Menzies B E, Kernodle D S
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Infect Immun. 1994 May;62(5):1843-7. doi: 10.1128/iai.62.5.1843-1847.1994.
Staphylococcus aureus alpha-toxin is a membrane-damaging exoprotein that oligomerizes to form transmembrane pores. Chemical modification of histidines with diethylpyrocarbonate has been shown to reduce the hemolytic activity of alpha-toxin, suggesting that one or more of the histidine residues is important for toxin function. To individually assess the functional importance of each of the four histidine residues (residues 35, 48, 144, and 259), we used oligonucleotide-directed mutagenesis of the cloned alpha-toxin gene to replace each histidine with leucine. The mutant toxins were expressed in S. aureus and evaluated for hemolytic activity in vitro and for lethality in an intraperitoneal murine model. Substitution of histidine 35 with leucine produced a mutant toxin (H35L) without hemolytic or lethal activity. Mutant toxins H48L, H144L, and H259L exhibited 7, 16, and 46%, respectively, of the hemolytic activity of wild-type toxin. Immunoblotting of purified H35L toxin incubated with liposomal membranes demonstrated intact membrane binding and hexamer formation that was clearly detectable but reduced compared with that of the wild-type toxin. This suggests that hexamer formation alone is not sufficient for the expression of alpha-toxin activity. The nature of the defect underlying the lack of activity of the H35L mutant toxin remains to be elucidated but may involve failure of the hexamer to span the lipid bilayer to form a transmembrane pore or a change in the internal surface and permeability characteristics of the pore.
金黄色葡萄球菌α毒素是一种能破坏细胞膜的外蛋白，它会寡聚化形成跨膜孔道。已证明用焦碳酸二乙酯对组氨酸进行化学修饰可降低α毒素的溶血活性，这表明一个或多个组氨酸残基对毒素功能很重要。为了单独评估四个组氨酸残基（第35、48、144和259位残基）各自的功能重要性，我们利用克隆的α毒素基因进行寡核苷酸定向诱变，将每个组氨酸替换为亮氨酸。突变毒素在金黄色葡萄球菌中表达，并在体外评估其溶血活性，在腹腔内小鼠模型中评估其致死性。将第35位组氨酸替换为亮氨酸产生了一种无溶血或致死活性的突变毒素（H35L）。突变毒素H48L、H144L和H259L分别表现出野生型毒素溶血活性的7%、16%和46%。用脂质体膜孵育纯化的H35L毒素后的免疫印迹显示，其膜结合完整且形成六聚体，这清晰可测，但与野生型毒素相比有所减少。这表明仅六聚体形成不足以表达α毒素活性。H35L突变毒素缺乏活性背后的缺陷性质尚待阐明，但可能涉及六聚体无法跨越脂质双层形成跨膜孔道，或者孔道内表面及通透性特征发生改变。
