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 生物武器有可能造成大量伤亡。 鼠疫菌可以对大多数抗生素产生抗药性， 通过气雾剂输送。因此，为了保护军队和平民， 来自地方性和重组Y.鼠疫，新生物 必须制定针对主要病原体的对策 漏洞鼠疫耶尔森鼠疫III型分泌（TTS）系统使 细菌通过递送抗宿主药物来破坏或破坏真核细胞 效应蛋白TTS过程的中断使Y。鼠疫无毒， 这表明TTS过程代表了一个有吸引力的目标， 治疗干预TTS装置的组装需要 参与至少21种Ysc（耶尔森氏菌分泌）蛋白，Ysc蛋白 被假设为组装成超分子结构， 一种蛋白质分泌注射装置，我们建议调查 TTS装置的组装和结构。具体目标1： 被描述为识别参与组装的蛋白质相互作用， TTS综合体免疫沉淀法和酵母二和三- 杂交研究将鉴定直接相互作用的Ysc蛋白。 具体目标2的总体目标是建立增溶条件， 组装或部分组装的TTS的纯化和可视化 复杂.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/或分离的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， 实验旨在 鉴定和表征染色体编码蛋白质 描述了TTS复合体的组装和功能所需的材料。 这些具体目标的完成不仅将提供有关 Y.鼠疫TTS复合物，但也可能促进 开发和/或设计靶向这种必需的新疗法， 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.