Fellowship extension request for F32GM130003 "The Molecular Mechanisms of Spore Germination"

F32GM130003“孢子萌发的分子机制”的研究金延期申请

• 批准号: 10457032
• 负责人: JEREMY David AMON
• 金额: $ 3.52万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10457032
• 关键词: Alanine; Amino Acid Substitution; Anti-Bacterial Agents; Antibiotic Resistance; Bacillus subtilis; Binding; Biochemical; Biological Assay; Cell Wall; Cells; Cleaved cell; Complement; Complex; Coupled; Crystallization; Deacetylation; Detection; Detergents; Endospore-Forming Bacteria; Environmental Monitoring; Enzymes; Excision; Family; Fellowship; Genes; Germination; Growth; Hydration status; Hydrolase; Ligands; Lipoprotein (a); LytA enzyme; Membrane; Membrane Proteins; Metabolic; Modeling; Molecular; Mutagenesis; Nutrient; Operon; Pathogenesis; Pathogenicity; Pathway interactions; Peptidoglycan; Preparation; Process; Proteins; Publishing; Radiolabeled; Recombinants; Reproduction spores; Resistance; Resolution; Set protein; Signal Transduction; Specificity; Starvation; Stress; Structure; Substrate Specificity; Testing; Therapeutic Intervention; Thick; Variant; Water; cell type; dipicolinic acid; experimental study; human pathogen; in vivo; pathogenic bacteria; premature; prevent; programs; protein protein interaction; receptor; response; small molecule; therapy development; transposon sequencing; ultraviolet irradiation; uptake; virtual

PROJECT SUMMARY In response to starvation, bacterial species of the orders Bacillales and Clostridiales differentiate into spores. These stress-resistant cell types are metabolically inactive and can remain dormant for years but rapidly germinate and resume growth upon sensing that proper nutrient conditions have returned. Many of these species are human pathogens, and in their dormant state are highly resistant to antibiotics and can withstand otherwise sterilizing treatments like heat and UV irradiation. To better understand how to prevent pathogenic spore-formers from entering this highly durable state, the molecular mechanisms underlying virtually every step in the sporulation pathway have been characterized. However, the equally important process of germination remains less well understood. A more complete molecular characterization of germination will facilitate the development of treatments that can prevent exit from dormancy or trigger premature germination, leaving cells vulnerable to antibacterial therapies. Most endospore-forming bacteria follow a similar germination program that involves a conserved set of factors. The first step involves environmental monitoring by a large family of putative germinant receptors. In Bacillus subtilis, the prototypical receptor is composed of the products of the gerA operon – GerAA, GerAB, and GerAC. The GerA proteins are required for spore germination in response to L-alanine. How they function and whether or not they act as a nutrient receptor has not been established. Upon sensing germinants, receptors act by an unknown mechanism to release large stores of the small molecule dipicolinic acid (DPA) from the spore core. Water replaces the DPA resulting in partial spore hydration. A conserved membrane complex is required for DPA release but how it is activated is unknown. Finally, conserved cell wall hydrolases packaged in the spore are activated and degrade a thick layer of specialized peptidoglycan known as the spore cortex. Removal of this protective layer allows further core hydration, the onset of metabolic activity, and the resumption of growth. How these enzymes are activated and their substrate specificities remain poorly understood. This proposal seeks to define the molecular underpinning of all three steps in the germination pathway, working from the last temporal step to the first. The specific aims are: Aim 1: Determine the mechanism that activates the cortex-degrading enzyme CwlJ and define its substrate specificity. Aim 2: Investigate the mechanism of germinant detection and signal transduction.

项目概要 为了应对饥饿，芽孢杆菌目和梭菌目细菌种类分化成孢子。 这些抗应激细胞类型的代谢不活跃，可以保持休眠状态数年，但很快 当感觉到适当的营养条件已经恢复时，发芽并恢复生长。其中许多 物种是人类病原体，在休眠状态下对抗生素具有高度抵抗力，可以承受 其他灭菌处理，如加热和紫外线照射。为了更好地了解如何预防病原体 孢子形成体进入这种高度持久的状态，几乎每一步的分子机制 已对孢子形成途径进行了表征。然而，同样重要的发芽过程 仍然不太了解。更完整的发芽分子特征将有助于 开发可以防止退出休眠或触发过早发芽的治疗方法，使细胞 容易受到抗菌治疗的影响。 大多数内生孢子形成细菌遵循类似的萌发程序，其中涉及一组保守的因素。 第一步涉及通过一大群推定的萌芽受体进行环境监测。在芽孢杆菌中 枯草芽孢杆菌的原型受体由 gerA 操纵子的产物组成——GerAA、GerAB 和 杰拉克。 GerA 蛋白是孢子萌发响应 L-丙氨酸所必需的。它们如何运作以及 它们是否充当营养受体尚未确定。在感应到萌芽后，受体 通过未知机制作用，从细胞中释放大量小分子吡啶二羧酸 (DPA) 孢子核心。水取代了 DPA，导致孢子部分水合。保守的膜复合物是 DPA 发布所需，但如何激活尚不清楚。最后，包装保守的细胞壁水解酶 孢子中的肽被激活并降解一层厚厚的特殊肽聚糖，称为孢子皮层。 去除该保护层可以进一步实现核心水合作用、代谢活动的开始以及 恢复增长。这些酶是如何被激活的，其底物特异性仍然很差 明白了。该提案旨在定义发芽过程中所有三个步骤的分子基础 路径，从最后一个时间步骤到第一个时间步骤。具体目标是： 目标 1：确定激活皮质降解酶 CwlJ 的机制并定义其 底物特异性。 目标 2：研究萌发检测和信号转导的机制。

项目成果

The SpoVA membrane complex is required for dipicolinic acid import during sporulation and export during germination.

• DOI: 10.1101/gad.349488.122
• 发表时间: 2022-05-01
• 期刊: GENES & DEVELOPMENT
• 影响因子: 10.5
• 作者: Gao, Yongqiang;Barajas-Ornelas, Rocio Del Carmen;Amon, Jeremy D.;Ramirez-Guadiana, Fernando H.;Alon, Assaf;Brock, Kelly P.;Marks, Debora S.;Kruse, Andrew C.;Rudner, David Z.
• 通讯作者: Rudner, David Z.