Institute of Toxicology, Hannover Medical School, Hannover, Germany.
Division of Molecular Cell Biology, Zoological Institute, Braunschweig, Germany.
Cell Microbiol. 2018 Oct;20(10):e12865. doi: 10.1111/cmi.12865. Epub 2018 Jun 28.
Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species-mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC-5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC-5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.
产毒 A 和产毒 B（TcdA/TcdB）是艰难梭菌产生的大型葡糖基转移酶。当高浓度应用时，TcdB 而不是 TcdA 会诱导活性氧介导的早期细胞死亡（ECD）。我们发现，非糖基化 Rac1 是诱导 ECD 的必要条件，因为 Rac1 的抑制会阻碍这种效应。只有当 TcdB 被快速内吞时，ECD 才会发生。这是通过使用来自高毒力株的 TcdB 主干生成嵌合体来证明的。已经描述了来自高毒力株的 TcdB 从内体在更高的 pH 值下易位，因此比参考型 TcdB 更快。相应地，通过来自高毒力株的 TcdB 主干将参考型 TcdB 的葡糖基转移酶结构域递送至细胞内增加了 ECD。此外，质子转运体，如钠/质子交换器（NHE）或 ClC-5 阴离子/质子交换器，两者都有助于内体酸化，也影响 TcdB 的细胞毒性效力：NHE 的特异性抑制降低了细胞毒性，而用内体阴离子/质子交换器 ClC-5 转染细胞增加了 TcdB 的细胞毒性。我们的数据表明，TcdB 进入细胞质的摄取率和非糖基化 Rac1 的状态是决定是否触发 ECD 或延迟细胞凋亡的关键决定因素。
