School of Bioengineering, East China University of Science and Technology, Shanghai, China.
PLoS One. 2013;8(3):e58634. doi: 10.1371/journal.pone.0058634. Epub 2013 Mar 6.
Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 - 1852, designated as ?97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP6), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.
艰难梭菌毒素 B（TcdB）通过葡萄糖基化 Rho GTPases 使靶细胞中毒。TcdB（269 kDa）至少由 4 个功能域组成，包括葡萄糖基转移酶结构域（GTD）、半胱氨酸蛋白酶结构域（CPD）、转运结构域（TD）和受体结合结构域（RBD）。TD 是 TcdB 中最大的片段，占蛋白质的近 50%，其功能和分子作用模式在很大程度上仍然未知。在这里，我们表明 TD 羧基末端的 97 个氨基酸片段（AA1756-1852，命名为？97 或 D97）与 RBD 相邻，对于 TcdB 的细胞活性是必需的。TcdB 中该片段的缺失（命名为 TxB-D97）并没有改变毒素的酶活性或其细胞结合和摄取能力。TxB-D97 以类似于 TcdB 全毒素的方式结合并进入细胞。野生型和突变型毒素在肌醇六磷酸（InsP6）存在下以相似的方式释放其 GTD，并在无细胞葡萄糖基化测定中表现出相似的葡萄糖基转移酶活性。尽管存在这些相似之处，但与野生型毒素相比，TxB-D97 的细胞毒性活性降低了 5 个对数级，这支持了 TxB-D97 无法使靶细胞中的 Rac1 葡萄糖基化的事实。此外，突变型毒素未能在巨噬细胞中引发肿瘤坏死因子-α（TNF-α），这一过程依赖于毒素的葡萄糖基转移酶活性。暴露于毒素的细胞的细胞分级显示，TxB-D97 无法有效地将 GTD 释放到细胞质中。因此，我们得出结论，TcdB TD 羧基末端的 97 个氨基酸区域紧邻 RBD，对于 TcdB 的毒性是必需的。
