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 pHs. 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.

中文翻译：

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（p）ppGpp合成酶RSH介导艰难梭菌中的固定相发作和抗生素应激生存。

人类病原体艰难梭状芽胞杆菌对多种抗生素的耐受性越来越高，并导致高复发率感染，迫切需要新的预防和治疗策略。严格的反应是对细胞外应激的普遍细菌反应，它控制着各种生物中的抗生素存活和发病机理，但以前在艰难梭菌中没有被表征过。在这里，我们报告艰难梭菌（p）ppGpp合成酶RSH无法利用GTP或GMP作为底物，但很容易从GDP合成ppGpp。该酶还利用许多结构多样的金属辅因子进行反应催化，并在各种环境pH值下保持功能稳定。转录静止期发作和暴露于克林霉素和甲硝唑会刺激rsh。ppGpp类似物乳胶蛋白对RSH的化学抑制作用增加了流行性艰难梭菌R20291中的抗生素敏感性，这表明RSH抑制剂可能是抵抗艰难梭菌感染的药物开发的可行策略。最后，rsh的转录抑制也增加了细菌对抗生素的敏感性，这表明RSH有助于艰难梭菌对抗生素的耐受性和存活率。