Nagahama Masahiro, Takehara Masaya, Takagishi Teruhisa, Seike Soshi, Miyamoto Kazuaki, Kobayashi Keiko
Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan
Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan.
Infect Immun. 2017 Mar 23;85(4). doi: 10.1128/IAI.00966-16. Print 2017 Apr.
C2 toxin consists of an enzyme component (C2I) and a binding component (C2II). Activated C2II (C2IIa) binds to a cell receptor, giving rise to lipid raft-dependent oligomerization, and it then assembles with C2I. The whole toxin complex is then endocytosed into the cytosol, resulting in the destruction of the actin cytoskeleton and cell rounding. Here, we showed that C2 toxin requires acid sphingomyelinase (ASMase) activity during internalization. In this study, inhibitors of ASMase and lysosomal exocytosis blocked C2 toxin-induced cell rounding. C2IIa induced Ca influx from the extracellular medium to cells. C2 toxin-induced cell rounding was enhanced in the presence of Ca ASMase was released extracellularly when cells were incubated with C2IIa in the presence of Ca Small interfering RNA (siRNA) knockdown of ASMase reduced C2 toxin-induced cell rounding. ASMase hydrolyzes sphingomyelin to ceramide on the outer leaflet of the membrane at acidic pH. Ceramide was detected in cytoplasmic vesicles containing C2IIa. These results indicated that ASMase activity is necessary for the efficient internalization of C2 toxin into cells. Inhibitors of ASMase may confer protection against infection.
C2毒素由一种酶成分（C2I）和一种结合成分（C2II）组成。活化的C2II（C2IIa）与细胞受体结合，引发脂筏依赖性寡聚化，然后与C2I组装。整个毒素复合物随后被内吞到细胞质中，导致肌动蛋白细胞骨架破坏和细胞变圆。在此，我们表明C2毒素在内化过程中需要酸性鞘磷脂酶（ASMase）活性。在本研究中，ASMase抑制剂和溶酶体胞吐作用阻断了C2毒素诱导的细胞变圆。C2IIa诱导Ca从细胞外介质流入细胞。在有Ca的情况下，C2毒素诱导的细胞变圆增强。当细胞在有Ca的情况下与C2IIa一起孵育时，ASMase被释放到细胞外。ASMase的小干扰RNA（siRNA）敲低减少了C2毒素诱导的细胞变圆。ASMase在酸性pH值下将膜外小叶上 的鞘磷脂水解为神经酰胺。在含有C2IIa的细胞质小泡中检测到神经酰胺。这些结果表明，ASMase活性是C2毒素有效内化进入细胞所必需的。ASMase抑制剂可能提供抗感染保护。
