Molecular basis of spore germination

孢子萌发的分子基础

• 批准号: 10659224
• 负责人: Andrew Kruse
• 金额: $ 84.72万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-05 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659224
• 关键词: Address; Alanine; Amino Acid Substitution; Amino Acids; Anti-Bacterial Agents; Antibiotics; Bacillus megaterium; Bacillus subtilis; Bacteria; Behavior; Binding; Biochemical; Cations; Cell Wall; Cells; Chemicals; Clostridium difficile; Complex; Cytology; Data; Detection; Disease; Exposure to; Family; Food; Food Industry; Genetic; Germination; Glucose; Growth; Hydration status; Hydrolase; Infection; Ion Channel; Ion Channel Gating; Ions; Leucine; Life; Ligand Binding; Ligands; Lipid Bilayers; Mediating; Membrane; Metabolic; Methods; Modeling; Molecular; Monovalent Cations; Mutagenesis; Nucleosides; Nutrient; Nutritional Requirements; Operon; Pathway interactions; Peptidoglycan; Proline; Proteins; Reproduction spores; Resolution; Series; Signal Transduction; Signal Transduction Pathway; Specificity; Sterilization; Structural Models; Structure; Testing; Variant; Work; crosslink; detection of nutrient; dipicolinic acid; foodborne illness; in vivo; insight; pathogen; prevent; programs; protein complex; receptor; response; small molecule; sugar; therapy development

PROJECT SUMMARY/ABSTRACT Bacteria in the orders Bacillales and Clostridiales cause over a million infections each year and are responsible for huge monetary losses to the food industry. These bacteria can resist antibiotics and sterilization by entering a highly durable spore state. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients they rapidly resume vegetative growth and cause food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, germination, is a key target in addressing these diseases. The germination program of most spore-forming bacteria involves a common series of chemical steps and a small set of highly conserved factors. GerA-family receptors embedded in the spore membrane are required for nutrient sensing. The presence of germinants triggers the release of monovalent cations from the spore core, which is rapidly followed by the expulsion of large stores of dipicolinic acid (DPA) likely mediated by a putative transporter complex encoded by the spoVA (5A) operon. This activates cell wall hydrolases that degrade the specialized spore cortex peptidoglycan, allowing rehydration of the spore core, macromolecular synthesis, and resumption of growth. The mechanisms behind each of these steps are almost entirely unknown. We seek to define the germination signal transduction pathway in molecular terms, taking an integrative approach that combines genetic, biochemical, computational, and structural methods. The aims are: (1) Elucidate the mechanisms of nutrient detection and signal transduction. We will determine how GerA- family receptors detect amino acids, sugars, and inorganic cations to trigger germination. We will test the hypothesis that the germination receptors oligomerize forming a membrane pore that functions as a ligand- gated ion channel that releases monovalent cations in response to nutrients. (2) Determine the mechanism of DPA release from the spore core. We will investigate the model that two subunits encoded by the 5A locus form a membrane channel and a third component functions a cytosolic plug that keeps the channel closed. We will test the model that this complex transports DPA and is activated by cation release. If successful, the proposed work will provide molecular-level insight into how spores detect nutrients, trigger ion release, and activate export of DPA, providing the mechanistic and structural framework needed for discovery and optimization of small molecule modulators of the germination pathway. Our work will enable the development of treatments that either inappropriately induce germination, leaving cells vulnerable to standard antibacterial therapies, or block it, directly preventing disease.

项目摘要/摘要 命令中的细菌每年会引起超过一百万个感染，并负责 对于食品行业的巨大货币损失。这些细菌可以通过进入 高度耐用的孢子状态。孢子在代谢上是非活性的，几十年来可能保持休眠状态 暴露于营养素中，他们迅速恢复营养生长，并导致食物变质，食物疾病或 威胁生命的疾病。从休眠，发芽中退出是解决这些疾病的关键目标。 大多数形成孢子的细菌的发芽计划涉及一系列常见的化学步骤和A 一组高度保守的因素。需要嵌入孢子膜中的GERA家庭受体 营养感应。发芽剂的存在触发了从孢子核中释放单价阳离子， 迅速驱逐大型二吡啶酸（DPA）可能是由推定的 由Spova（5A）操纵子编码的转运蛋白配合物。这激活了降解的细胞壁水解酶 专门的孢子皮质肽聚糖，允许孢子核，大分子合成和 恢复生长。这些步骤中每个步骤背后的机制几乎完全未知。 我们试图用分子术语定义发芽信号转导途径，采用综合性 结合遗传，生化，计算和结构方法的方法。目的是： （1）阐明营养检测和信号转导的机制。我们将确定如何 家族受体检测氨基酸，糖和无机阳离子以触发发芽。我们将测试 假设发芽受体使形成膜孔的偏成亲聚体，该膜起着配体的作用 封闭的离子通道，该通道响应养分而释放单价阳离子。 （2）确定从孢子芯释放DPA的机理。我们将研究两个模型 由5A基因座编码的亚基形成膜通道，第三个组件功能胞质塞 这使通道关闭。我们将测试该复合物传输DPA并被激活的模型 阳离子释放。 如果成功，建议的工作将为孢子如何检测营养，触发离子提供分子水平的见解 释放并激活DPA的出口，提供发现所需的机械和结构框架 以及优化发芽途径的小分子调节剂。我们的工作将使发展 不适当地诱导发芽的治疗方法，使细胞容易受到标准抗菌的影响 疗法或阻止它直接预防疾病。