EndD mediates butyrate-dependent toxin release and sporulation in Clostridioides difficile

Clostridioides difficile is an urgent threat to human health. Current treatments for C. difficile infections (CDIs) are antibiotics and microbiome restoration therapy (MRT) for recurrent cases. However, antibiotics contribute to antibiotic resistance and recurrent CDIs, and the long-term sustainability and accessibility of MRTs remain to be determined. Since a dysbiotic gut microbiome is the primary risk factor for CDI, a better understanding of the interactions between C. difficile, the microbiome, and the host will aid development of treatments with improved precision. Emerging evidence supports that butyrate, a prominent end product of gut microbiome metabolism, is a key determinant of C. difficile pathogenesis. Notably, C. difficile releases more of its toxins (TcdA and TcdB) in butyrate-rich environments. Here, we demonstrate that butyrate-dependent toxin release is not driven by two previously characterized modes of toxin release (e.g., TcdE-dependent secretion or Cwp19-dependent autolysis). Instead, butyrate enhances the expression of a broadly conserved endolysin (EndD), which is responsible for butyrate-dependent toxin release. We additionally demonstrate that endD-dependent toxin release does not universally occur under all growth conditions, that its expression is dependent on the late-stage sporulation sigma factor SigK, and that endD enhances butyrate-dependent sporulation. Overall, our findings provide deeper insight into butyrate-dependent effects on C. difficile pathogenesis and set the stage for future work to better understand the molecular and genetic underpinnings of endD regulation.IMPORTANCEClostridioides difficile is a leading cause of infectious diarrhea. The gut microbiome-derived short-chain fatty acid butyrate triggers C. difficile to release its major virulence factors, toxins TcdA and TcdB. Here, we show that butyrate-dependent toxin release is independent of classical secretion or autolytic pathways and is instead mediated by EndD, an endolysin broadly conserved among C. difficile clinical isolates. We also demonstrate that endD expression is governed by the sporulation factor SigK and enhances sporulation, directly linking transmission and virulence. Uncovering how C. difficile couples metabolic sensing to EndD-mediated toxin release and sporulation advances our understanding of pathogen-microbiome interactions and highlights potential targets for non-antibiotic therapeutics.