Impacts of perR on oxygen sensitivity, gene expression, and murine infection in Clostridioides difficile 630Δ erm

Clostridioides difficile infection (CDI), characterized by colitis and diarrhea, afflicts approximately half a million people in the United States every year, burdening both individuals and the healthcare system. C. difficile 630Δ erm is an erythromycin-sensitive variant of the clinical isolate C. difficile 630 and is commonly used in the C. difficile research community due to its genetic tractability. 630Δ erm possesses a point mutation in perR , an autoregulated transcriptional repressor that regulates oxidative stress resistance genes. This point mutation results in a constitutively de-repressed PerR operon in 630Δ erm . To address the impacts of perR on phenotypes relevant for oxygen tolerance and relevant to a murine model of CDI, we corrected the point mutant to restore PerR function in 630Δ erm (herein, 630Δ erm perR ^WT ). We demonstrate that there is no difference in growth between 630Δ erm and a 630Δ erm perR ^WT under anaerobic conditions or when exposed to concentrations of O ₂ that mimic those found near the surface of the colonic epithelium. However, 630Δ erm perR ^WT is more sensitive to ambient oxygen than 630Δ erm , which coincides with alterations in expression of a variety of perR -dependent and perR -independent genes. Finally, we show that 630Δ erm and 630Δ erm perR ^WT do not differ in their ability to infect and cause disease in a well-established murine model of CDI. Together, these data support the hypothesis that the perR mutation in 630Δ erm arose as a result of exposure to ambient oxygen and that the perR mutation in 630Δ erm is unlikely to impact CDI-relevant phenotypes in laboratory studies.