Institute of Infectious Diseases, Medical Faculty, University of Bern, Bern, Switzerland.
PLoS One. 2013 May 29;8(5):e64644. doi: 10.1371/journal.pone.0064644. Print 2013.
Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").
产气荚膜梭菌β 毒素（CPB）是一种β-桶状孔形成毒素，也是 C. perfringens 型 C 菌株的一种重要毒力因子，该菌株可导致动物和人类致命性出血性肠炎。我们之前已经表明 CPB 与受影响猪和人类肠道内的内皮细胞结合，并且 CPB 对体外原代猪内皮细胞（pEC）具有高度毒性。本研究的目的是研究 CPB 在这些细胞中诱导的细胞死亡类型，并研究该过程中涉及的潜在宿主细胞机制。CPB 可迅速诱导乳酸脱氢酶（LDH）释放、碘化丙啶摄取、ATP 耗竭、钾离子外流、细胞内钙离子 [Ca(2+)]i 显著升高、高迁移率族蛋白 B1（HMGB1）释放，并导致坏死细胞死亡的典型超微结构变化。尽管 caspase-3 有一定程度的激活，但未检测到明显的 DNA 片段化。CPB 诱导的 LDH 释放和碘化丙啶摄取被 necrostatin-1 和两种不同的钙蛋白酶抑制剂 PD150606 和 calpeptin 抑制。同样，抑制钾离子外流、螯合细胞内钙并用环孢菌素 A 处理 pEC 也显著抑制 CPB 诱导的 LDH 释放。我们的结果表明，rCPB 主要在 pEC 中诱导坏死性细胞死亡，并且坏死性细胞死亡不仅仅是由毒素诱导的膜破坏引起的被动事件，而是通过由细胞内钙升高激活的宿主细胞依赖性生化途径传播的，可被 necrostatin-1 抑制，与新兴的程序性坏死（“坏死性细胞凋亡”）概念一致。
