Schöttelndreier Dennis, Seeger Katrin, Grassl Guntram A, Winny Markus R, Lindner Robert, Genth Harald
Institute for Toxicology, Hannover Medical School, Hanover, Germany.
Institute of Medical Microbiology and Hospital Epidemiology and DZIF Partner Site Hannover-Braunschweig, Hannover Medical School, Hanover, Germany.
Front Microbiol. 2018 Jul 4;9:1483. doi: 10.3389/fmicb.2018.01483. eCollection 2018.
Toxin-producing strains of and cause infections of the gastrointestinal tract in humans and ruminants, with the toxins being major virulence factors, essential for the infection, and responsible for the onset of severe symptoms. toxin A (TcdA) and toxin B (TcdB), and the large cytotoxin (TpeL) from are single chain bacterial protein toxins with an AB-like toxin structure. The C-terminal delivery domain mediates cell entry of the N-terminal glycosyltransferase domain by receptor-mediated endocytosis. Several cell surface proteins have been proposed to serve as toxin receptors, including chondroitin-sulfate proteoglycan 4 (CSPG4), poliovirus receptor-like 3 (PVRL3), and frizzled-1/2/7 (FZD1/2/7) for TcdB and LDL-receptor-related protein-1 (LRP1) for TpeL. The expression of the TcdB receptors was investigated in human intestinal organoids (HIOs) and in cultured cell lines. HIOs from four human donors exhibited a comparable profile of receptor expression, with PVRL3, LRP1, and FZD7 being expressed and CSPG4 and FZD2 not being expressed. In human epithelial Caco-2 cells and HT29 cells as well as in immortalized murine fibroblasts, either receptor FZD2/7, CSPG4, PVRL3, and LRP1 was expressed. The question whether the toxins take advantage of the normal turnover of their receptors (i.e., constitutive endocytosis and recycling) from the cell surface or whether the toxins activity induce the internalization of their receptors has not yet been addressed. For the analysis of receptor internalization, temperature-induced uptake of biotinylated toxin receptors into immortalized mouse embryonic fibroblasts (MEFs) and Caco-2 cells was exploited. Solely LRP1 exhibited constitutive endocytosis from the plasma membrane to the endosome, which might be abused by TpeL (and possibly TcdB as well) for cell entry. Furthermore, internalization of CSPG4, PVRL3, FZD2, and FZD7 was observed neither in MEFs nor in Caco-2 cells. FZD2/7, CSPG4, and PVRL3 did thus exhibit no constitutive recycling. The presence of TcdB and the p38 activation induced by anisomycin were not able to induce or enhance CSPG4 or PVRL3 uptake in MEFs. In conclusion, FZD2/7, CSPG4, and PVRL3 seem to serve as cell surface binding receptors rather than internalizing receptors of TcdB.
[某种细菌名称]和[另一种细菌名称]的产毒素菌株可导致人类和反刍动物的胃肠道感染，毒素是主要的毒力因子，对感染至关重要，并导致严重症状的发作。[某种细菌名称]的毒素A（TcdA）和毒素B（TcdB）以及大细胞毒素（TpeL）是具有AB样毒素结构的单链细菌蛋白毒素。C末端递送结构域通过受体介导的内吞作用介导N末端糖基转移酶结构域进入细胞。已经提出几种细胞表面蛋白作为毒素受体，包括硫酸软骨素蛋白聚糖4（CSPG4）、脊髓灰质炎病毒受体样3（PVRL3）以及TcdB的卷曲蛋白-1/2/7（FZD1/2/7）和TpeL的低密度脂蛋白受体相关蛋白-1（LRP1）。在人肠道类器官（HIOs）和培养的细胞系中研究了TcdB受体的表达。来自四名人类供体的HIOs表现出可比的受体表达谱，其中PVRL3、LRP1和FZD7表达，而CSPG4和FZD2不表达。在人上皮Caco-2细胞和HT29细胞以及永生化小鼠成纤维细胞中，FZD2/7、CSPG4、PVRL3和LRP1这几种受体中的任何一种都有表达。毒素是否利用其受体从细胞表面的正常周转（即组成型内吞作用和再循环），或者毒素活性是否诱导其受体的内化，这个问题尚未得到解决。为了分析受体内化，利用温度诱导生物素化的毒素受体摄取到永生化小鼠胚胎成纤维细胞（MEFs）和Caco-2细胞中。仅LRP1表现出从质膜到内体的组成型内吞作用，这可能被TpeL（也可能还有TcdB）用于进入细胞。此外，在MEFs和Caco-2细胞中均未观察到CSPG4、PVRL3、FZD2和FZD7的内化。因此，FZD2/7、CSPG4和PVRL3似乎作为TcdB的细胞表面结合受体而非内化受体。
