Receptor Binding Domains of TcdB from for Chondroitin Sulfate Proteoglycan-4 and Frizzled Proteins Are Functionally Independent and Additive.

Toxin B (TcdB) produced by is a main pathogenicity factor that affects a variety of different cell types within the colonic mucosa. TcdB is known to utilize frizzled-1,2,7 and chondroitin sulfate proteoglycan-4 (CSPG4) as protein receptors. By using human cervical cancer cell line HeLa CSPG4 knockout (CSPG4) cells as well as TcdB mutants which do not bind to either CSPG4 or frizzled-1,2,7, or both, we evaluated the impact of the individual receptors for cytopathic and cytotoxic effects of TcdB. We compared TcdB from the reference strain VPI10463 (TcdB) and the endemic strain R20291 (TcdB) which does not interact with frizzled-1,2,7. TcdB devoid of CSPG4 binding (TcdB ΔCROP) shows identical cytopathic potency as full-length TcdB in HeLa CSPG4 cells, indicating that interaction with frizzled proteins is not affected in the presence of the C-terminal CROP domain. We validated CSPG4 as cellular receptor for both TcdB toxinotypes in HeLa and HEp-2 cells. By exchange of a single phenylalanine residue, 1597 with serine, we generated a mutated TcdB variant (TcdB F1597S) that in accordance with TcdB lacks binding to frizzled-1,2,7 and showed identical potency as TcdB on HeLa cells. This enabled us to estimate the respective share of CSPG4 and frizzled-1,2,7 in the cytotoxic and cytopathic effect induced by TcdB. Our data reveal that binding to frizzled-1,2,7 and to CSPG4 occurs independently and in an additive manner.