Impacts of perR on oxygen sensitivity, gene expression, and murine infection in Clostridioides difficile 630?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 630?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. 630?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 630?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 630?erm (herein, 630?erm perRWT). We demonstrate that there is no difference in growth between 630?erm and a 630?erm perRWT under anaerobic conditions or when exposed to concentrations of O2 that mimic those found near the surface of the colonic epithelium. However, 630?erm perRWT is more sensitive to ambient oxygen than 630?erm, which coincides with alterations in expression of a variety of perR-dependent and perR-independent genes. Finally, we show that 630?erm and 630?erm perRWT 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 630?erm arose as a result of exposure to ambient oxygen and that the perR mutation in 630?erm is unlikely to impact CDI-relevant phenotypes in laboratory studies Overall design: C. difficile 630?erm and 630?erm perRWT overnight cultures were back-diluted 1:100 into 35 mL of pre-reduced mRCM in Erlenmeyer flasks and incubated anaerobically at 37°C until cultures reached mid-log phase (OD600=0.3-0.4). At mid-log phase, 5 mL aliquots of the cultures were diluted 1:1 in chilled 1:1 ethanol:acetone and stored at -20° to preserve RNA for 0 minute timepoints. The remaining cultures were aerobically shaken (220 rpms) at 37°C for 60 minutes. After 60 minutes, 5 mL aliquots were diluted 1:1 in chilled 1:1 ethanol:acetone and stored at -20° to preserve RNA for the 60 minute timepoints. RNA was extracted by centrifuging samples at 3,000 xg for 5 minutes at 4°C. Pellets were washed with 5 mL cold, nuclease free PBS, and centrifuged at 3,000 xg for 5 minutes at 4°C. The supernatant was removed, and remaining pellets were resuspended in 1 mL TRIzol and processed using a TRIzol Plus RNA Purification Kit (Thermo) with on-column DNase treatment. Purified RNA integrity was confirmed via 2100 Agilent BioAnalyzer and frozen at -80°C. RNA-seq was performed by Microbial Genome Sequencing Center (MiGS) on high-quality rRNA-depleted RNA extracts (12 M paired end reads per sample).