A superoxide reductase contributes toClostridioides difficileresistance to oxygen

AbstractClostridioides difficilecauses a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, mechanistic details ofC. difficileintestinal colonization remain undefined.C. difficileis highly sensitive to oxygen and requires anaerobic conditions forin vitrogrowth. However, the mammalian gut is not entirely devoid of oxygen, andC. difficiletolerates moderate oxidative stressin vivo. TheC. difficilegenome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protectingC. difficileagainst oxygen exposure. Insertional inactivation ofsorrenderedC. difficilemore sensitive to superoxide indicating that SOR contributes to antioxidant defense. HeterologousC. difficile sorexpression inEscherichia coliconferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, aC. difficileSOR mutant exhibited global proteome changes under oxygen stress when compared to its parent strain. Collectively, our data establish the enzymatic activity ofC. difficileSOR, confirm its role in protection against oxidative stress, and demonstrate its broader impacts on the vegetative cell proteome.ImportanceClotridioides difficileis an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobein vitro, C. difficilehas been shown to tolerate low levels of oxygen in its mammalian host. Among other well-characterized antioxidant proteins, theC. difficilegenome includes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of the enzymatic activity of the putative SOR protein, including confirmation of its role in protection ofC. difficileagainst oxidative stress. This furthers our understanding ofC. difficilepathogenesis and presents a potential new avenue for targeted therapies.