Spo0A suppresses sin locus expression in Clostridioides difficile

Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with the production of toxins, and spores are responsible for the transmission and persistence of the organism. Previously we characterized sin locus regulators SinR and SinR prime, where SinR is the regulator of toxin production and sporulation, while the SinR prime acting as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, regulates SinR, by regulating the expression of its antagonist sinI. However, the role of Spo0A in the expression of sinR and sinR prime in C. difficile is not yet reported. In this study, we tested spo0A mutants in three different C. difficile strains R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. The qRT-PCR analysis for its expression further supported this data. By carrying out genetic and biochemical assays, we have shown that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile.