Identification of a Bile Acid-Binding Transcription Factor in Clostridioides difficile Using Chemical Proteomics

Clostridioides difficile is a Gram-positive anaerobic bacterium that is the leading cause of hospital-acquired gastro-enteritis in the US. In the gut milieu, C. difficile encounters microbiota-derived, growth-inhibiting bile acids that are thought to be a significant mechanism of colonization resistance. While the levels of certain bile acids in the gut correlate with susceptibility to C. difficile infection, their molecular targets in C. difficile remain unknown. In this study, we sought to use chemical proteomics to identify bile acid-interacting proteins in C. difficile. Using photoaffinity bile acid probes and chemical proteomics, we identified a previously uncharacterized MerR family protein, CD3583 (now BapR), as a putative bile acid-sensing transcription regulator. Our data indicate that BapR specifically binds to and is stabilized by lithocholic acid (LCA) in C. difficile. Although loss of BapR did not affect C. difficile's sensitivity to LCA, Delta bapR cells elongated more in the presence of LCA compared to wild-type cells. Transcriptomics revealed that BapR regulates several gene clusters, with the expression of the mdeA-cd3573 locus being specifically de-repressed in the presence of LCA in a BapR-dependent manner. Electrophoretic mobility shift assays revealed that BapR directly binds to the mdeA promoter region. Because mdeA is involved in amino acid-related sulfur metabolism and the mdeA-cd3573 locus encodes putative transporters, we propose that BapR senses a gastrointestinal tract-specific small molecule, LCA, as an environmental cue for metabolic adaptation.