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.

项目总结/摘要 萌发对于孢子形成专性厌氧菌（如医院病原体）的生命周期至关重要， 艰难梭菌尽管这一过程很重要，但细菌感染的分子机制 孢子物理检测触发萌发的小分子萌发剂仍然知之甚少。 此外，还探讨了C.艰难梭菌孢子感生和萌发信号是独特的 在孢子形成细菌中，因为（i）C.艰难梭菌孢子对胆汁酸萌发剂的反应， 迄今为止研究的所有孢子形成者使用的典型营养萌发剂（例如糖），和（ii）C.艰难缺乏 由几乎所有其他孢子形成者编码的跨膜萌发受体。相反，C.艰难梭菌是 人们认为使用可溶性蛋白质CspC假蛋白酶来检测胆汁酸并触发蛋白水解信号传导 级联导致发芽。 我们最近解决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直接感知胆汁酸萌发物的流行模型， CspC不仅整合来自胆汁酸萌发物的信号，还整合来自氨基酸和钙共刺激物的信号， 萌芽。由于这些意想不到的发现，拟议的研究将解决以下问题：（i） 是C.艰难孢子以及（ii）CspC如何促进 和共同萌发的信号（共）萌发感测仍然不清楚，所以我们对CspC和 胆汁酸结合蛋白的鉴定将为C.艰难 孢子萌发。此外，由于孢子萌发是C.很难引发感染，我们的研究 可能有助于确定预防~ 224，000 C.仅在美国每年就有300万人感染艰难梭菌。 除了增进我们对C.艰难的萌芽，这个项目将为我提供 全面的学术和职业培训。我的赞助商，Aimee Shen博士，致力于教我 技术技能，以完成拟议的实验和学术技能，有效地分享我的工作， 科学界。正如我的培训计划所概述的，我们为写作培训设定了具体目标， 演讲，教学和指导对我实现我的总体职业目标至关重要， 学术研究员职位。沈博士和我召集了一群合作者， 生物化学，晶体学和化学生物学，我将抓住机会学习许多 从他们的实验方法。我将从沈博士和我的合作者那里得到的各种指导 最终将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