A rubrerythrin locus of Clostridioides difficile efficiently detoxifies reactive oxygen species
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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 % O 2 ) 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 H 2 O 2 and O 2 ●- . 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 H 2 O 2 reduction potential was demonstrated for Rbr, Rbo and glutamate dehydrogenase. Rbr and glutamate dehydrogenase proved to synergistically detoxify H 2 O 2 very efficiently. Furthermore, Rbo was verified as a O 2 ●- 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 O 2 ●- and H 2 O 2 to water and could thus be vital for C. difficile to establish an infection in the host.
