Physiological role and complex regulation of O₂-reducing enzymes in the obligate anaerobe Clostridioides difficile

Clostridioides difficile, the major cause of antibiotic-associated diarrhea, is a strict anaerobic, sporulating Firmicutes. However, during its infectious cycle, this anaerobe is exposed to low oxygen (O₂) tensions, with a longitudinal decreasing gradient along the gastrointestinal tract and a second lateral gradient with higher O₂ tensions in the vicinity of the cells. A plethora of enzymes involved in oxidative stress detoxication has been identified in C. difficile, including four O₂-reducing enzymes: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). Here, we investigated the role of the four O₂-reducing enzymes in the tolerance to increasing physiological O₂ tensions and air. The four enzymes have different, yet overlapping, spectra of activity. revRbr2 is specific to low O₂ tensions (<0.4%), FdpA to low and intermediate O₂ tensions (0.4%–1%), revRbr1 has a wider spectrum of activity (0.1%–4%), and finally FdpF is more specific to tensions > 4% and air. These different O₂ ranges of action partly arise from differences in regulation of expression of the genes encoding those enzymes. Indeed, we showed that revrbr2 is under the dual control of σ^A and σ^B. We also identified a regulator of the Spx family that plays a role in the induction of fdp and revrbr genes upon O₂ exposure. Finally, fdpF is regulated by Rex, a regulator sensing the NADH/NAD⁺ ratio. Our results demonstrate that the multiplicity of O₂-reducing enzymes of C. difficile is associated with different roles depending on the environmental conditions, stemming from a complex multi-leveled network of regulation. IMPORTANCE The gastrointestinal tract is a hypoxic environment, with the existence of two gradients of O₂ along the gut, one longitudinal anteroposterior decreasing gradient and one proximodistal increasing from the lumen to the epithelial cells. O₂ is a major source of stress for an obligate anaerobe such as the enteropathogen C. difficile. This bacterium possesses a plethora of enzymes capable of scavenging O₂ and reducing it to H₂O. In this work, we identified the role of the four O₂-reducing enzymes in the tolerance to the physiological O₂ tensions faced by C. difficile during its infectious cycle. These four enzymes have different spectra of action and protect the vegetative cells over a large range of O₂ tensions. These differences are associated with a distinct regulation of each gene encoding those enzymes. The complex network of regulation is crucial for C. difficile to adapt to the various O₂ tensions encountered during infection.