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.

项目总结/摘要 芽孢杆菌目和梭菌目的细菌每年造成超过一百万例感染， 造成食品行业巨大的金钱损失。这些细菌可以通过进入 一种非常持久的孢子状态孢子在新陈代谢上是不活跃的，可以休眠几十年， 暴露于营养物质后，它们迅速恢复营养生长，并导致食物腐败，食源性疾病，或 危及生命的疾病。从休眠中退出，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包括一系列共同的化学步骤和一个 一小部分高度保守的因子。GerA家族受体嵌入孢子膜中， 养分传感萌发剂的存在触发了一价阳离子从孢子核的释放， 随后迅速排出大量储存的吡啶二羧酸（DPA），这可能是由假定的 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， 特殊的孢子皮层肽聚糖，允许孢子核心的再水化，大分子合成， 恢复增长。这些步骤背后的机制几乎完全未知。 我们试图在分子水平上定义发芽信号转导途径， 结合遗传学、生物化学、计算和结构方法的方法。其目标是： (1)阐明了营养物质检测和信号转导的机制。我们将决定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作为配体的膜孔- 门控离子通道，释放一价阳离子以响应营养物。 (2)确定DPA从孢子核释放的机制。我们将研究两个模型 由5A基因座编码的亚基形成膜通道，第三组分起胞质塞的作用 保持频道关闭我们将测试该复合体传输DPA并被激活的模型 阳离子释放。 如果成功的话，这项工作将提供分子水平的见解，了解孢子如何检测营养物质，触发离子， 发布和激活DPA的导出，提供发现所需的机制和结构框架 和萌发途径的小分子调节剂的优化。我们的工作将使发展 不适当地诱导发芽，使细胞容易受到标准抗菌剂的影响， 治疗，或阻止它，直接预防疾病。