Receptor-mediated uptake of Clostridioides difficile Toxins A and B

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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.