Action on membrane microdomain and structural study of Clostridium perfringens beta-toxin.

产气荚膜梭菌β毒素是坏死性肠炎的重要致病因子。在测试的10个细胞系中，只有HL-60细胞系对β毒素敏感。毒素引起细胞肿胀和溶解。用毒素处理细胞后，钾离子外流，钙、钠、氯离子内流。就在细胞被毒素溶解之前，这些事件达到了最大值。毒素与细胞孵育形成约191 kDa和228 kDa的毒素复合体，定位于满足脂筏标准的结构域。孵育30min后观察到228 kDa的复合体，60min后仅保留191 kDa的复合体。用甲基-β-环糊精或胆固醇氧化酶处理细胞，可阻止毒素与木筏的结合，并阻止毒素引起的K外流和肿胀。当聚乙二醇水动力直径从200增加到400时，毒素诱导的钙内流和形态改变被抑制，而聚乙二醇600和1000显著或完全抑制毒素诱导的钙内流和形态变化。然而，这些聚乙二醇组分对毒素诱导的钾外流没有影响。该毒素可诱导含有羧基荧光素的磷脂酰胆碱-胆固醇脂质体释放羧基荧光素，并以剂量依赖的方式形成228 kDa的低聚体，但不与191 kDa的复合体形成低聚体。结论：该毒素通过与脂筏结合形成228 kDa的功能性寡聚体作用于HL-60细胞。

Clostridium perfringens beta toxin is an important agent of necrotic enteritis. Of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin. The toxin induced swelling and lysis of the cell. Treatment of the cells with the toxin resulted in K+ efflux from the cells and Ca2+, Na+, and Cl-influxes. These events reached a maximum just before the cells were lysed by the toxin. Incubation of the cells with the toxin showed the formation of toxin complexes of about 191 and 228 kDa, which were localized in the domains that fulfilled the criteria of lipid rafts. The complex of 228 kDa was observed until 30 min after incubation, and only the complex of 191 kDa was remained after 60 min. Treatment of the cells with methyl-beta-cyclodextrin or cholesterol oxidase blocked binding of the toxin to the rafts and the toxin-induced K+ efflux and swelling. The toxin-induced Ca2+ influx and morphological changes were inhibited by an increase in the hydrodynamic diameter of polyethylene glycols from 200 to 400 and markedly or completely inhibited by polyethylene glycol 600 and 1000. However, these polyethylene glycols had no effect on the toxin-induced K+ efflux. The toxin induced carboxy-fluorescein release from phosphatidyl-choline-cholesterol liposomes containing carboxyfluorescein and formed an oligomer with 228 kDa in a dose-dependent manner but did not form an oligomer with the 191-kDa complex. We conclude that the toxin acts on HL 60 cells by binding to lipid rafts and forming a functional oligomer with 228 kDa.