralizing activity were obtained. Furthermore, non-immunized IgMs also showed weak binding activity to VMH which was suspected to bind to sialic acid on the IgM molecule. The IgM antibodies, however, showed neutralizing activity to VMH purified by a modified method without ethylene glycol, suggesting the conformational change of recognition site for sialic acid of the membrane surface by ethylene glycol, although it is not so distinguish detectable by physico-chemical methods such as fluorescence analysis. Informations on mechanism of binding of VMH molecule to target cells obtained in this project are useful for understanding of the mechanism of gastroenteritis due to the vibrio.