The critical role of <i>hcpR</i> in regulating nitrosative stress defense in <i>Clostridioides difficile</i>

<i>Clostridioides difficile</i> 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 <i>C. difficile</i> 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 <i>hcpR</i>, 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 <i>C. difficile</i>. Our results revealed that <i>hcpR</i> is critical for nitrosative stress adaptation, with the <i>hcpR</i>::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 <i>hcpR</i> regulates <i>hcp</i>, which is involved in NO detoxification, and <i>frdX</i>, an iron-sulfur binding protein. Knockdown of <i>hcp</i> and <i>frdX</i> individually conferred NO sensitivity similar to the <i>hcpR</i>-inactivated mutant. Kyoto Encyclopedia of Genes and Genomes pathway analysis of <i>hcpR</i>::Tn transcriptome revealed elevated expression of genes associated with butanoate metabolism. Furthermore, the <i>hcpR</i>::Tn strain showed increased TcdA/TcdB toxin levels compared to the wild type. Targeted metabolomics revealed that <i>hcpR</i> inactivation causes metabolic remodeling, shifting toward enhanced amino acid fermentation and increased short-chain fatty acid production, including butyrate. These findings demonstrate that <i>hcpR</i> is essential for nitrosative stress defense and contributes to virulence regulation through metabolic remodeling in <i>C. difficile</i>.IMPORTANCEWithin the host gastrointestinal tract, <i>Clostridioides difficile</i> 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 <i>hcpR</i> as a key factor in <i>C. difficile</i> ability to withstand nitrosative stress. Mutants lacking an intact <i>hcpR</i>, or the knockdown of its downstream targets <i>hcp</i> and <i>frdX</i>, showed increased sensitivity to RNS, confirming their roles in nitrosative stress adaptation. The <i>hcpR</i> 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 <i>hcpR</i> as an important nitrosative stress defense regulator linking stress adaptation and virulence modulation through coordinated metabolic and transcriptional responses.