Identification of DraRS in C. difficile, a two-component regulatory system that responds to lipid II interacting antibiotics

Clostridioides difficile is a Gram-positive opportunistic pathogen that results in 220,000 infections, 12,000 deaths, and upwards of $1 billion in medical costs in the US each year. C. difficile is highly resistant to a variety of antibiotics, but we have a poor understanding of how C. difficile senses and responds to antibiotic stress, and how such sensory systems affect clinical outcomes. There has been recent interest in using a daptomycin analog, Surotomycin, to treat C. difficile infections. We have identified a spontaneous C. difficile mutant that displays increased daptomycin resistance. We performed whole-genome sequencing that this mutant possessed a nonsense mutation, S605*, in draS which encodes a putative sensor histidine kinase of a two-component system (TCS). The draSS605* mutant has approximately a 4-8-fold increase in daptomycin MIC compared to WT. We found that expression of constitutively active DraRD54E in WT increases daptomycin resistance 8-16-fold and increases bacitracin resistance ~4-fold. We found that a selection of lipid II inhibiting compounds leads to increased activity of the luciferase-based reporter, PdraR-sLucopt including vancomycin, bacitracin, ramoplanin, and daptomycin. Using RNA-seq we identified the DraRS regulon. Interestingly, we found that DraRS can induce expression of the previously identified hex locus required for synthesis of a novel glycolipid produced in C. difficile. Our data suggest that induction of the hex locus by DraR explains some, but not all, of the DraR-induced daptomycin and bacitracin resistance. Overall design: RNA-seq experiments, controls and experimental conditions all done in biological triplicate