Structure of the Yersinia pestis type III export complex

鼠疫耶尔森氏菌 III 型输出复合体的结构

• 批准号: 6414649
• 负责人: Gregory V Plano
• 金额: $ 25.51万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6414649
• 关键词: Yersinia pestis; bacteria infection mechanism; bacterial proteins; bioterrorism /chemical warfare; immunoprecipitation; microorganism culture; protein protein interaction; protein purification; protein structure function; scanning electron microscopy; transmission electron microscopy; yeast two hybrid system

DESCRIPTION (provided by applicant): The use of aerosolized Yersinia pestis as a biological weapon could potentially produce massive numbers of casualties. Weaponized plague could be rendered resistant to most antibiotics and delivered via an aerosol. Thus, in order to protect the military and civilian populations from both endemic and recombinant Y. pestis, new biological countermeasures must be developed that target essential pathogen vulnerabilities. The Y. pestis type III secretion (TTS) system enables the bacterium to subvert or destroy eukaryotic cells via the delivery of anti-host effector proteins. Disruption of the TTS process renders Y. pestis avirulent, indicating that the TTS process represents an attractive target for therapeutic intervention. Assembly of the TTS apparatus requires the participation of at least 21 Ysc (Yersinis secretion) proteins, Ysc proteins are hypothesized to assemble into a supramolecular structure that functions as a protein secretion and injection device. We propose to investigate the assembly and structure of the TTS apparatus. In Specific Aim 1, experiments are described to identify the protein interactions involved in the assembly of the TTS complex. Immunoprecipitation methodologies and yeast two- and three- hybrid studies will identify Ysc proteins that directly interact with one another. The overall objective of Specific Aim 2 is to establish conditions for the solubilization, purification and visualization of the assembled or partially assembled TTS complex. Environmental scanning electron microscopy (ESEM) of fixed samples and/or transmission electron microscopy (TEM) of negatively-stained samples will be used to visualize the surface-exposed and/or isolated TTS complex and/or partially assembled intermediates of this complex. In Specific Aim 3, experiments designed to identify and characterize chromosomally-encoded proteins that are required for the assembly and function of the TTS complex are described. Completion of these Specific Aims will not only provide essential information about the assembly and function of the Y. pestis TTS complex, but may also facilitate the development and/or design of novel therapeutics that target this essential virulence system.

描述（由申请人提供）：雾化鼠疫耶尔森氏菌作为疫苗的用途 生物武器可能会造成大量人员伤亡。 武器化的瘟疫可能会对大多数抗生素产生耐药性 通过气雾剂输送。因此，为了保护军人和平民 来自地方性和重组鼠疫耶尔森氏菌的种群，新生物 必须制定针对主要病原体的对策 漏洞。鼠疫耶尔森氏菌 III 型分泌 (TTS) 系统能够 细菌通过递送抗宿主来颠覆或破坏真核细胞 效应蛋白。 TTS 过程的破坏使鼠疫耶尔森氏菌无毒， 表明 TTS 过程是一个有吸引力的目标 治疗干预。 TTS 设备的组装需要 至少21种Ysc（耶尔森氏菌分泌）蛋白的参与，Ysc蛋白 假设组装成超分子结构，其功能如下 蛋白质分泌和注射装置。我们建议调查 TTS装置的组装和结构。在具体目标 1 中，实验 被描述为鉴定参与组装的蛋白质相互作用 TTS 复合体。免疫沉淀方法和酵母二和三 杂交研究将鉴定出彼此直接相互作用的 Ysc 蛋白。 具体目标 2 的总体目标是为溶解创造条件， 组装或部分组装的 TTS 的纯化和可视化 复杂的。固定样品的环境扫描电子显微镜 (ESEM) 和/或负染色样品的透射电子显微镜 (TEM) 将用于可视化表面暴露和/或隔离的 TTS 复合体 和/或该复合物的部分组装的中间体。在具体目标 3 中， 旨在识别和表征染色体编码蛋白质的实验 描述了 TTS 复合体的组装和功能所需的信息。 完成这些具体目标不仅会提供有关 鼠疫耶尔森氏菌 TTS 复合体的组装和功能，但也可能促进 针对这一基本要素的新型疗法的开发和/或设计 毒力系统。