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、皮肤坏死和致死作用。它被认为与膜结合，组装成形成孔的六角体，最终导致细胞的胶体渗透溶解。我们以前的研究表明，葡萄球菌α-毒素特异性地结合到胆碱型磷脂上，并且膜流动性的增加促进了毒素在靶膜上的六聚化。在本项目中，我们制备了α-毒素片段，并分析了它们的结合和成孔能力。结果表明：(1)Alal-Glu71、Gly72-Asn293、Thr9-Lys131和Ile132-Asn293等片段保持了与磷脂酰胆碱-胆固醇脂质体和红细胞膜的结合能力；(2)这些片段虽然部分组装成二聚体，但不形成膜孔；(3)较大尺寸的片段(Thr9-Asn293)虽然保持了结合和寡聚能力，但对脂质体和红细胞几乎没有成孔活性。这些数据表明，α-毒素的N-末端和C-末端都是其成孔能力所必需的。因此，我们的数据与先前关于不同的α-毒素结构域负责不同的功能的建议是不一致的(例如，据报道毒素的C-末端18-kDa片段是溶血的)。我们的结果可能表明，当α-毒素单体分子组装成六聚体时，它们以头对头的方式连接在一起。

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.