A rubrerythrin locus of Clostridioides difficile encodes enzymes that efficiently detoxify reactive oxygen species

The microaerophilic conditions in the large intestine and reactive oxygen species (ROS) produced by the immune system represent a challenge for the strictly anaerobic pathogen Clostridioides difficile, which protects itself by a variety of oxidative stress proteins. Four of these are encoded in an operon that has been implicated in the detoxification of H₂O₂ and O₂^●-. In this study, proteins of this operon, i. e. a rubrerythrin (Rbr), a superoxide reductase (Sor) and a putative glutamate dehydrogenase (CD630_08280) were investigated for their ROS detoxifying activity in vitro.

Recombinant proteins were overexpressed in C. difficile and purified anaerobically by affinity chromatography. The H₂O₂-reductase activity was determined by measuring the NADH consumption after peroxide addition. Superoxide detoxification potential of Sor was detected colorimetrically using a xanthine/xanthine oxidase system with cytochrome c as analytical probe.

Proposed roles of the investigated proteins in the detoxification pathways of ROS could partially be demonstrated. Specifically, Rbr and glutamate dehydrogenase synergistically detoxify H₂O₂, although with a very low turnover. Furthermore, Sor was shown to scavenge O₂^●- by superoxide dismutase activity and its activity was compared to superoxide dismutase of Escherichia coli.

The investigated gene locus codes for an oxidative stress operon whose members have the potential to neutralize O₂^●- and H₂O₂ to water and thus complements the arsenal of ROS detoxifying mechanisms that are already known in C. difficile. However, full activity with adequate physiological electron transfer partners still needs to be demonstrated.