Mechanisms of Clostridioides difficile germinant and co-germinant sensing

艰难梭菌萌发和共萌发传感机制

基本信息

批准号：
10320344
负责人：
Emily Rachel Forster
金额：
$ 4.18万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10320344
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/antagonist innovation insight light scattering molecular mass novel novel strategies pathogen prevent receptor response sensor skills small molecule stoichiometry sugar

项目摘要

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.

项目概要/摘要发芽对于形成孢子的专性厌氧菌（如医院病原体）的生命周期至关重要，艰难梭菌。尽管这个过程很重要，但细菌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，艰难梭菌孢子感知和转导萌发信号的机制是独特的在孢子形成细菌中，因为 (i) 艰难梭菌孢子对胆汁酸萌发剂而不是胆汁酸萌发剂做出反应迄今为止研究的所有孢子形成体所使用的规范营养萌发剂（例如糖），以及 (ii) 艰难梭菌缺乏由几乎所有其他孢子形成体编码的跨膜萌发受体。相反，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导致发芽的级联。我们最近解决了这种假定的萌芽受体的结构。我们后续的结构-功能分析挑战了 CspC 直接感知胆汁酸起始物的流行模型，并令人惊讶地发现 CspC 不仅整合来自胆汁酸起始物的信号，还整合来自氨基酸和钙共体的信号萌芽。由于这些意外的发现，拟议的研究将解决以下问题：(i) 艰难梭菌孢子中的胆汁酸萌芽体是直接传感器吗？ (ii) CspC 如何转导萌芽和共同萌芽信号？（共）萌发传感仍然没有明确定义，因此我们对 CspC 和胆汁酸结合蛋白的鉴定将为艰难梭菌的机制提供分子洞察孢子发芽。此外，由于孢子萌发对于艰难梭菌发起感染至关重要，我们的研究仅在美国，可能有助于确定每年预防约 224,000 例艰难梭菌感染的新策略。除了加深我们对艰难梭菌萌发的理解之外，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 Aimee Shen 博士致力于教我完成拟议实验的技术技能和有效分享我的工作的学术技能科学界。正如我的培训计划中所述，我们以书面形式设定了具体的培训目标，演讲、教学和指导对于我实现持有学位的总体职业目标至关重要学术研究教职。沉博士和我组建了一群具有以下专业知识的合作者生物化学、晶体学、化学生物学，我会抓住机会学习很多他们的实验方法。我将从沉博士和我的合作者那里得到多样化的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人研究员。