c-di-AMP signaling is required for bile salts resistance and long-term colonization by Clostridioides difficile

In this study, we showed for the first time that c-di-AMP is produced by C. difficile and controls the uptake of potassium, making it essential for growth. We found that c-di-AMP is involved in biofilm formation, cell wall homeostasis, osmotolerance as well as detergent and bile salt resistance in C. difficile. We identified BusR as a new regulator that binds c-di-AMP and represses the expression of the compatible solute transporter BusAA-AB. Interestingly, a busR mutant is highly resistant to a hyperosmotic or bile salt stress compared to the parental strain while a busAA mutant is more susceptible. A short exposure of C. difficile cells to bile salts resulted in a decrease of the c-di-AMP concentrations reinforcing the hypothesis that changes in membrane characteristics due to variations of the cellular turgor or membrane damages constitute a signal for the adjustment of the intracellular c-di-AMP concentration. In a colonization mouse model, a strain producing elevated c-di-AMP concentrations failed to persist in the gut in contrast to the parental strain. Thus, c-di-AMP is a signaling molecule with pleiotropic effects that controls osmolyte uptake to confer osmotolerance and bile salt resistance in C. difficile and that is important for colonization of the host. Strain 630∆erm∆gdpP served as the control strain. Expression in 630∆erm∆gdpP∆busR was compared to this control strain. Four independent replicates are included.