The human pathogen Clostridioides difficile is increasingly tolerant of multiple antibiotics and causes infections with a high rate of recurrence, creating an urgent need for new preventative and therapeutic strategies. The stringent response, a universal bacterial response to extracellular stress, governs antibiotic survival and pathogenesis in diverse organisms but has not previously been characterized in C. difficile. Here, we report that the C. difficile (p)ppGpp synthetase RSH is incapable of utilizing GTP or GMP as a substrate but readily synthesizes ppGpp from GDP. The enzyme also utilizes many structurally diverse metal cofactors for reaction catalysis and remains functionally stable at a wide range of environmental pH. Transcription of rsh is stimulated by stationary phase onset and by exposure to the antibiotics clindamycin and metronidazole. Chemical inhibition of RSH by the ppGpp analog Relacin increases antibiotic susceptibility in epidemic C. difficile R20291, indicating that RSH inhibitors may be a viable strategy for drug development against C. difficile infection. Finally, transcriptional suppression of rsh also increases bacterial antibiotic susceptibility, suggesting that RSH contributes to C. difficile antibiotic tolerance and survival. Importance Clostridioides difficile infection (CDI) is an urgent public health threat with a high recurrence rate, in part because the causative bacterium has a high rate of antibiotic survival. The (p)ppGpp-mediated bacterial stringent response (SR) plays a role in antibiotic tolerance in diverse pathogens and is a potential target for development of new antimicrobials because the enzymes that metabolize (p)ppGpp have no mammalian homologs. We report that stationary phase onset and antibiotics induce expression of the clostridial ppGpp synthetase RSH and that both chemical inhibition and translational suppression of RSH increases C. difficile antibiotic susceptibility. This demonstrates that development of RSH inhibitors to serve as adjuvants to antibiotic therapy is a potential approach for the development of new strategies to combat CDI.

Despite the role of the stringent response in antibiotic survival and recurrent infections, it has been a challenging target for antibacterial therapies because it is so ubiquitous. This is an especially relevant consideration for the treatment of Clostridioides difficile infection (CDI), as exposure to broad-spectrum antibiotics that harm commensal microbes is a major risk factor for CDI.

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The magic spot alarmones (pp)pGpp, previously implicated in Clostridioides difficile antibiotic survival, are synthesized by CdRSH and CdRelQ. These enzymes are transcriptionally activated by diverse environmental stresses, but both exclusively synthesize pGpp rather than ppGpp as has previously been reported. While direct synthesis of pGpp from a GMP substrate and (p)ppGpp hydrolysis into pGpp by NUDIX hydrolases have previously been reported, there is no precedent for a bacterium synthesizing pGpp exclusively. Hydrolysis of the 5-prime phosphate or pyrophosphate from GDP or GTP substrates is necessary for activity by the clostridial enzymes, neither of which can utilize GMP as a substrate. Both enzymes are remarkably insensitive to the size of their metal ion cofactor, tolerating a broad array of metals that do not allow activity in (pp)pGpp synthetases from other organisms. It is clear that while C. difficile utilizes magic spot signaling, its mechanisms of alarmone synthesis are not directly homologous to those in more completely characterized organisms.