Mechanism of membrane damage induced by Clostridium perfringens α toxin

我们报道了产气荚膜荚膜杆菌α毒素激活兔红细胞内源性磷脂酶C （PLC）和磷脂酶D (PLD)，毒素通过gtp结合蛋白（g蛋白）激活PLC。然而，关于α -毒素处理的兔红细胞PLD的活化，我们知之甚少。近年来，有报道称，在激素和生长因子的作用下，多种哺乳动物细胞中的PLDs通过低分子g蛋白被激活。利用兔网织细胞的m-RNA cDNA，通过PCR扩增构建Rho和Rho- gdi基因，并将其连接到pGEX-5X载体上。这些蛋白的表达和纯化是用携带Rho和Rho- gdi基因的大肠杆菌jm109转化子进行的。Rho- gdi对毒素诱导的溶血和PLD活化有抑制作用，而Rho对其无刺激作用。据报道，从细胞质中分离的Rho是一种无活性形式，而突变体Rho的Gly-14被Val取代为一种活性形式。突变体Rho （G14V）通过位点定向诱变取代残基，刺激了毒素诱导的溶血和PLD激活。由此可见，毒素通过Rho激活PLD在兔红细胞溶血过程中起重要作用。

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.