Receptor-mediated uptake of Clostridioides difficile Toxins A and B

大葡糖基转移酶TcdA和TcdB在结肠感染期间由致病性艰难梭菌释放。这两种毒素都是导致C.艰难梭菌相关性腹泻和伪膜性结肠炎。毒素是约300 kDa的大蛋白质，其修饰其靶细胞内的信号蛋白。由于几乎任何细胞类型对这两种毒素都敏感，因此在发病过程中肠上皮、肠神经系统和肠免疫细胞都会受到影响。每种毒素中的两个单独的受体结合结构域使毒素能够通过不同的结合结构影响广谱的靶细胞。TcdB的细胞表面受体是硫酸软骨素蛋白聚糖4（CSPG 4）和Wnt受体Frizzled 1、2和7。对于TcdA，寡糖（Gal-1，3-）Gal-1，4-GlcNAc和最近的硫酸化糖胺聚糖（主要是硫酸软骨素和硫酸乙酰肝素）被鉴定为结合结构。初步结果表明，TcdB中的两个受体结合结构域独立地起作用，彼此不受影响，从而显示出相加的功能。由于TcdA显示不同的行为相比，TcdB构象依赖性的过程，如自蛋白水解裂解或细胞表面结合，在受体结合结构域的层次结构可以假定为TcdA。TcdA和TcdB的位点特异性和缺失突变体将评估特异性受体结合结构域在结合和摄取到细胞中的影响。受体敲除细胞将用于补充实验。由于TcdB受体蛋白CSPG 4也可能通过其硫酸软骨素基团潜在地充当TcdA的结合结构，因此还将研究TcdA和TcdB在受体水平上的相互作用。通过应用TcdA或TcdB的酶缺陷突变体来促进或抑制同时应用的相反活性毒素，显示了两种毒素的协同或竞争效应。该项目旨在鉴定TcdA中的特异性糖胺聚糖结合位点，并更深入地了解这两种毒素的构象依赖性结合和转运过程。此外，将评估TcdA和TcdB中的神经痛位点是否有效中和毒素。

The large glucosyltransferases TcdA and TcdB are released by pathogenic Clostridioides difficile during infection of the colon. Both toxins are responsible for the clinical symptoms of C. difficile-associated diarrhoea and pseudomembranous colitis. The toxins are about 300 kDa large proteins that modify signaling proteins within their target cells. Since almost any cell type is sensitive towards both toxins, the intestinal epithelium, enteric nervous system and intestinal immune cells are affected during pathogenesis. Two separate receptor binding domains in each toxin enable the toxins to affect a broad spectrum of target cells via different binding structures. The cell surface receptors for TcdB are chondroitin sulfate proteoglycan 4 (CSPG4) and the Wnt receptors Frizzled 1,2 and 7. For TcdA the oligosaccharides (Gal-1,3-)Gal-1,4-GlcNAc and very recently the sulfated glycosamine glycans, mainly chondroitin sulfate and heparan sulfate, were identified as binding structures.The appearance of two separate receptor binding domains in each toxin raises the question about interference of receptor binding resulting in competition or synergistic effects. Preliminary results indicate that the two receptor binding domains in TcdB act independently and unaffected by each other, thereby showing additive function. Since TcdA shows different behavior in comparison with TcdB regarding conformation-dependent processes such as autoproteolytic cleavage or cell surface binding, a hierarchy in the receptor binding domains can be postulated for TcdA. Site specific and deletion mutants of TcdA and TcdB will evaluate the impact of specific receptor binding domains in binding and uptake into cells. Receptor knock out cells will be used in complementary experiments. Since the TcdB receptor protein CSPG4 might potentially serve as binding structure for TcdA via its chondroitin sulfate group as well, the interaction of TcdA and TcdB on receptor level will also be investigated. Synergistic or competition effects of both toxins are shown by the application of enzyme deficient mutants of TcdA or TcdB to facilitate or inhibit simultaneously applied opposite active toxins. By this, the project aims at identification of the specific glycosamine glycan binding site in TcdA and at a deeper understanding of the conformation-dependent binding and translocation process of both toxins into cells. Moreover, neuralgic sites in TcdA and TcdB will be evaluated for efficient neutralization of toxins.