Xtr-mediated regulation of 4Fe-4S binding protein gene confers resistance to oxidative and antibiotic stress in Clostridioides difficile.

BACKGROUND

Xenobiotic response element (XRE) family transcriptional regulators play crucial roles in diverse bacterial physiological processes and environmental adaptation. However, the functional characterization of these regulators in Clostridioides difficile remains unclear.

METHODS

In this study, we employed the Allele-coupled exchange (ACE) gene knockout technique to construct knockout (Δxtr) and complemented (::xtr) mutants of the xtr gene, which encodes a member of the XRE family protein in C. difficile, and systematically analyzed the resulting phenotypic alterations. Subsequently, transcriptome sequencing, EMSA, and reporter gene assays were utilized to investigate how xtr regulates 4Fe-4S binding protein gene (4Fe-4S_BP, CD17960).

RESULTS

The Δxtr mutant exhibited a statistically significant reduction in hydrogen peroxide tolerance, as well as decreased susceptibility to vancomycin and metronidazole. Concomitantly, the expression of virulence-related genes and cellular toxicity, as well as growth rate and autolysis, were all notably diminished. Transcriptomic analysis revealed a significant downregulation of 4Fe-4S_BP in the Δxtr mutant (3.69-fold). Overexpression of the 4Fe-4S_BP in the Δxtr background restored hydrogen peroxide tolerance to wild-type levels. Electrophoretic Mobility Shift Assays (EMSA) confirmed that the recombinant Xtr protein directly binds to the promoter region of 4Fe-4S_BP. Reporter gene assays utilizing gusA demonstrated that Xtr activates transcription from the 4Fe-4S_BP promoters.

CONCLUSION

These findings elucidate a molecular mechanism by which Xtr positively regulates the expression of 4Fe-4S_BP, thereby modulating C. difficile tolerance to hydrogen peroxide, metronidazole, and vancomycin. This work provides novel experimental evidence for a deeper understanding of the environmental adaptation strategies and virulence regulatory networks employed by C. difficile.