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

项目摘要/摘要 艰难梭状芽胞杆菌感染(CDI)是世界上最常见和最昂贵的医院感染。 美国。负责任的病原体是一种极具弹性的细菌，对多种 抗生素的种类。因此，CDI的复发率非常高，为20%-35%。而当 (PP)pGpp‘armones’介导的严格反应(SR)对生存和毒力至关重要 在许多细菌病原体中，这在艰难梭菌中以前没有被研究过。我们的 初步研究表明艰难梭菌利用警报信号来协调 它对抗生素诱导的压力的反应。我们进一步确定化学抑制或 梭状芽胞杆菌丙二醛合成酶基因敲除增加艰难梭菌抗生素 敏感度。静止期开始、酸应激和氧化应激被确认为 丙酮合成的诱导剂。此外，氧化应激刺激艰难梭菌生物被膜 形成，一种对抗应激源的保护机制。因为有限的养分供应引发了 (Sr)在许多病原体中，并刺激艰难梭菌的产孢量和毒素合成，我们的 假设是警报信号协调梭状芽胞杆菌对营养的反应， 免疫和抗生素应激源，并促成了这种极端的弹性 病原体。我们预计SR将影响运动性、生物膜的形成、孢子形成和/或 毒素的产生以及抗生素的存活。出乎意料的是，我们发现艰难梭菌 发现只合成pGpp而不是典型的(P)ppGpp丙酮，必须 水解两个磷酸键以进行此合成。这在细菌物种中是没有先例的。 具有特征性的SRS。因为设计针对CDI的疗法的挑战是平衡 致命性和特异性为了避免损害有益的共生微生物区系， 梭状芽胞杆菌合成酶与其他生物中保守机制的差异 为艰难梭菌特异性SR抑制剂的设计提供了一个有吸引力的靶点 减少抗生素的存活率。珀塞尔实验室已经建立了一个强有力的研究计划， 培养了几名研究生和本科生。我们建议探索营养素的作用 以及压力感应在调节艰难梭菌在宿主内的生理和行为方面的作用 确定SR在调节与疾病相关的细菌过程中的作用。此外， 艰难梭菌中调节丙氨酸新陈代谢的酶将被鉴定。这项建议 将使我们能够扩大我们的培训活动，以便让更多的学生参与到有意义的 艰难梭菌应激生存机制的研究及其启示 用于CDI持久性和重复性。

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.