Toxin producing Clostridioides difficile strains cause gastrointestinal infections with the large glucosylating protein toxins A (TcdA) and B (TcdB) being major virulence factors responsible for the onset of symptoms. TcdA and TcdB enter their target cells via receptor-mediated endocytosis. Inside the cell, the toxins glucosylate and thereby inactivate small GTPases of the Rho-/Ras subfamilies resulting in actin reorganization and cell death. The receptors of TcdA are still elusive, glycoprotein 96 (gp96), the low density lipoprotein receptor family (LDLR) and sulfated glycosaminoglycans (sGAGs) have most recently been suggested as receptors for TcdA. In this study, we provide evidence on rapid endocytosis of Low density lipoprotein Receptor-related Protein-1 (LRP1) into fibroblasts and Caco-2 cells by exploiting biotinylation of cell surface proteins. In contrast, gp96 was not endocytosed either in the presence or absence of TcdA. The kinetics of internalization of TfR and LRP1 were comparable in the presence and the absence of TcdA, excluding that TcdA facilitates its internalization by triggering internalization of its receptors. Exploiting fibroblasts with a genetic deletion of LRP1, TcdA was about one order of magnitude less potent in LRP1-deficient cells as compared to the corresponding control cells. In contrast, TcdB exhibited a comparable potency in LRP1-proficient and -deficient fibroblasts. These findings suggested a role of LRP1 in the cellular uptake of TcdA but not of TcdB. Correspondingly, binding of TcdA to the cell surface of LRP1-deficient fibroblasts was reduced as compared with LRP1-proficient fibroblasts. Finally, TcdA bound to LRP1 ligand binding type repeat cluster II (amino acid 786–1,165) and cluster IV (amino acid 3332-3779). In conclusion, LRP1 appears to serve as an endocytic receptor and gp96 as a non-endocytic receptor for TcdA.

产生毒素 艰难梭菌菌株引起胃肠道感染，其中大的糖基化蛋白毒素A（TcdA）和B（TcdB）是导致症状发作的主要毒力因子。TcdA和TcdB进入目标细胞通过受体介导的内吞作用。在细胞内部，毒素会糖化葡萄糖，从而使Rho- / Ras亚家族的小GTP酶失活，从而导致肌动蛋白重组和细胞死亡。TcdA的受体仍然难以捉摸，糖蛋白96（gp96），低密度脂蛋白受体家族（LDLR）和硫酸化的糖胺聚糖（sGAGs）最近被建议用作TcdA的受体。在这项研究中，我们通过利用细胞表面蛋白的生物素化技术，为低密度脂蛋白受体相关蛋白1（LRP1）快速内吞到成纤维细胞和Caco-2细胞中提供了证据。相反，在存在或不存在TcdA的情况下，gp96均未被内吞。在存在和不存在TcdA的情况下，TfR和LRP1内在化的动力学相当，排除TcdA通过触发其受体的内在化促进其内在化。利用具有LRP1基因缺失的成纤维细胞，与相应的对照细胞相比，TcdA在LRP1缺陷细胞中的效价低约一个数量级。相比之下，TcdB在LRP1熟练和缺乏的成纤维细胞中显示出可比的效力。这些发现提示LRP1在TcdA而不是TcdB的细胞摄取中的作用。相应地，与缺乏LRP1的成纤维细胞相比，TcdA与缺乏LRP1的成纤维细胞的细胞表面结合减少。最后，与LRP1配体结合类型结合的TcdA重复簇II（氨基酸786-1,165）和簇IV（氨基酸3332-3779）。总之，LRP1似乎充当TcdA的内吞受体，而gp96充当非内吞受体。