A rubrerythrin locus of Clostridioides difficile efficiently detoxifies reactive oxygen species

As an intestinal human pathogen, Clostridioides difficile is the main cause of antibiotic-associated diarrhoea. Endospores of this gram-positive bacterium enter the intestinal tract via faecal-oral transmission, germinate into vegetative and toxin-producing cells and can trigger a Clostridioides difficile infection. The microaerophilic conditions (0.1 to 0.4 % O₂) of the large intestine represent a challenge for the strictly anaerobic organism, which protects itself by a variety of oxidative stress proteins. Four of these are encoded in an operon that is assumed to be involved in the detoxification of H₂O₂ and O₂^●-. This operon encodes a rubrerythrin (rbr), its own transcriptional repressor PerR (perR), a desulfoferrodoxin (rbo) and a putative glutamate dehydrogenase (CD630_08280) with an N-terminal rubredoxin domain, which is only expressed under high oxidative stress conditions. In this study, the enzyme activity of Rbr, Rbo and CD630_08280 was tested in-vitro. Recombinant proteins were overexpressed in C. difficile and purified anaerobically by affinity chromatography. A H₂O₂ reduction potential was demonstrated for Rbr, Rbo and glutamate dehydrogenase. Rbr and glutamate dehydrogenase proved to synergistically detoxify H₂O₂ very efficiently. Furthermore, Rbo was verified as a O₂^●- reductase and its activity compared to the superoxide dismutase of E. coli. The investigated gene locus codes for an oxidative stress operon whose members are able to completely neutralize O₂^●- and H₂O₂ to water and could thus be vital for C. difficile to establish an infection in the host.