How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O(2) Tensions

Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O(2)) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O(2)) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O(2) detoxification and/or protection. Tolerance of C. difficile to low O(2) tensions requires the presence of the alternative sigma factor, σ(B), involved in the general stress response. Among the genes positively controlled by σ(B), four encode proteins likely involved in O(2) detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O(2)- and H(2)O(2)-reductase activities in vitro, while purified FdpA mainly acts as an O(2)-reductase. The growth of a fdpA mutant is affected at 0.4% O(2), while inactivation of both revRbrs leads to a growth defect above 0.1% O(2). O(2)-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O(2) tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O(2) tensions such as those encountered in the colon.