Assembly of Staphy lococcus aureus alpha-toxin on target membranes

金黄色葡萄球菌α -毒素是一种33 kda的多肽，具有溶血、皮肤坏死和致死活性。它被认为与膜结合，组装成形成孔的六聚体，最终导致细胞的胶体渗透裂解。我们之前的研究表明，葡萄球菌α -毒素与胆碱型磷脂特异性结合，并且膜流动性的增加促进了毒素在靶膜上的六聚体化。在这个项目中，我们制备了α -毒素的片段，并测定了它们的结合和成孔能力。结果表明：(1)一些片段，如Alal-Glu71、Gly72-Asn293、Thr9-Lys131和Ile132-Asn293保留了与磷脂酰胆碱-胆固醇脂质体和红细胞膜结合的能力；(2)这些片段虽然在膜上部分组装成二聚体，但不形成膜孔；(3)较大的片段（Thr9-Asn293）对脂质体和红细胞几乎没有成孔活性。虽然它保留了结合和寡聚的能力。这些数据表明α -毒素的n端和c端对其成孔能力都是不可或缺的。因此，我们的数据与之前提出的α -毒素的不同结构域负责不同功能的建议不一致（例如，据报道毒素的c -末端18kda片段具有溶血作用）。我们的结果可能表明，当α -毒素的单体分子组装成六聚体时，它们以头对头的方式连接。

Staphylococcus aureus alpha-toxin is a 33-kDa polypeptide endowed with hemolytic, dermonecrotic and lethal activities. It is thought to bind to membranes, assemble into pore-forming hexamers, eventually causing colloid osmotic lysis of cells. Our previous studies showed that staphylococcal alpha-toxin binds specifically to the choline-type phospholipids, and that the increase in membrane fluidity promotes hexamerization of the toxin on target membranes. In this project, we prepared fragments of alpha-toxin and assayd their binding and pore-forming abilities. The results indicated (1) that several fragments, such as Alal-Glu71, Gly72-Asn293, Thr9-Lys131 and Ile132-Asn293 retained ability to bind to phosphatidylcholine-cholesterol liposomes and erythrocyte membranes, (2) that these fragments did not form membrane pores, although they assembled partially into dimers on the membranes, and (3) that a larger-sized fragment (Thr9-Asn293) showed little pore-forming activity towards liposomes and erythrocytes, although it retained the binding and oligomerizing abilities. These data suggest that both of the N-terminal and the C-terminal portions of alpha-toxin are indispensable for its pore-forming ability. Thus, our data are not consistent with the previous proposal that different domains of alpha-toxin are responsible for defferent functions (e.g., C-Terminal 18-kDa fragment of the toxin was reported to be hemolytic). Our results may suggest that monomer molecules of alpha-toxin are linked in a head-to-head manner when they assemble into hexamers.