Spo0A Suppresses Locus Expression in 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 toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized locus regulators SinR and SinR' (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In , Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, However, the role of Spo0A in the expression of and in had not yet been reported. In this study, we tested mutants in three different strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in locus expression. Western blot analysis revealed that mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In , the locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.