Development of Clostridium difficile R20291ΔPaLoc model strains and in vitro methodologies reveals CdtR is required for the production of CDT to cytotoxic levels

•Model strains to study the C. difficile binary toxin, CDT, were generated by deleting the PaLoc in R20291.•The gene encoding CdtR was deleted and re-integrated on the chromosome in the model strains.•In vitro Cytotoxicity assays were established to assess CDT-mediated virulence.•CdtR was required for the production of CDT to cytotoxic levels towards Vero cells.

Assessing the regulation of Clostridium difficile transferase (CDT), is complicated by the presence of a Pathogenicity locus (PaLoc) which encodes Toxins A and B. Here we developed R20291ΔPaLoc model strains and cell-based assays to quantify CDT-mediated virulence. Their application demonstrated that the transcriptional regulator, CdtR, was required for CDT-mediated cytotoxicity.