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 630Δerm 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. 630Δerm 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 630Δerm. 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 630Δerm and a 630Δerm 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 C. difficile 630Δerm and 630Δerm 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).