Functional Analyses Of Epidemic Clostridioides Difficile Toxin B Variants Reveal Their Divergence In Utilizing Receptors And Inducing Pathology

Clostridioides difficile toxin B (TcdB) is a key virulence factor that causes C. difficile associated diseases (CDAD) including diarrhea and pseudomembranous colitis. TcdB can be divided into multiple subtypes/variants based on their sequence variations, of which four (TcdB1-4) are dominant types found in major epidemic isolates. Here, we find that these variants are highly diverse in their receptor preference: TcdB1 uses two known receptors CSPG4 and Frizzled (FZD) proteins, TcdB2 selectively uses CSPG4, TcdB3 prefers to use FZDs, whereas TcdB4 uses neither CSPG4 nor FZDs. By creating chimeric toxins and systematically switching residues between TcdB1 and TcdB3, we determine that regions in the N-terminal cysteine protease domain (CPD) are involved in CSPG4-recognition. We further evaluate the pathological effects induced by TcdB1-4 with a mouse intrarectal installation model. TcdB1 leads to the most severe overall symptoms, followed by TcdB2 and TcdB3. When comparing the TcdB2 and TcdB3, TcdB2 causes stronger oedema while TcdB3 induces severer inflammatory cell infiltration. These findings together demonstrate divergence in the receptor preference and further lead to colonic pathology for predominant TcdB subtypes.Author summaryClostridioides difficile is a major cause of nosocomial and community-associated gastrointestinal infections. The bacterium produces three exotoxins including TcdA, TcdB, and CDT, of which TcdB is known as a key virulence factor causing the diseases. Since C. difficile was first linked to antibiotic-associated infections in 1978, a large number of clinically relevant strains were characterized and many of them were found to harbor some variant forms of TcdB. In this study, we examined four predominant TcdB variants from epidemic C. difficile strains. We found that these variants are highly diverse in preference to the known receptors, CSPG4 and Frizzled proteins. By conducting a systematically designed mutagenesis study, we determined that TcdB interacts with CSPG4 via regions across multiple domains. We also found that TcdB variants could induce distinguishable pathological phenotypes in a mouse model, suggesting C. difficile strains harboring divergent TcdB variants might exhibit different disease progression. Our study provides new insights into the toxicology and pathology of C. difficile toxin variants.