Chassin C, Bens M, de Barry J, Courjaret R, Bossu J L, Cluzeaud F, Ben Mkaddem S, Gibert M, Poulain B, Popoff M R, Vandewalle A
Institut National de la Santé et de la Recherche Médicale U773, Centre de Recherche Biomédicale Bichat-Beaujon CRB3, Paris, France.
Am J Physiol Renal Physiol. 2007 Sep;293(3):F927-37. doi: 10.1152/ajprenal.00199.2007. Epub 2007 Jun 13.
Clostridium perfringens epsilon toxin (ET) is a potent pore-forming cytotoxin causing fatal enterotoxemia in livestock. ET accumulates in brain and kidney, particularly in the renal distal-collecting ducts. ET binds and oligomerizes in detergent-resistant membranes (DRMs) microdomains and causes cell death. However, the causal linkage between membrane permeabilization and cell death is not clear. Here, we show that ET binds and forms 220-kDa insoluble complexes in plasma membrane DRMs of renal mpkCCD(cl4) collecting duct cells. Phosphatidylinositol-specific phospholipase C did not impair binding or the formation of ET complexes, suggesting that the receptor for ET is not GPI anchored. ET induced a dose-dependent fall in the transepithelial resistance and potential in confluent cells grown on filters, transiently stimulated Na+ absorption, and induced an inward ionic current and a sustained rise in [Ca2+]i. ET also induced rapid depletion of cellular ATP, and stimulated the AMP-activated protein kinase, a metabolic-sensing Ser/Thr kinase. ET also induced mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of apoptosis-inducing factor, a potent caspase-independent cell death effector. Finally, ET induced cell necrosis characterized by a marked reduction in nucleus size without DNA fragmentation. DRM disruption by methyl-beta-cyclodextrin impaired ET oligomerization, and significantly reduced the influx of Na+ and [Ca2+]i, but did not impair ATP depletion and cell death caused by the toxin. These findings indicate that ET causes rapid necrosis of renal collecting duct cells and establish that ATP depletion-mediated cell death is not strictly correlated with the plasma membrane permeabilization and ion diffusion caused by the toxin.
产气荚膜梭菌ε毒素（ET）是一种强效的成孔细胞毒素，可导致家畜致命性肠毒血症。ET在脑和肾中蓄积，尤其是在肾远曲集合管中。ET在耐去污剂膜（DRM）微结构域中结合并寡聚化，导致细胞死亡。然而，膜通透性与细胞死亡之间的因果联系尚不清楚。在此，我们表明ET在肾mpkCCD（cl4）集合管细胞的质膜DRM中结合并形成220 kDa的不溶性复合物。磷脂酰肌醇特异性磷脂酶C不影响ET复合物的结合或形成，这表明ET的受体不是糖基磷脂酰肌醇锚定的。ET导致在滤膜上生长的汇合细胞的跨上皮电阻和电位呈剂量依赖性下降，短暂刺激Na+吸收，并诱导内向离子电流和[Ca2+]i持续升高。ET还导致细胞内ATP迅速消耗，并激活AMP活化蛋白激酶，一种代谢感应丝氨酸/苏氨酸激酶。ET还诱导线粒体膜通透性增加和凋亡诱导因子的线粒体-核转位，凋亡诱导因子是一种强效的不依赖半胱天冬酶的细胞死亡效应器。最后，ET诱导细胞坏死，其特征是细胞核大小显著减小且无DNA片段化。甲基-β-环糊精破坏DRM会损害ET寡聚化，并显著减少Na+和[Ca2+]i的内流，但不影响毒素引起的ATP消耗和细胞死亡。这些发现表明ET导致肾集合管细胞快速坏死，并证实ATP消耗介导的细胞死亡与毒素引起的质膜通透性和离子扩散并不严格相关。
