Temporally segregated subpopulations of CDT and TcdA producer cells of .

Most strains of produce two toxins, TcdA and TcdB, which are mainly responsible for the disease symptoms. TcdA and TcdB are coded for by genes in the pathogenicity locus (PaLoc). Some epidemic strains, however, such as R20291, of ribotype 027, additionally produce a binary toxin, CDT, coded for by genes in the CDT locus (CdtLoc). We analyzed the relationship between transcription of the TcdA and CDT encoding genes in strain R20291 using single-cell analysis with orthogonal fluorescence reporters. We found that most of the cells express whereas a much smaller fraction expresses and most of those also express . Expression of begins during exponential growth and persists during the stationary phase of growth, while the main period of transcription occurs as cells enter the stationary phase. This translates into early synthesis and release of CDT from the cells, whereas production of TcdA is detected mainly from the onset of the stationary phase. Both the PaLoc and CdtLoc also code for regulatory proteins, TcdR, a sigma factor, and CdtR, a response regulator of the LytR family, required for expression of the toxin-encoding genes, respectively. While CdtR contributes to the expression of , as shown before, we found reduced expression of in the absence of TcdR. Hence, TcdR and CdtR cross-regulate the PaLoc and the CdtLoc. Finally, and unlike the case for TcdA, we found no evidence for the association of CDT with mature spores.IMPORTANCEThe enteropathogen causes a spectrum of intestinal diseases, ranging from mild diarrhea to severe conditions such as intestinal inflammation, perforation, and sepsis, that may lead to death, primarily through the production of two cytotoxins, TcdA and TcdB. Certain strains, however, such as those of ribotypes 027 and 078, additionally produce a binary toxin, CDT. Here, we employed single-cell analysis to investigate toxin gene expression in epidemic strain R20297 (RT027), commonly associated with severe infections. We found that CDT is synthesized early during growth, while TcdA is produced at the onset of the stationary phase, and that the two populations partially overlap. We also identify cross-regulation between two key regulatory proteins, TcdR and CdtR, which control TcdA/TcdB and CDT production. These insights into the mechanisms of toxin production at the population level may contribute to the development of targeted therapies for managing infections and the resulting complications.