F. tularensis Virulence Protein Structure and Function

描述（由申请人提供）：土拉热弗朗西丝菌被认为是A类生物武器，因为它易于传播，与肺炎土拉菌病相关的感染剂量低，死亡率高，并且在几个国家的生物武器计划中已经进行了深入研究和开发。目前对兔热病的发病机制知之甚少，主要是由于缺乏F。tularensis研究。克洛泽实验室已经确定了两个F。土拉热菌基因产物，IglC和IglD是毒力所必需的。这些蛋白质与任何其他已知蛋白质没有显著的同源性，它们的确切功能目前尚不清楚。然而，它们在逃避吞噬溶酶体融合、吞噬体逃逸和诱导感染的巨噬细胞内的细胞死亡中发挥作用，这些属性对于弗朗西斯菌引起疾病的能力至关重要。该项目是UTSA的Klose实验室和UTHSCSA的哈特实验室之间的合作奋进，以研究F.土拉热菌IglC和IglD。 在第一个具体目标中，F.将测定土拉热菌IglC和IglD。IglC和IglD蛋白将被过表达、纯化，并使用单晶X射线衍射的完善工具，辅以NMR光谱来确定其结构。该目标将由UTHSCSA的哈特实验室执行，目标2的结果将在出现时纳入该目标。在第二个具体目标中，F.土拉热菌IglC和IglD将通过细菌遗传学和生物化学的组合来表征。利用GSK标记的蛋白质，将在感染的巨噬细胞内确定IglC和IglD的分泌性质。IglC和IglD功能所必需的氨基酸残基将通过遗传筛选来鉴定。这些蛋白质与其他细菌和/或宿主蛋白质之间的相互作用将通过免疫沉淀来确定。这一具体目标将由UTSA的克洛泽实验室执行，目标1的结果将在出现时纳入这一目标。 这些研究将大大增加我们对这种潜在生物武器及其发病机制的了解。这些结果将有助于开发新的治疗方法和亚单位疫苗。 关于土拉热弗朗西丝菌如何引起疾病知之甚少，这阻碍了针对这种潜在生物武器的治疗方法和疫苗的开发。这些研究的重点是两个F。土拉热菌基因产物IglC和IglD对毒力至关重要。这些研究将大大增加我们对这种生物体及其如何引起疾病的了解。这些结果将有助于开发新的治疗方法和亚单位疫苗。

DESCRIPTION (provided by applicant): Francisella tularensis is considered a Category A bioweapon due to the ease of transmission, the low infectious dose and high mortality associated with pneumonic tularemia, and the fact that it has been intensively studied and developed in bioweapons programs in several countries. Very little is known about the pathogenesis of tularemia, primarily due to lack of F. tularensis research. The Klose laboratory has identified two F. tularensis gene products, IglC and IglD that are essential for virulence. These proteins have no significant homology with any other known proteins, and their exact function is currently unknown. However, they play roles in evasion of phagolysosome fusion, phagosome escape, and induction of cell death within infected macrophages, attributes that are critical for Francisella's ability to cause disease. This project is a collaborative endeavor between the Klose laboratory at UTSA and the Hart laboratory at UTHSCSA to study the structure and function of F. tularensis IglC and IglD. In the first specific aim, the three-dimensional structures of F. tularensis IglC and IglD will be determined. The IglC and IglD proteins will be overexpressed, purified, and their structures determined using the well established tools of single crystal X-ray diffraction, supplemented by NMR spectroscopy. This aim will be performed by the Hart laboratory at UTHSCSA, and the results of Aim 2 will be incorporated into this aim as they arise. In the second specific aim, the functions of F. tularensis IglC and IglD will be characterized through a combination of bacterial genetics and biochemistry. Utilizing GSK-tagged proteins, the secreted nature of IglC and IglD will be determined within infected macrophages. Amino acid residues necessary for the functions of IglC and IglD will be identified by a genetic screen. Interactions between these proteins and other bacterial and/or host proteins will be determined by immunoprecipitation. This specific aim will be performed by the Klose laboratory at UTSA, and the results of Aim 1 will be incorporated into this aim as they arise. These studies will dramatically increase our knowledge of this potential bioweapon and its mechanism of pathogenesis. The results will assist in the development of novel therapeutics and subunit vaccines. Very little is known about how Francisella tularensis causes disease, which has hampered the development of therapeutics and vaccines against this potential bioweapon. The focus of these studies is two F. tularensis gene products, IglC and IglD, that are essential for virulence. These studies will dramatically increase our knowledge of this organism and how it causes disease. The results will assist in the development of novel therapeutics and subunit vaccines.