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

(p)ppGpp 介导的严格反应在艰难梭菌对哺乳动物宿主体内常见的营养和免疫应激的恢复能力中的作用

• 批准号: 10514305
• 负责人: Erin Bridget Purcell
• 金额: $ 39.01万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-18 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10514305
• 关键词: Acids; Active Sites; Affect; Antibiotic susceptibility; Antibiotics; Bacteria; Basic Science; Behavior; Behavioral Assay; Biological Assay; Cell Density; Cells; Chemicals; Clostridium difficile; Cytoplasm; Development; Disease; Environment; Environmental Risk Factor; Enzymes; Equilibrium; Exposure to; Foundations; Future; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Growth; Guanosine Triphosphate; Host Defense; Human; Hydrolase; Hydrolysis; Immune; Immune response; Immune system; In Vitro; Individual; Infection; Intestines; Ligase; Mediating; Metabolic; Metabolism; Metals; Microbial Biofilms; Mucous body substance; Mutate; Nosocomial Infections; Nutrient; Nutritional; Organism; Oxidative Stress; Paper; Pathogenicity; Peptides; Phase; Physiology; Play; Prevention strategy; Process; Production; Publishing; Rapid screening; Reactive Oxygen Species; Recurrence; Regulation; Reporter; Research; Research Personnel; Role; Signal Pathway; Signal Transduction; Specificity; Starvation; Stress; Students; Testing; Toxin; Training; Training Activity; United States; Virulence; Work; acid stress; antibiotic tolerance; cell motility; commensal microbes; cost; design; economic cost; enzyme activity; extracellular; graduate student; in vivo; inhibitor; inorganic phosphate; knock-down; novel therapeutic intervention; pathogen; pathogenic bacteria; programs; protein expression; protein purification; recurrent infection; resilience; response; stressor; therapy design; transcriptomics; tripolyphosphate; undergraduate student

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.

项目概要/摘要 艰难梭菌感染 (CDI) 是最常见且最昂贵的医院感染 美国。负责的病原体是一种生命力极强的细菌，能够耐受多种 抗生素类别。因此，CDI 的复发率非常高，可达 20-35%。虽然 (pp)pGpp“警报素”介导的严格反应（SR）对于生存和毒力至关重要 在许多细菌病原体中，此前尚未在艰难梭菌中进行过研究。我们的 初步研究表明，艰难梭菌利用警报信号传导来协调 它对抗生素引起的压力的反应。我们进一步确定化学抑制或 梭菌警报酮合成酶的基因敲除增加了艰难梭菌抗生素的含量 易感性。稳定期开始、酸应激和氧化应激被确定为 警报酮合成的诱导剂。此外，氧化应激刺激艰难梭菌生物膜 形成，对抗压力源的保护机制。由于有限的营养供应会引发 (SR) 在许多病原体中，并刺激艰难梭菌中的孢子形成和毒素合成，我们的 假设是警报信号传导协调梭菌对营养物的反应， 免疫和抗生素应激源，并有助于这种极端的恢复能力 病原。我们预计 SR 将影响运动性、生物膜形成、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 发现专门合成 pGpp 而不是典型的 (p)ppGpp 警报酮，并且必须 水解用于该合成的两个磷酸键。这在细菌物种中是没有先例的 具有特征 SR。由于设计针对 CDI 的疗法的挑战是平衡 致死性和特异性，以避免损害有益的共生微生物群， 梭菌合成酶与其他生物体中保守机制的差异 为设计针对艰难梭菌的 SR 抑制剂提供了一个有吸引力的目标 减少抗生素的存活率。珀塞尔实验室建立了强大的研究计划 培养了多名研究生和本科生。我们建议探索营养素的作用 和压力感应调节宿主内的艰难梭菌生理和行为，同时 确定 SR 在调节疾病相关细菌过程中的作用。更远， 将表征艰难梭菌中介导报警素代谢的酶。这个提议 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活动中 研究调查了艰难梭菌应激生存的未探索机制及其意义 对于 CDI 持续存在和复发。

项目成果

Host-defense piscidin peptides as antibiotic adjuvants against Clostridioides difficile.

• DOI: 10.1371/journal.pone.0295627
• 发表时间: 2024
• 期刊: PloS one
• 影响因子: 3.7