Naturally Occurring CodY Variants Alter Ligand Binding, DNA Target Affinity, and Virulence in Clostridioides difficile.

Clostridioides difficile is an important nosocomial pathogen and is the major cause of antibiotic-associated diarrhea and colitis. CodY is a global transcriptional regulator that coordinates metabolism and virulence in Gram-positive pathogens by sensing branched-chain amino acids and GTP. In C. difficile, CodY represses toxin production by inhibiting transcription of tcdR and by influencing c-di-GMP turnover. Here, we characterized two naturally occurring CodY variants, CodY(Y146N) and CodY(V58A), whose substitutions lie near the GTP- and ILV-binding sites, respectively. GTP-binding by CodY(Y146N) was severely compromised, while leucine binding was enhanced; CodY(V58A) showed reduced leucine binding. Both variants exhibited reduced ligand-dependent binding to the tcdR promoter and failed to repress toxin production as effectively as CodY(WT). Expression of virulence-associated genes (tcdR, pdcB) was elevated in strains producing either variant. In a hamster infection model, both variant-producing strains were significantly more virulent than the CodY(WT) strain. These findings demonstrate that single amino acid substitutions in this global regulator can alter ligand affinity and promoter binding, potentially rewiring gene regulatory networks to enhance the pathogenic potential of C. difficile.