Naturally occurring CodY variants alter ligand binding, DNA target affinity, and virulence in Clostridioides difficile

Clostridioides difficile is an important nosocomial pathogen that has been classified as an “urgent threat” by the CDC. Antibiotic use is the primary risk factor for the development of C. difficile -associated disease because it disrupts healthy protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. Nutrient availability and other environmental factors greatly influence toxin production in C. difficile . CodY is a global transcriptional regulator that coordinates metabolism and virulence in Gram-positive pathogens in response to nutrient availability, primarily through sensing branched-chain amino acids (isoleucine, leucine, valine; ILV) and GTP. In C. difficile , CodY indirectly represses toxin production by inhibiting transcription of the positive regulator tcdR and influences sporulation through unknown mechanisms. Here, we characterized two naturally occurring CodY variants, CodY(Y146N) and CodY(V58A). The Y146N substitution is located near the GTP-binding pocket, while V58A is near the ILV-binding site. Ligand binding assays revealed GTP binding ability of CodY(Y146N) is severely compromised. Electrophoretic mobility shift assays (EMSAs) demonstrated that ligand binding differentially influenced promoter binding; in the presence of GTP, CodY(Y146N) bound to the tcdR promoter less efficiently than CodY(WT). In C. difficile , production of either variant resulted in reduced repression of toxin production compared to CodY(WT). Subsequent in vivo experiments in a hamster infection model showed that strains producing CodY(Y146N) or CodY(V58A) were significantly more virulent than the CodY(WT) producing strain. These findings demonstrate that a single amino acid change in this global regulator can alter its ligand affinity and promoter-binding properties to potentially rewire the gene regulatory networks to enhance the pathogenic potential in C. difficile .