SRP345710 PRJNA779458 LL-37 activates glycine catabolism in C. difficile to promote virulence Clostridioides difficile (C. difficile) is the leading cause of antibiotic-associated diarrhea. This human pathogen can ferment amino acids to generate energy through Stickland reactions, in which a donor amino acid is oxidized while an acceptor amino acid is reduced. The glycine Stickland pathway is conserved among C. difficile strains, and represents one of the preferred acceptor amino acids in Stickland reactions. We have observed changes in C. difficile glycine Stickland transcription in response to the host peptide, LL-37. Even though it is known that Stickland metabolism is important for C. difficile growth in vitro, how this metabolism impacts pathogenesis and how it is regulated remains largely uncharacterized. We hypothesized that C. difficile colonization and pathogenesis is increased by glycine Stickland metabolism, and that this metabolic pathway is modulated in response to LL-37. To test this hypothesis, we generated a grdAB mutant and analyzed its growth in minimal media. Our data indicate that the amino acid glycine enhanced C. difficile growth through the glycine Stickland pathway. We also found that the ability to catabolize glycine promotes toxin production in minimal media. Using transcriptional reporter assays we showed that expression of the grd operon is enhanced by LL-37. We assessed transcription of the grd operon and investigated direct binding of the LL-37 regulator, ClnR, to the grdX promoter. Finally, we observed that glycine catabolism promotes C. difficile virulence in a hamster model of disease.