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.

项目概要/摘要 芽孢杆菌目和梭状芽胞杆菌目中的细菌每年引起超过一百万次感染，并对此负责 给食品工业带来巨大的金钱损失。这些细菌可以通过进入来抵抗抗生素和杀菌。 高度耐用的孢子状态。孢子的代谢不活跃，可以休眠数十年，但 接触营养物质后，它们会迅速恢复营养生长并导致食物腐败、食源性疾病或 危及生命的疾病。摆脱休眠、发芽是解决这些疾病的关键目标。 大多数孢子形成细菌的萌发程序涉及一系列常见的化学步骤和 一小组高度保守的因素。嵌入孢子膜中的 GerA 家族受体是 营养感应。萌芽体的存在会触发孢子核心释放单价阳离子， 随后迅速排出大量吡啶二羧酸（DPA），这可能是由假定的介导的 由 spoVA (5A) 操纵子编码的转运蛋白复合物。这会激活细胞壁水解酶，从而降解 特殊的孢子皮层肽聚糖，允许孢子核心再水化、大分子合成和 恢复增长。每个步骤背后的机制几乎完全未知。 我们寻求从分子角度定义发芽信号转导途径，采取综合的方法 结合遗传、生化、计算和结构方法的方法。目标是： (1)阐明营养物质检测和信号转导机制。我们将决定如何GerA- 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 假设萌发受体寡聚化形成膜孔，充当配体- 响应营养物质释放单价阳离子的门控离子通道。 (2)确定DPA从孢子核心释放的机制。我们将研究两个模型 由 5A 位点编码的亚基形成膜通道，第三个成分起到胞质塞的作用 使通道保持关闭状态。我们将测试该复合体运输 DPA 并被激活的模型 阳离子释放。 如果成功，拟议的工作将为孢子如何检测营养物质、触发离子提供分子水平的见解 发布并激活 DPA 的导出，提供发现所需的机制和结构框架 和发芽途径小分子调节剂的优化。我们的工作将促进发展 不恰当地诱导发芽，使细胞容易受到标准抗菌药物的影响 治疗或阻断它，直接预防疾病。