The σB signalling activation pathway in the enteropathogen Clostridioides difficile.

Clostridium difficile is the main cause of antibiotic-associated diarrhoea. Inside the gut, C. difficile must adapt to the stresses it copes with, by inducing protection, detoxification and repair systems that belong to the general stress response involving σB . Following stresses, σB activation requires a PP2C phosphatase to dephosphorylate the anti-anti-sigma factor RsbV that allows its interaction with the anti-sigma factor RsbW and the release of σB . In this work, we studied the signalling pathway responsible for the activation of σB in C. difficile. Contrary to other firmicutes, the expression of sigB in C. difficile is constitutive and not autoregulated. We confirmed the partner switching mechanism that involved RsbV, RsbW and σB . We also showed that CD2685, renamed RsbZ, and its phosphatase activity are required for RsbV dephosphorylation triggering σB activation. While CD0007 and CD0008, whose genes belong to the sigB operon, are not involved in σB activity, depletion of the essential iron-sulphur flavoprotein, CD2684, whose gene forms an operon with rsbZ, prevents σB activation. Finally, we observed that σB is heterogeneously active in a subpopulation of C. difficile cells from the exponential phase, likely leading to a 'bet-hedging' strategy allowing a better chance for the cells to survive adverse conditions.