| Summary: | Toxin B (TcdB) produced by <i>Clostridioides difficile</i> 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<sup>−/−</sup>) 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<sub>VPI</sub>) and the endemic strain R20291 (TcdB<sub>R20</sub>) which does not interact with frizzled-1,2,7. TcdB<sub>VPI</sub> devoid of CSPG4 binding (TcdB<sub>VPI</sub> ΔCROP) shows identical cytopathic potency as full-length TcdB in HeLa CSPG4<sup>−/−</sup> 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<sub>VPI</sub> variant (TcdB<sub>VPI</sub> F1597S) that in accordance with TcdB<sub>R20</sub> lacks binding to frizzled-1,2,7 and showed identical potency as TcdB<sub>R20</sub> 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.
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