MECHANISM OF ACTION OF PORE FORMING TOXINS

弧菌属细菌是自然环境水体中的常住菌。由于日本人普遍食用海产品，由致病性弧菌引起的胃肠道感染时有发生，因此弄清弧菌的感染机制是一个重要的问题。许多致病性弧菌产生溶血素是一种重要的致病因子。对溶血素作用机制的阐明，可望为弧菌感染的防治提供新的途径。我们选取了两种溶血弧菌——创伤弧菌溶血素（vibrio vulnificus hemolyysis， VVH）和拟弧菌溶血素（vumicus hemolyysis， VMH），对其体内外作用机制进行了对比研究。该项目始于1994年。不同菌株产生的溶血素性质的差异以及VMH作为致泻因子的作用在1994年和1995年被证实。1996年，开展了VMH的蛋白质化学和免疫化学研究，特别是结构和功能研究。以乙二醇为溶剂提纯VMH。制备了抗VMH的单克隆抗体（MAb），但只获得了与VMH有结合活性但无中和活性的IgM单克隆抗体。此外，未免疫的IgM对VMH的结合活性也较弱，怀疑VMH与IgM分子上的唾液酸结合。然而，IgM抗体对不含乙二醇的改进方法纯化的VMH表现出中和活性，这表明乙二醇对膜表面唾液酸识别位点的构象发生了变化，尽管用荧光分析等物理化学方法不能如此区分。本课题所获得的VMH分子与靶细胞结合的机制信息，对了解弧菌所致肠胃炎的机制有重要意义。

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.