Mechanism of membrane damage induced by Clostridium perfringens α toxin

我们报道了C.产气荚膜杆菌α毒素激活兔红细胞中的内源性磷脂酶C（PLC）和磷脂酶D（PLD），并且毒素通过GTP结合蛋白（G蛋白）激活PLC。然而，很少有人知道PLD的激活与α毒素处理的兔红细胞。最近，有报道称，各种哺乳动物细胞中的PLD通过用激素和生长因子处理的低分子G蛋白被激活。以兔网织红细胞cDNA为模板，经PCR扩增获得Rho和Rho-GDI基因，并将其克隆到pGEX-5X载体中。这些蛋白的表达和纯化使用E. coliJM 109转化子。Rho-GDI抑制毒素诱导的溶血和PLD激活，而Rho对溶血和PLD激活无刺激作用。据报道，从胞质溶胶中分离的Rho是无活性形式，其中Gly-14被瓦尔取代的突变体Rho是活性形式。通过定点突变将Rho（G14 V）替换为残基，刺激毒素诱导的溶血和PLD激活。这些结果表明，PLD激活Rho毒素激活兔红细胞溶血中起重要作用。

We reported that C. perfringens alpha-toxin activates endogenous phospholipase C (PLC) and phospholipase D (PLD) in rabbit erythrocytes, and that the toxin activates PLC through GTP-binding protein (G-protein). However, little is known about the activation of PLD in rabbit erythrocytes treated with alpha-toxin. Recently, it has been reported that PLDs in various mammalian cells are activated through low molecular G-proteins by treatment with hormones and growth factors. Rho and Rho-GDI genes were constructed from cDNA through m-RNA in rabbit reticulocytes by PCR amplification and these genes were ligated into pGEX-5X vector. Expression and purification of these proteins were performed using E. coli JM 109 transformants carrying Rho and Rho-GDI genes. Rho-GDI inhibited the toxin-induced hemolysis and PLD activation, However, Rho did not stimulate them. Rho isolated from cytosol is reported to be a inactive form and the mutant Rho in which is replaced Gly-14 with Val to be a active form. The mutant Rho (G14V) replaced the residue by site-directed mutagenesis stimulated the toxin-induced hemolysis and PLD activation. These observation suggested that PLD activation through Rho activated by the toxin plays a important role in hemolysis of rabbit erythrocytes.