Exogenous butyrate inhibits butyrogenic metabolism and alters expression of virulence genes inClostridioides difficile

Abstract

ABSTRACTThe gut microbiome engenders colonization resistance against the diarrheal pathogenClostridioides difficilebut the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance againstC. difficileis short chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness ofC. difficilein vitro and is correlated withC. difficile- inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition inC. difficileoccurs under conditions whereC. difficilealso produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized intoC. difficilecells, is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and (S)-3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of butyrogenic pathway(s) inC. difficilecoincides with alterations in toxin production and sporulation. Together, this work highlights butyrate as a signal of aC. difficileinhospitable environment to whichC. difficileresponds by producing its diarrheagenic toxins and producing environmentally-resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of howC. difficilegrowth is inhibited by butyrate and how butyrate serves as a signal to alterC. difficilevirulence in the face of a highly competitive and dynamic gut environment.IMPORTANCEThe gut microbiome engenders colonization resistance against the diarrheal pathogenClostridioides difficilebut the molecular basis of this colonization resistance is incompletely understood, which hinders the development of novel therapeutic interventions forC. difficileinfection (CDI). We investigated howC. difficileresponds to butyrate, an end-product of gut microbiome community metabolism which inhibitsC. difficilegrowth. We show that exogenously-produced butyrate is internalized intoC. difficile, which inhibitsC. difficilegrowth by interfering with its own butyrate production. This growth inhibition coincides with the expression of virulence-related genes. Future work to disentangle the molecular mechanisms underlying these growth and virulence phenotypes will likely lead to new strategies to restrictC. difficilegrowth in the gut and minimize its pathogenesis during CDI.