The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile

Abstract

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.

Unique features of magic spot metabolism in Clostridioides difficile

“…In our earlier work, we utilized an anaerobic fluorescent transcriptional reporter incorporating the oxygen-independent fluorescent flavoprotein phiLOV2.1 to monitor transcription of the clostridial rsh and relQ genes. 34 We observed the increased transduction of these genes upon exposure to sublethal concentrations of clindamycin and metronidazole in a strain-specific manner. 34 Here, we assessed the transcriptional response of C. difficile to vancomycin and fidaxomicin, the currently recommended antibiotic treatments for severe or recurring CDI.…”

“…50 There does not appear to be any precedent for the extremely broad range of metals successfully utilized as cofactors by both clostridial synthetases. 34 This could be a C. difficile adaptation to so-called 'nutritional immunity,' which is the ability of mammalian immune systems to chelate and sequester metal ions that are limiting factors for the growth of foreign pathogens. 64 65 As metal starvation can trigger the stringent response in some bacteria, [66][67] it is possible that promiscuous utilization of divalent cations in the enzymes that initiate the clostridial stringent response could provide a survival advantage during infection.…”

“…We have previously reported that CdRSH binds both GDP and GTP in vitro but exclusively utilizes GDP as a phosphoacceptor in order to synthesize ppGpp. 34 This is unusual among RSH family enzymes, which utilize both GDP and GTP with varying affinity to Here, we report that expression of clostridial (pp)pGpp synthetase genes is induced by antibiotic and acid stress. We further report that the RelQ enzyme utilizes both GDP and GTP as substrates, but that the only form of alarmone produced by either CdRSH or CdRelQ enzyme is pGpp.…”

“…We have previously reported that C. difficile encodes an RSH family enzyme and the SAS RelQ, and confirmed the enzymatic activity of RSH. 34 In addition to RelQ, C. difficile encodes another putative SAS enzyme, CDR20291_1607, which we have named RelC. RelC contains a putative SAS domain and a C-terminal 250 residue region that contains no conserved domain, and its only homologs are in Firmicutes species.…”

Pleiotropic Effects of Bacterial Small Alarmone Synthetases: Underscoring the Dual-Domain Small Alarmone Synthetases in Mycobacterium smegmatis

“…Clostridium difficile has a RelQ that is induced by antibiotic stress. There is a 2-fold upregulation of relQ after exposure to ampicillin and clindamycin, which explains the role of RelQ in antibiotic survival ( Pokhrel et al, 2020 ).…”