How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O ₂ Tensions

Abstract

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 (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 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 O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 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 O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

Responses of Clostridia to oxygen: from detoxification to adaptive strategies

“…The level of O 2 tolerance varies among clostridial vegetative cells and this tolerance is both strain-and mediumdependent within a species (Kawasaki et al, 1998;Hillmann et al, 2008;Edwards et al, 2016). Tolerance to O 2 with a maintained growth in liquid culture for several hours in the presence of low O 2 tension (0.2%-3%) has been described for C. acetobutylicum (O'Brien and Morris, 1971;Hillmann et al, 2008), Clostridium butyricum (Camerini et al, 2019) and C. difficile (Neumann-Schaal et al, 2018;Giordano et al, 2018b;Kint et al, 2020) even if a phase of latence is observed in C. butyricum (Kawasaki et al, 1998). Woundcolonizing clostridial species such as C. perfringens and C. tetani and Clostridium tertium isolated from gas gangrene wounds are considered to be aerotolerant (Trinh et al, 2000;Bruggemann et al, 2004;Fujitani et al, 2007).…”

“…In C . difficile , the two FDPs and revRbrs play a pivotal role in O 2 tolerance and their NAD(P)H‐dependent O 2 ‐reductase activities seem to be additive in vivo (Kint et al ., 2020). Indeed, while single mutations have no or only a weak effect on O 2 tolerance, a quadruple mutant has a strong growth defect when exposed to 0.1% O 2 and is completely unable to grow at 0.4% O 2 .…”

“…Representation of the flavodiiron proteins and rubrerythrins found in several clostridial genomes. The substrates and products displayed are represented for the respective proteins and are based on those determined by in vitro experiments with the enzymes of C. difficile and C. acetobutylicum(Kawasaki et al, 2009;Riebe et al, 2009;Folgosa et al, 2018a;Folgosa et al, 2018b;Kint et al, 2020) except for those shown for canonical rubrerythrins that are determined only for C. acetobutylicum(Riebe et al, 2009). The diiron catalytic centres of both canonical FDPs and Rbrs are shown in the correspondent panel.…”

Impact of subinhibitory concentrations of metronidazole on proteome of Clostridioides difficile strains with different levels of susceptibility

“…A recent study showed that RevRbrs are O 2 - and H 2 O 2 -reductases, which have a key role to the ability of C . difficile to grow in the presence of low oxygen tension [ 54 ]. On the contrary, for CD17-146, their amount did not change at MIC/4 and decreased by half at MIC/2 ( Table 2 ).…”

A short chain fatty acid–centric view of Clostridioides difficile pathogenesis

“…However, unlike the Enterobacteriaceae, C . difficile is an obligate anaerobe (i.e., it cannot use O 2 as a terminal electron acceptor) and is only capable of growing in the presence of relatively low levels of oxygen (<3% O 2 ), likely due to a suite of enzymes involved in detoxifying reactive oxygen species [ 97 – 99 ]. So, we hypothesize that if inflammation ( C .…”