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

Abstract

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.

TheClostridioides difficilespecies problem: global phylogenomic analysis uncovers three ancient, toxigenic, genomospecies

“…We have previously demonstrated that common laboratory diagnostic assays may be challenged by changes in the PaLoc of C-I strains 21 . The same might be true for monoclonal antibody-based treatments for CDI such as bezlotoxumab, known to have distinct neutralizing activities against different TcdB subtypes 57 .…”

Functional analyses of epidemic Clostridioides difficile toxin B variants reveal their divergence in utilizing receptors and inducing pathology

“…TcdB1-4 are major variants that together were found in over 99.6% of the pathogenic C . difficile strains [ 15 ]. To determine the functional contribution of CSPG4 and FZD1/2/7 in mediating the toxin entry of these TcdB variants into host cells, we compared the activities of TcdB variants using the standard cytopathic cell-rounding assay on the HeLa-Cas9 (parental cell line, referred to as the wildtype (WT) HeLa thereafter) cells versus CSPG4 knockout ( CSPG4 –/– ) and FZD1/2/7 knockout ( FZD1/2/7 –/– ) HeLa cells, which were generated previously via CRISPR/Cas9 approach [ 30 ].…”

“…The high diversity of receptor preference may potentially benefit the pathogen on its host adaption. Although the driven force remains unknown, TcdB appears to be rapidly evolving [ 15 ] which may provide flexibility to attack a wide range of host cells and tissues. On the other hand, natural toxin variants provide optimal tools to study their contributions to disease progressions.…”

“…Recombinant full-length TcdB proteins were expressed in Bacillus subtilis SL401 and purified as previously described [ 15 ]. In brief, expression was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at 25°C for 16 hours.…”

“…Although the prototypical TcdB (TcdB1) is a major form expressed in the clinical C . difficile isolates such as VPI10463 and 630, natural variants of TcdB are widespread and can be grouped into 8 subtypes/variants [ 15 ]. Notably, some variants are expressed in strains particularly important to the epidemiology of CDI.…”