Investigating the role of selenium-dependent Stickland reactions in Clostridioides difficile bioenergetics

Clostridioides difficile is an anaerobic, spore-forming bacterium and one of the leading causes of antibiotic- associated diarrhea. Colonization depends on the metabolism of certain nutrients in the host gut, namely amino acids and carbohydrates. C. difficile ferments amino acids as an integral part of its metabolism via Stickland reactions, utilizing the selenoproteins D-proline reductase (Prd) and glycine reductase (Grd) to generate energy. Specificity of selenocysteine incorporation reflects the intracellular levels of selenophosphate, the activated selenium donor synthesized by selenophosphate synthetase (SelD). SelD is coincidentally a selenoprotein itself, containing a UGA codon at position 17 (Sec-17). The functional and physiological importance of Sec-17 has not been previously investigated in C. difficile. Furthermore, while the role of selenoproteins in C. difficile amino acid catabolism is well known, recent in vivo experiments suggest a preference for proline and glycine, as well as certain sugars during infection. In this study, we generated a deletion mutant in the hypervirulent strain R20291 unable to produce selenoproteins (∆selAB), as well as created strains with a mutated Sec-17 in the SelD protein. We performed physiological and metabolic assays in defined minimal media supplemented with different carbohydrate sources and found that selenoprotein deficiency decreased viability and motility, demonstrating a selenium-dependent phenotype related to carbohydrate metabolism. This implies that the oxidation of carbohydrates may be coupled to the reduction of different substrates depending on the carbohydrate source. Moreover, we found that mutating Sec-17 to a cysteine residue in SelD decreased Grd levels in a minimal medium and impacted amino acid and carbohydrate metabolic pathways. These results provide a direct link between carbohydrate and selenium-dependent Stickland reactions in C. difficile, broadening the field’s insight into the complex metabolism of this pathogen.