The role of the (p)ppGpp-mediated stringent response in Clostridioides difficile's resilience to nutrient and immune stresses typically found within a mammalian host

项目总结/摘要 艰难梭菌感染（CDI）是美国最常见和最昂贵的医院感染。 美国的负责的病原体是一种非常有弹性的细菌，能够耐受多种细菌。 抗生素的种类因此，CDI具有非常高的20-35%复发率。而 由（pp）pGpp `alarmones'介导的严格反应（SR）对于存活和毒力是至关重要的 在许多细菌病原体中，这在C.很难我们 初步研究表明，C. difficile利用报警信号来协调 它对南极引起的压力的反应。我们进一步确定，化学抑制或 梭菌警报素合成酶的遗传敲低增加了C.难治性抗生素 易感性稳定期开始、酸应激和氧化应激被确定为 alarmone合成的诱导剂。此外，氧化应激刺激C。艰难生物膜 形成，对压力源的保护机制。由于有限的养分供应引发了 (SR)在许多病原体中，并刺激C. difficile，our 假设是警报素信号传导协调梭菌对营养物的反应， 免疫和抗生素压力源，并有助于这种极端的弹性， 病原体我们预期SR将影响运动性、生物膜形成、孢子形成和/或细胞增殖。 毒素产生以及抗生素存活。出乎意料的是，我们发现C.艰难梭状芽抱 发现专门合成pGpp而不是典型的（p）ppGpp alarmones，并且必须 水解两个磷酸键进行合成。这在细菌物种中没有先例 具有特征的SR。由于设计针对CDI的疗法的挑战是平衡 为了避免对有益的肠道微生物群的损害， 梭菌合成酶与其他生物体中保守机制的差异 提出了一个有吸引力的目标，为设计的SR抑制剂，具体到C。难为 减少抗生素存活率。珀塞尔实验室已经建立了一个强大的研究计划， 培养了几名研究生和本科生。我们建议探索营养素的作用 和应激感受调节C.在宿主体内的艰难生理和行为， 确定SR在调节疾病相关细菌过程中的作用。此外，本发明还 在C.将表征艰难。这项建议 将使我们能够扩大我们的培训活动，以使更多的学生参与有意义的 一项研究探讨了C.艰难应激生存与影响 CDI的持续性和复发性。

Project Summary/Abstract Clostridioides difficile infection (CDI) is the most common and costly nosocomial infection in the United States. The responsible pathogen is an extremely resilient bacterium, tolerant of multiple classes of antibiotics. As a result, CDI has a very high 20-35% recurrence rate. While the stringent response (SR) mediated by (pp)pGpp `alarmones' is crucial for survival and virulence in a number of bacterial pathogens, this had not previously been studied in C. difficile. Our preliminary research demonstrated that C. difficile utilizes alarmone signaling to coordinate its response to antibiotic-induced stresses. We further determined that chemical inhibition or genetic knockdown of a clostridial alarmone synthetase enzyme increases C. difficile antibiotic susceptibility. Stationary phase onset, acid stress, and oxidative stress were identified as inducers of alarmone synthesis. Additionally, oxidative stress stimulates C. difficile biofilm formation, a protective mechanism against stressors. As limited nutrient availability triggers the (SR) in a number of pathogens, and stimulates sporulation and toxin synthesis in C. difficile, our hypothesis is that alarmone signaling coordinates clostridial responses to nutrient, immune, and antibiotic stressors and contributes to the extreme resilience of this pathogen. We anticipate that the SR will influence motility, biofilm formation, sporulation, and/or toxin production as well as antibiotic survival. Unexpectedly, we have found that C. difficile was found to exclusively synthesize pGpp rather than canonical (p)ppGpp alarmones and must hydrolyze two phosphate bonds for this synthesis. This is without precedent in bacterial species with characterized SRs. As the challenge of designing therapies against CDI is to balance lethality and specificity in order to avoid damage to beneficial commensal microbiota, the divergence of clostridial synthetases from mechanisms conserved in other organisms presents an attractive target for the design of SR inhibitors specific to C. difficile for reducing antibiotic survival. The Purcell lab has established a robust research program and trained several graduate and undergraduate students. We propose exploring the role of nutrient and stress sensing in regulating C. difficile physiology and behavior within the host while determining the role of the SR in regulating disease-relevant bacterial processes. Further, enzymes that mediate alarmone metabolism in C. difficile will be characterized. This proposal will allow us to expand our training activities in order to involve more students in meaningful research investigating an unexplored mechanism of C. difficile stress survival with implications for CDI persistence and recurrence.