Mechanisms of Clostridioides difficile germinant and co-germinant sensing

艰难梭菌萌发和共萌发传感机制

• 批准号: 10462682
• 负责人: Emily Rachel Forster
• 金额: $ 2.15万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462682
• 关键词: Academic skills; Address; Adopted; Affinity; Amino Acids; Anaerobic Bacteria; Bacteria; Bacterial Spores; Behavior; Bile Acids; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Calcium; Calorimetry; Cessation of life; Chemicals; Chemistry; Clostridium difficile; Collaborations; Communities; Critical Thinking; Crystallography; Data; Deuterium; Development; Disease; Drug Targeting; Educational process of instructing; Exhibits; Fostering; Future; Genetic; Genetic Screening; Genetic study; Germination; Goals; Healthcare Systems; Hydrogen; Immunoprecipitation; Infection; Label; Learning; Mass Spectrum Analysis; Measures; Mentorship; Methods; Modeling; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Nutritional; Pathogenesis; Pathway interactions; Positioning Attribute; Process; Proteins; Recurrence; Reproduction spores; Research Personnel; Signal Transduction; Specificity; Structure; Taurine Cholate; Technical Expertise; Testing; Titrations; Training; Ultraviolet Rays; Universities; Vermont; Work; Writing; X-Ray Crystallography; bile acid binding proteins; career; chenodeoxycholate; cost; crosslink; experimental study; faculty research; gastrointestinal infection; genetic analysis; inhibitor; innovation; insight; light scattering; molecular mass; novel; novel strategies; pathogen; prevent; receptor; response; sensor; skills; small molecule; stoichiometry; sugar

PROJECT SUMMARY / ABSTRACT Germination is essential for the lifecycle of spore-forming obligate anaerobes like the nosocomial pathogen, Clostridioides difficile. Despite the importance of this process, the molecular mechanisms by which bacterial spores physically detect the small molecule germinants that trigger germination remain poorly understood. Furthermore, the mechanism by which C. difficile spores sense and transduce germinant signals is unique among spore-forming bacteria because (i) C. difficile spores respond to bile acid germinants rather than the canonical nutritional germinants (e.g. sugars) used by all spore-formers studied to date, and (ii) C. difficile lacks the transmembrane germinant receptors encoded by almost all other spore-formers. Instead, C. difficile is thought to use a soluble protein, the CspC pseudoprotease, to detect bile acids and trigger a proteolytic signaling cascade that leads to germination. We recently solved the structure of this putative germinant receptor. Our subsequent structure-function analyses challenge the prevailing model that CspC directly senses bile acid germinants and surprisingly revealed that CspC not only integrates signals from bile acid germinants but also from amino acid and calcium co- germinants. Because of these unexpected findings, the proposed studies will address questions such as (i) what are the direct sensors of bile acid germinants in C. difficile spores? and (ii) How does CspC transduce germinant and co-germinant signals? (Co-)germinant sensing remains poorly defined, so our analyses of CspC and identification of bile acid-binding proteins will provide molecular insight into the mechanisms by which C. difficile spores germinate. Furthermore, since spore germination is essential for C. difficile to initiate infection, our studies may help identify novel strategies for preventing the ~224,000 C. difficile infections per year in the US alone. In addition to advancing our understanding of C. difficile germination, this project will provide me with comprehensive academic and career training. My sponsor, Dr. Aimee Shen, is committed to teaching me the technical skills to complete the proposed experiments and the academic skills to effectively share my work with the scientific community. As outlined in my training plan, we have set specific goals for training in writing, presentation, teaching, and mentorship that are critical for me to obtain my overall career goal of holding an academic research faculty position. Dr. Shen and I have assembled a group of collaborators with expertise in biochemistry, crystallography, and chemical biology, and I will seize the opportunity to learn numerous experimental approaches from them. The diverse mentorship I will receive from Dr. Shen and my collaborators will ultimately foster my critical thinking skills, technical capabilities, and development into an independent researcher.

项目摘要/摘要 萌发对芽胞形成专性厌氧菌的生命周期是必不可少的，比如医院病原体， 艰难梭状芽胞杆菌尽管这一过程很重要，但细菌通过的分子机制 孢子物理检测触发萌发的小分子发芽物质仍然知之甚少。 此外，艰难梭菌孢子感知和传递萌发信号的机制是独特的 在芽胞形成细菌中，因为(I)艰难梭菌的孢子对胆汁酸萌发产生反应，而不是对 迄今研究过的所有芽胞形成者所使用的典型营养发芽剂(如糖)，以及(Ii)艰难梭菌缺乏 几乎所有其他芽胞形成者编码的跨膜萌发受体。相反，艰难梭菌是 被认为使用一种可溶的蛋白质，CSPC假酶，来检测胆汁酸并触发蛋白水解性信号 导致种子萌发的级联反应。 我们最近解开了这个假定的萌发受体的结构。我们随后的结构-功能 分析挑战了CSPC直接感知胆汁酸发芽的主流模式，并令人惊讶地揭示 CSPC不仅整合了胆汁酸发芽的信号，而且还整合了氨基酸和钙联合作用的信号。 萌芽动物。由于这些意想不到的发现，拟议的研究将解决以下问题：(I)什么 艰难梭菌孢子中的胆汁酸发芽的直接感受器吗？和(Ii)CSPC是如何转导发芽的 以及共同萌发的信号呢？(共同)萌芽感知仍然定义不清，因此我们对CSPC和 胆汁酸结合蛋白的鉴定将为艰难梭菌感染的机制提供分子基础 孢子萌发。此外，由于孢子萌发是艰难梭菌发起感染的关键，我们的研究 可能有助于确定新的策略，仅在美国就可以预防每年约22.4万例艰难梭菌感染。 除了增进我们对艰难梭菌萌发的了解外，这个项目还将为我提供 全面的学术和职业培训。我的赞助人沈爱梅博士致力于教我 完成建议的实验所需的技术技能和有效分享工作的学术技能 科学界。正如我在培训计划中概述的那样，我们已经制定了具体的书面培训目标， 演讲、教学和指导，这些对我实现持有 学术研究教员职位。沈博士和我已经召集了一个小组，他们在 生物化学、结晶学和化学生物学，我会抓住机会学习无数 来自他们的实验方法。我将从沈博士和我的合作者那里得到多样化的指导 最终会培养我的批判性思维能力、技术能力，并发展成为一个独立的 研究员。