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

Abstract

Toxin B (TcdB) produced by Clostridioides difficile 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 (TcdBVPI) and the endemic strain R20291 (TcdBR20) which does not interact with frizzled-1,2,7. TcdBVPI devoid of CSPG4 binding (TcdBVPI 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 TcdBVPI variant (TcdBVPI F1597S) that in accordance with TcdBR20 lacks binding to frizzled-1,2,7 and showed identical potency as TcdBR20 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.

Invisible steps for a global endemy: molecular strategies adopted by Clostridioides difficile

“…The binding of TcdB to CSPG4 receptor induces cell rounding and apoptosis in HeLa and HT29 cells at picomolar concentrations of TcdB, while binding of TcdB to CSPG4 receptor mediate necrotic effects at higher concentration of TcdB. 70 – 72 The binding of TcdB to PVRL3 receptor induces necrosis cell death at high concentrations (the nanomolar range) of TcdB. 70 , 72 , 73 The FZD functions as an alternative receptor to CSPG4; indeed the binding of TcdB to FZD receptor induces cytopathic effects and apoptosis at picomolar concentrations of TcdB.…”

“… 70 – 72 The binding of TcdB to PVRL3 receptor induces necrosis cell death at high concentrations (the nanomolar range) of TcdB. 70 , 72 , 73 The FZD functions as an alternative receptor to CSPG4; indeed the binding of TcdB to FZD receptor induces cytopathic effects and apoptosis at picomolar concentrations of TcdB. 70 , 72 , 74 Another important peculiarity of TcdB is that TcdB can bind to the membrane receptor with amino acid sequences that extend beyond the CROP sequences.…”

“… 70 , 72 , 74 Another important peculiarity of TcdB is that TcdB can bind to the membrane receptor with amino acid sequences that extend beyond the CROP sequences. 70 , 72 …”

“… 62 , 70 , 72 – 74 Then, TcdB may utilize multiple receptors with different binding sites to broaden the selection of mammalian cells it can target. 62 , 70 , 72 – 74 Moreover, TcdB variants are highly diverse for their receptor preference, with relevant implications on the colonic pathology. 45 , 53 , 56 , 62 , 72 …”

Plakoglobin and HMGB1 mediate intestinal epithelial cell apoptosis induced by Clostridioides difficile TcdB

“…Thus, it is believed that TcdB could use multiple receptors at the same time or have specific receptors for different cell types. Recent studies also found TcdB variants derived from different strains show varied binding affinities to the above receptors (Chung et al, 2018;Henkel et al, 2020;Lopez-Urena et al, 2019;Mileto et al, 2020). Surprisingly, TcdB from hypervirulent 027 strain has low affinity to FZDs and PVRL3, which are expressed in epithelial cells, leading to the hypothesis that this TcdB variant may use other unknown receptors for cell entry.…”