Mechanism of membrane damage induced by Clostridium perfringens α toxin

产气荚膜梭菌α毒素诱导细胞膜损伤的机制

• 批准号: 09670305
• 负责人: SAKURAI Jun
• 金额: $ 2.3万
• 依托单位: Tokushima Bunri University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670305/
• 关键词: Clostridium perfringens; Alpha-toxin; Phospholipase C; phospholipase D; Hemolysis; Site-directed-mutagenesis; Rho; Rho-GDI; 低分子量Gタンパク質; ガス壊疸; ガス破疽; 細菌内情報伝達系; ARF; Rho-GDI

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.

我们报道了产气荚膜弧菌α-毒素激活兔红细胞内源性磷脂酶C(PLC)和磷脂酶D(PLD)，并通过GTP结合蛋白(G-蛋白)激活PLC。然而，对经α-毒素处理的兔红细胞中PLD的激活知之甚少。最近有报道称，在激素和生长因子作用下，多种哺乳动物细胞中的PLD通过低分子G蛋白被激活。通过聚合酶链式反应从兔网织红细胞m-RNA中获得Rho和Rho-GDI基因，并将其连接到pGEX-5X载体中。用携带Rho和Rho-GDI基因的JM109转化子对这些蛋白进行了表达和纯化。Rho-GDI可抑制毒素诱导的溶血和PLD激活，但Rho对其无刺激作用。据报道，从胞浆中分离的Rho是一种非活性形式，而用Val取代Gly-14的突变体Rho是一种活性形式。突变体Rho(G14V)通过定点突变取代残基，刺激毒素诱导的溶血和PLD激活。提示该毒素通过Rho激活PLD在兔红细胞溶血中起重要作用。

项目成果

J.Sakurai,M.Nagahama and S.Dchi: "Major toxins of Clostridium perfringens"J.Toxicol.-Toxin Rev.. 16. 195-214 (1997)

J.Sakurai、M.Nagahama 和 S.Dchi：“产气荚膜梭菌的主要毒素”J.Toxicol.-Toxin Rev.. 16. 195-214 (1997)

櫻井 純: "細菌性食中毒講座(6)Gram陽性菌桿菌4.Clostridium perfringens" J.Antibact. Antifung. Agents. 25. 651-657 (1997)

Jun Sakurai：“细菌性食物中毒课程 (6) 革兰氏阳性杆菌 4.产气荚膜梭菌”J.Antibact。 25. 651-657 (1997)

M. Nagahama et al.: "Characterization of the enzymatic component of Clostridiums perfringens iota-toxin"J. Bacteriol.. 182 (In press). (2000)

M. Nagahama 等人：“产气荚膜梭菌 iota 毒素酶成分的表征”J。

M.Nagahama,Y.Sakaguchi,K.Kobayashi,S.Ochi and J.Sakurai: "Characterization of the enzymatic component of clostridium perfringens iota-toxin"J.Bacteriol.. 182(In press). (2000)

M.Nagahama、Y.Sakaguchi、K.Kobayashi、S.Ochi 和 J.Sakurai：“产气荚膜梭菌 iota 毒素酶成分的表征”J.Bacteriol.. 182（印刷中）。

M. Nagahama et al.: "Assembly of Clostridium perfringens opsilon-toxin on MDCK cell membrane"J. Nat. Toxin. 7. 291-302 (1998)

M. Nagahama 等人：“产气荚膜梭菌 opsilon 毒​​素在 MDCK 细胞膜上的组装”J。