Stabilization and regulation of a Bacillus anthracis spore lytic enzyme

炭疽芽孢杆菌孢子裂解酶的稳定和调节

• 批准号: 8623183
• 负责人: DAVID L POPHAM
• 金额: $ 22.36万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8623183
• 关键词: Address; Bacillus anthracis; Bacillus anthracis spore; Bacteria; Bacterial Spores; Biochemical; Biochemical Genetics; Biological Assay; Bypass; Chemicals; Chimeric Proteins; Commit; Communicable Diseases; Complement; Complex; Data; Decontamination; Disease; Effectiveness; Environment; Enzymes; Event; Food; Genes; Genetic; Genetic Screening; Germination; Goals; Homology Modeling; Hydrolysis; Immune Sera; Industrial Product; Infectious Agent; Knowledge; Left; LytA enzyme; Lytic; Methods; Modeling; Mutagenesis; Mutation; Organism; Peptide Hydrolases; Peptides; Peptidoglycan; Pharmaceutical Preparations; Pharmacologic Substance; Play; Poisoning; Population; Process; Production; Property; Protein Region; Proteins; Regulation; Reproduction spores; Resistance; Role; Signal Pathway; Site; Structure; Technology; Vaccines; Variant; Work; X-Ray Crystallography; antimicrobial; cell type; crosslink; his6 tag; improved; in vivo; innovation; killings; prevent; protein complex; protein function; public health relevance; structural biology

Elimination of spores of Bacillus anthracis and other disease-causing bacteria from contaminated sites or materials is challenging due to the extreme resistance of spores to conventional antimicrobial treatments. Activation of the B. anthracis spore cortex peptidoglycan lytic machinery results in rapid loss of spore dormancy and resistance properties. This machinery can thus be targeted for germination-activating and spore-killing treatments. The cortex lytic enzyme SleB is stored in an inactive and highly stable form in the dormant spore and becomes active early during the germination process. The YpeB protein is required for incorporation of SleB into the dormant spore, and during germination, proteolytic cleavage of YpeB releases SleB to depolymerize the cortex. Aim 1 of the proposed work includes biochemical, genetic, and structural biology methods to reveal protein-protein contacts formed by YpeB and SleB in the dormant spore. Protein complexes containing YpeB or SleB will be purified from spores and co-purified proteins will be identified. Altered forms of YpeB will be expressed in vivo to identify key regions of the protein involved in function and protein interaction. The YpeB structure will be determined using X-ray crystallography in order to understand the effects of mutations. This work will clarify the mechanism of SleB stabilization, which may be adaptable to the production of stable protein pharmaceuticals, vaccines, and other industrial products. Aim 2 is the identification of the protease that processes YpeB and thus activates SleB. Knowledge of this protease and its regulation will suggest methods for the activation of germination via YpeB and SleB. The proposed work will contribute to two major translational goals: Activation of spore germination in order to improve decontamination and understanding of a mechanism of extreme protein stabilization.

消除炭疽杆菌和其他致病细菌的孢子 由于孢子具有极强的抵抗力，污染场地或材料具有挑战性 常规抗菌治疗。炭疽芽孢杆菌孢子皮层肽聚糖的激活 裂解机制导致孢子休眠和抗性特性迅速丧失。这 因此，机械可以用于发芽激活和孢子杀死处理。这 皮质裂解酶 SleB 以非活性且高度稳定的形式储存在休眠孢子中， 在发芽过程的早期就变得活跃。 YpeB 蛋白是必需的 SleB 并入休眠孢子，在萌发过程中，蛋白水解裂解 YpeB 释放 SleB 来解聚皮质。拟议工作的目标 1 包括 生物化学、遗传和结构生物学方法揭示蛋白质-蛋白质接触形成 由休眠孢子中的 YpeB 和 SleB 产生。含有 YpeB 或 SleB 的蛋白质复合物将 从孢子中纯化的产物和共纯化的蛋白质将被鉴定。 YpeB 的改变形式将是 体内表达以识别参与功能和蛋白质的蛋白质的关键区域 相互作用。 YpeB 结构将使用 X 射线晶体学测定，以便 了解突变的影响。这项工作将阐明 SleB 稳定的机制， 它可能适用于生产稳定的蛋白质药物、疫苗和 其他工业产品。目标 2 是鉴定处理 YpeB 和 从而激活SleB。对这种蛋白酶及其调节的了解将为我们提供以下方法： 通过 YpeB 和 SleB 激活发芽。拟议的工作将有助于两个主要方面 转化目标：激活孢子萌发以改善净化和 了解极端蛋白质稳定的机制。