Abstract
Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101–1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin–receptor interaction.
Highlights
The two large glucosyltransferases TcdA and TcdB from Clostridioides difficile (C. difficile) are the main pathogenicity factors leading to the clinical symptoms associated with C. difficile infections (CDI) (Just and Gerhard, 2004; Voth and Ballard, 2005)
We used HEp-2 cells, since these cells show transcriptome for all known TcdB receptors and are well described for early cell death induced by TcdB (Beer et al, 2018)
We wanted to learn about the role of the prominent cysteine 2232 which is conserved in TcdA and TcdB, but is exchanged for tyrosine in TcdB from hypervirulent strains
Summary
The two large glucosyltransferases TcdA and TcdB from Clostridioides difficile (C. difficile) are the main pathogenicity factors leading to the clinical symptoms associated with C. difficile infections (CDI) (Just and Gerhard, 2004; Voth and Ballard, 2005). These toxins glucosylate cytosolic Rho GTPases which are master regulators of the actin cytoskeleton. Since the intestinal epithelium is the first line of target for TcdA and TcdB, these toxins induce loss of barrier function.
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