Structure of the Yersinia pestis type III export complex

描述(由申请人提供)：使用雾化鼠疫耶尔森氏菌作为 生物武器可能会造成大量人员伤亡。 武器化的鼠疫可以对大多数抗生素产生抗药性 通过气雾剂传播。因此，为了保护军民 来自地方性和重组鼠疫杆菌的种群，新的生物学 必须制定针对主要病原体的对策 漏洞。鼠疫杆菌III型分泌物(TTS)系统使 细菌通过递送抗宿主来颠覆或摧毁真核细胞 效应器蛋白。TTS过程的中断使鼠疫杆菌变得无毒， 表明TTS流程是一个有吸引力的目标 治疗性干预。TTS设备的组装需要 至少21个YSC(耶尔森氏菌分泌物)蛋白的参与，YSC蛋白 假设组装成一种超分子结构，其功能是 一种蛋白质分泌注射装置。我们建议调查 TTS装置的装配和结构。在具体目标1中，实验 以确定在组装过程中涉及的蛋白质相互作用 TTS综合体。免疫沉淀法和酵母菌二三- 杂交研究将确定直接相互作用的YSC蛋白质。 具体目标2的总体目标是为增溶创造条件， 组装或部分组装的TTS的提纯和可视化 很复杂。固定样品的环境扫描电子显微镜(ESEM) 和/或负染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/或该复合体的部分组装的中间体。在具体目标3中， 用于鉴定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组件。 完成这些具体目标不仅将提供有关 鼠疫杆菌TTS复合体的组装和功能，但也可能促进 开发和/或设计以此为主要目标的新疗法 毒力系统。

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.