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 i… Show more
“…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 .…”
Clostridium difficile induces antibiotic-associated diarrhea due to the release of toxin A (TcdA) and toxin B (TcdB), the latter being its main virulence factor. The epidemic strain NAP1/027 has an increased virulence attributed to different factors. We compared cellular intoxication by TcdBNAP1 with that by the reference strain VPI 10463 (TcdBVPI). In a mouse ligated intestinal loop model, TcdBNAP1 induced higher neutrophil recruitment, cytokine release, and epithelial damage than TcdBVPI. Both toxins modified the same panel of small GTPases and exhibited similar in vitro autoprocessing kinetics. On the basis of sequence variations in the frizzled-binding domain (FBD), we reasoned that TcdBVPI and TcdBNAP1 might have different receptor specificities. To test this possibility, we used a TcdB from a NAP1 variant strain (TcdBNAP1v) unable to glucosylate RhoA but with the same receptor-binding domains as TcdBNAP1. Cells were preincubated with TcdBNAP1v to block cellular receptors, prior to intoxication with either TcdBVPI or TcdBNAP1. Preincubation with TcdBNAP1v blocked RhoA glucosylation by TcdBNAP1 but not by TcdBVPI, indicating that the toxins use different host factors for cell entry. This crucial difference might explain the increased biological activity of TcdBNAP1 in the intestine, representing a contributing factor for the increased virulence of the NAP1/027 strain.
“…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 .…”
Clostridium difficile induces antibiotic-associated diarrhea due to the release of toxin A (TcdA) and toxin B (TcdB), the latter being its main virulence factor. The epidemic strain NAP1/027 has an increased virulence attributed to different factors. We compared cellular intoxication by TcdBNAP1 with that by the reference strain VPI 10463 (TcdBVPI). In a mouse ligated intestinal loop model, TcdBNAP1 induced higher neutrophil recruitment, cytokine release, and epithelial damage than TcdBVPI. Both toxins modified the same panel of small GTPases and exhibited similar in vitro autoprocessing kinetics. On the basis of sequence variations in the frizzled-binding domain (FBD), we reasoned that TcdBVPI and TcdBNAP1 might have different receptor specificities. To test this possibility, we used a TcdB from a NAP1 variant strain (TcdBNAP1v) unable to glucosylate RhoA but with the same receptor-binding domains as TcdBNAP1. Cells were preincubated with TcdBNAP1v to block cellular receptors, prior to intoxication with either TcdBVPI or TcdBNAP1. Preincubation with TcdBNAP1v blocked RhoA glucosylation by TcdBNAP1 but not by TcdBVPI, indicating that the toxins use different host factors for cell entry. This crucial difference might explain the increased biological activity of TcdBNAP1 in the intestine, representing a contributing factor for the increased virulence of the NAP1/027 strain.
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Clostridioides difficile toxins (TcdA and TcdB) are major exotoxins responsible for C. difficile infection (CDI) associated diseases. The previously reported TcdB variants showed distinct biological features, immunoactivities, and potential pathogenicity in disease progression. Here, we performed global comparisons of amino acid sequences of both TcdA and TcdB from 3,269 C. difficile genomes and clustered them according to the evolutionary relatedness. We found that TcdB was much diverse and could be divided into eight subtypes, of which four were first described. Further analysis indicates that the tcdB gene undergoes accelerated evolution to maximize diversity. By tracing TcdB subtypes back to their original isolates, we found that the distribution of TcdB subtypes was not completely aligned with the phylogeny of C. difficile. These findings suggest that the tcdB genes not only frequently mutate, but also continuously transfer and exchange among C. difficile strains.
“…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] .…”
AbstractClostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB) is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.
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