Inactivation of the riboswitch-controlled GMP synthase GuaA in Clostridioides difficile is associated with severe growth defects and poor infectivity in a mouse model of infection.

Clostridioides difficile is the main cause of nosocomial antibiotic-associated diarrhoea. There is a need for new antimicrobials to tackle this pathogen. Guanine riboswitches have been proposed as promising new antimicrobial targets, but experimental evidence of their importance in C. difficile is missing. The genome of C. difficile encodes four distinct guanine riboswitches, each controlling a single gene involved in purine metabolism and transport. One of them controls the expression of guaA, encoding a guanosine monophosphate (GMP) synthase. Here, using in-line probing and GusA reporter assays, we show that these riboswitches are functional in C. difficile and cause premature transcription termination upon binding of guanine. All riboswitches exhibit a high affinity for guanine characterized by Kd values in the low nanomolar range. Xanthine and guanosine also bind the guanine riboswitches, although with less affinity. Inactivating the GMP synthase (guaA) in C. difficile strain 630 led to cell death in minimal growth conditions, but not in rich medium. Importantly, the capacity of a guaA mutant to colonize the mouse gut was significantly reduced. Together, these results demonstrate the importance of de novo GMP biosynthesis in C. difficile during infection, suggesting that targeting guanine riboswitches with analogues could be a viable therapeutic strategy.