The critical role of hcpR in regulating nitrosative stress defense in Clostridioides difficile.

Clostridioides difficile is an anaerobic, toxin-producing pathogen that colonizes the host gastrointestinal tract. Within this hostile environment, the bacterium encounters stressors such as reactive nitrogen species (RNS), which impose nitrosative stress that must be mitigated for survival. This study aimed to elucidate the molecular mechanisms by which C. difficile defends against nitrosative stress. We screened an unordered transposon mutant library of the epidemic strain R20291 using a nitric oxide (NO) donor and identified a nitrosative stress-sensitive mutant with an inactivated hcpR, a transcriptional regulator of the Crp/Fnr family. Transcriptomic and metabolomic analyses were conducted, alongside the assessment of toxin production, a key virulence factor in C. difficile. Our results revealed that hcpR is critical for nitrosative stress adaptation, with the hcpR::Tn mutant displaying eightfold increased sensitivity to NO. The mutant also showed sensitivity to other RNS, but not to reactive oxygen species. RNA-seq analysis showed that hcpR regulates hcp, which is involved in NO detoxification, and frdX, an iron-sulfur binding protein. Knockdown of hcp and frdX individually conferred NO sensitivity similar to the hcpR-inactivated mutant. Kyoto Encyclopedia of Genes and Genomes pathway analysis of hcpR::Tn transcriptome revealed elevated expression of genes associated with butanoate metabolism. Furthermore, the hcpR::Tn strain showed increased TcdA/TcdB toxin levels compared to the wild type. Targeted metabolomics revealed that hcpR inactivation causes metabolic remodeling, shifting toward enhanced amino acid fermentation and increased short-chain fatty acid production, including butyrate. These findings demonstrate that hcpR is essential for nitrosative stress defense and contributes to virulence regulation through metabolic remodeling in C. difficile.IMPORTANCEWithin the host gastrointestinal tract, Clostridioides difficile encounters various toxic compounds, including reactive nitrogen species (RNS), which induce nitrosative stress. To survive in this hostile environment, the bacterium must mount an effective defense against these damaging agents. In this study, we identified the transcriptional regulator hcpR as a key factor in C. difficile ability to withstand nitrosative stress. Mutants lacking an intact hcpR, or the knockdown of its downstream targets hcp and frdX, showed increased sensitivity to RNS, confirming their roles in nitrosative stress adaptation. The hcpR mutant also produced significantly elevated levels of toxins (TcdA/TcdB), highlighting its influence on virulence. In addition, the mutant demonstrated significant metabolic changes, including increased production of short-chain fatty acids, such as butyrate, which is known to enhance toxin production. Together, these findings underscore hcpR as an important nitrosative stress defense regulator linking stress adaptation and virulence modulation through coordinated metabolic and transcriptional responses.