MECHANISM OF ACTION OF PORE FORMING TOXINS

弧菌属细菌是天然环境水体中的正常栖息地。在日本，由于海产品的普遍食用，致病性弧菌引起的胃肠道感染频繁爆发，因此弄清弧菌的感染机制是一个重要的问题。许多致病弧菌产生溶血素作为重要致病因子。通过对溶血素作用机制的阐明，有望为弧菌感染的防治提供新的思路和方法。我们选择了创伤弧菌溶血素(VVH)和拟态弧菌溶血素(VMH)两种弧菌溶血素，并进行了体内外作用机制的比较研究。该项目始于1994年。1994年和1995年，不同菌株产生的溶血素的性质差异以及VMH作为腹泻因子的作用被证明。1996年，人们对VMH的结构和功能进行了蛋白质化学和免疫化学研究。用乙二醇作溶剂提纯VMH。制备了抗VMH的单抗，但仅获得了与VMH有结合活性但不具有中和活性的IgM单抗。此外，未免疫的IgM还表现出与VMH的弱结合活性，VMH可能与IgM分子上的唾液酸结合。然而，IgM抗体对改良的不含乙二醇法纯化的VMH显示出中和活性，这表明乙二醇膜表面唾液酸的识别位点发生了构象变化，尽管用荧光分析等物理化学方法检测不到这种变化。本项目中获得的VMH分子与靶细胞结合机制的信息有助于理解弧菌引起的胃肠炎的机制。

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.