McClane B A, Wnek A P
Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pennsylvania 15261.
Infect Immun. 1990 Sep;58(9):3109-15. doi: 10.1128/iai.58.9.3109-3115.1990.
The cytotoxicity of Clostridium perfringens enterotoxin (CPE) was completely blocked in Vero cells continuously CPE treated at 4 degrees C. [125I]CPE-specific binding to either Vero cells or isolated rabbit intestinal brush border membranes (BBMs) was lower at 4 degrees C than at 24 or 37 degrees C, but reduced enterotoxin binding could not totally explain the loss of cytotoxicity at low temperature. Insertion of enterotoxin into Vero cell membranes or BBMs was temperature independent. However, CPE complex formation (A. P. Wnek and B. A. McClane, Infect. Immun. 57:574-581, 1989) in BBMs and Vero cells was blocked at 4 degrees C. When Vero cells were CPE treated at 4 degrees C, washed to remove unbound toxin, and then shifted to 37 degrees C, complex formation and cytotoxicity were rapidly detected. When CPE binding and complex formation were permitted for 2 min at 37 degrees C, and the Vero cells were then shifted to 4 degrees C, cytotoxicity was detectable at 4 degrees C. These results are consistent with complex formation, rather than complex activity, being the temperature-sensitive step in CPE action which is blocked at 4 degrees C. These studies demonstrate a strong correlation between complex formation and cytotoxicity and are consistent with complex involvement in CPE cytotoxicity. These studies also strongly suggest that CPE insertion precedes both complex formation and induction of small-molecule permeability changes.
在4℃持续用产气荚膜梭菌肠毒素（CPE）处理的Vero细胞中，CPE的细胞毒性被完全阻断。[125I]CPE与Vero细胞或分离的兔肠刷状缘膜（BBM）的特异性结合在4℃时低于24℃或37℃，但肠毒素结合减少并不能完全解释低温下细胞毒性的丧失。肠毒素插入Vero细胞膜或BBM与温度无关。然而，BBM和Vero细胞中CPE复合物的形成（A. P. Wnek和B. A. McClane，《感染与免疫》57:574 - 581，1989）在4℃时被阻断。当Vero细胞在4℃用CPE处理，洗涤以去除未结合的毒素，然后转移到37℃时，迅速检测到复合物的形成和细胞毒性。当在37℃允许CPE结合和复合物形成2分钟，然后将Vero细胞转移到4℃时，在4℃可检测到细胞毒性。这些结果与复合物形成而非复合物活性是CPE作用中对温度敏感的步骤一致，该步骤在4℃被阻断。这些研究表明复合物形成与细胞毒性之间有很强的相关性，并且与复合物参与CPE细胞毒性一致。这些研究还强烈表明，CPE的插入先于复合物形成和小分子通透性变化的诱导。
