AUTHOR=Chung Soo-Young , Schöttelndreier Dennis , Tatge Helma , Fühner Viola , Hust Michael , Beer Lara-Antonia , Gerhard Ralf 

TITLE=The Conserved Cys-2232 in Clostridioides difficile Toxin B Modulates Receptor Binding

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.02314

DOI=10.3389/fmicb.2018.02314

ISSN=1664-302X

ABSTRACT=<p><italic>Clostridioides difficile</italic> toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in <italic>C. difficile</italic>-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 TcdB<sub>V PI10463</sub> 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, TcdB<sub>R20291</sub> 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 TcdB<sub>R20291</sub> to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101–1836) of TcdB<sub>V PI10463</sub> and TcdB<sub>R20291</sub>, 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.</p>