The selenophosphate synthetase, selD, is important for Clostridioides difficile physiology

The endospore-forming pathogen, Clostridioides difficile, is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and healthcare. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using RNAseq, we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways.