Exogenous butyrate inhibits butyrogenic metabolism and alters virulence phenotypes in Clostridioides difficile
The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile-inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells and 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) in C. difficile coincides with alterations in toxin release and sporulation. Together, this work highlights butyrate as a marker of a C. difficile inhospitable environment to which C. difficile responds by releasing 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 how C. difficile growth is inhibited by butyrate and how butyrate alters C. difficile virulence in the face of a highly competitive and dynamic gut environment. Overall design: C. difficile 630 overnight cultures were back-diluted 1:50 in 20mL mRCM in 50mL conical tubes with 50 mM sodium butyrate or 50 mM sodium chloride adjusted to pH=6.5. Harvest was performed at OD600=0.3-0.5 for mid-log phase samples, at 24 hours post inoculation (early stationary phase) or at 48 hours post inoculation (late stationary phase). At the appropriate time points, 5mL aliquots of the cultures were diluted 1:1 in chilled 1:1 Ethanol:Acetone to preserve RNA. These samples were then centrifuged for 5 minutes at 3000 x g at room temperature and cell pellets were frozen at -80°C. Immediately prior to RNA extraction, cell pellets were centrifuged at 4°C at 3000 x g for 1minute. Residual supernatant was removed from the cell pellets, which were subsequently washed with 5mL nuclease free PBS. Washed pellets were centrifuged at 4°C at 3000 x g for 1minute, the supernatant was removed, and the resulting pellet was resuspended in 1mL TRIzol and processed using a TRIzol Plus RNA Purification Kit (Thermo) with on-column DNase treatment according to the manufacturer's instructions. Purified RNA was frozen at -80C and RNA integrity was confirmed via BioAnalyzer (Agilent) prior to proceeding. RNA-seq on high quality rRNA-depleted RNA extracts (12M paired end reads per sample) and transcript level quantification, count normalization, and differential expression analysis were performed using the C. difficile 630 reference genome (GCF_000009205.2_ASM920v2) for sequence alignments (SeqCenter, Pittsburgh, Pennsylvania, USA).