Staphylococcal pore-forming toxins: Mechanism of pore-forming and recognition of the target cells

葡萄球菌LukF和Hlg2是溶血素的水溶性单体，它们聚集在红细胞膜上的低聚孔中。LukF的结构表明，在组装过程中，向帽结构域向后折叠的茎前结构域可能插入到膜中。在这里，我们创建了双半胱氨酸LukF突变体，其中单二硫键连接前茎结构域和帽结构域（V12C-T136C, cap -stem），或两个b链连接前茎结构域（T117C-T136C,Stem-Stem），以研究孔组装中前茎结构域的结构变化。两种突变体在红细胞溶解中都不活跃，但如果二硫键减少，则获得完全的溶血活性。二硫键既不阻碍膜的结合能力，也不阻碍中间预孔的寡聚，但有效地抑制了预孔向功能孔的转变。Cap-Stem突变体形成的sds不稳定预孔和Stem-Stem突变体形成的环形sds稳定预孔分别属于预孔形成的“早期”和“晚期”状态。两种突变体在2℃下的膜结合和巯基乙醇诱导的孔形成表明，只有寡聚化是一个温度依赖的步骤。因此，二硫键形成和低温对不同步骤孔隙组装的抑制证明了LukF在孔隙组装过程中需要在茎前结构域发生结构变化。

Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-hemolysin that assemble into an oligomeric pore on the erythrocyte membrane. The structure of LukF suggests that the pre-stem domain, which folds back toward the cap domain, may insert into the membrane during assembly. Here, we created double-cysteine LukF mutants in which single disulfide bonds connect either the pre-stem domain and the cap domain(V12C-T136C,Cap-Stem), or two b-strands within the pre-stem domain(T117C-T136C,Stem-Stem), to study structural changes of the pre-stem domain in pore assembly. Both mutants are inactive in erythrocyte lysis, however gain full hemolytic activity if the disulfide bonds are reduced. The disulfide bonds block neither the membrane binding ability nor the intermediate pre-pore oligomerization, but efficiently inhibit the transition from pre-pores to functional pores. An SDS-unstable pre-pore formed by the Cap-Stem mutant and the ring-shaped, SDS-stable pre-pore formed by the Stem-Stem mutant are ascribed to "early" and "late" states of pre-pore formation, respectively. The membrane binding and the mercaptoethanol-induced pore formation of both mutants even at 2℃ indicate that only oligomerization is a temperature-dependent step. Thus, the inhibition of pore assembly at various steps by disulfide bond formation and low temperature proves the requirement for structural changes in the pre-stem domain of LukF during pore assembly.