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

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.

Toxin B Variants from Clostridium difficile Strains VPI 10463 and NAP1/027 Share Similar Substrate Profile and Cellular Intoxication Kinetics but Use Different Host Cell Entry Factors

“…The TcdB fragment, referred to by the authors as FZD-binding domain (FBD), covers amino acid residues 1285–1804 and matches nearly exactly the delimitations of the additional receptor-binding domain (residues 1349–1811) discovered previously [26]. Recently, Chung et al have shown that TcdB NAP1 exhibited only weak binding to FZD2 [33]. In line with this observation, the authors showed that a fragment of TcdB VPI covering amino acid residues 1101–1836 (and which largely overlaps with the FBD described above) was capable of competitively inhibiting TcdB VPI but not TcdB NAP1 .…”

Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B

“…There is currently no standard nomination for TcdB variants, miscellaneous names referred to same toxin variants were sometimes assigned in different studies. For example, TcdB variant initially identified from NAP1/BI/027 strains was later called TcdB 027 28,29 , TcdB HV 22,30 , TcdB NAP1 15 , TcdB2 31 , TcdB R20291 32,33 , or TcdB-R20291 34 . In 2005, Rupnik et al 35 proposed nomenclature of C. difficile toxin variants by adding the bacterial strain in which the toxin was originally found.…”

“…The classical TcdB (TcdB1) uses chondroitin sulfate proteoglycan 4 36 , poliovirus receptor-like 3 37 , and frizzled proteins (FZDs) 38 as cellular receptors; and can glucosylate small GTPase such as RhoA/B/C, Rac1, Cdc42, TC10, TCL, RhoG, Rap, and Ras [39][40][41] . Recent studies showed that TcdB2 only weakly bound to FZDs when compared with TcdB1 32,42 , likely owing to discrepancies between FZD-binding sequences of TcdB1 and TcdB2 43,44 . Also, when compared with TcdB1, both TcdB3 and TcdB4 showed a drastically reduced ability to glucosylate RhoA and Cdc42 15,23 , which was also supported by our sequence analyses (Fig.…”

Phylogenomics of 8,839Clostridioides difficilegenomes reveals recombination-driven evolution and diversification of toxin A and B

“…However, sequence variations in the toxin genes exist across C. difficile strains and could affect receptor-binding specificity, preferences toward distinct small GTPases, overall toxicity, and antigenicity. For instance, strains such as R20291 (belonging to RT027) produces a TcdB variant with ~8% of residue differences from the reference TcdB, which exhibited a significant impact on its immunogenicity: mice immunized with the reference TcdB developed resistance to the same TcdB, but all died when challenged with this variant TcdB 34 , and several antibodies raised against the reference TcdB, including the FDA approved therapeutic antibody bezlotoxumab, either do not recognize or have lower efficacy against this TcdB variant [34][35][36] . Furthermore, this TcdB variant also loses the ability to recognize frizzled (FZD) proteins, which are one of the major receptors for the reference TcdB, due to residue changes at the FZD-binding interface 35,[37][38][39][40] .…”