F. tularensis Virulence Protein Structure and Function

描述(申请人提供)：由于图拉氏方济氏菌易于传播，与肺炎图拉热症相关的低感染剂量和高死亡率，以及它在几个国家的生物武器计划中得到了深入的研究和开发，因此被认为是A类生物武器。关于图拉热症的发病机制知之甚少，主要是由于缺乏对图拉氏菌的研究。克洛泽实验室已经确定了图拉氏F菌的两种基因产物，IglC和IglD，它们是毒力所必需的。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，它们在逃避吞噬小体融合、吞噬小体逃逸和诱导受感染的巨噬细胞内的细胞死亡方面发挥作用，这些属性是方济各氏菌致病能力的关键。该项目是UTSA的克洛泽实验室和UTHSCSA的HART实验室合作开展的，旨在研究F.tularensis IglC和IglD的结构和功能。 在第一个具体目标中，我们将确定图拉夫酵母IglC和IglD的三维结构。IglC和IglD蛋白将被过度表达、纯化，并使用成熟的单晶X射线衍射工具和核磁共振光谱来确定它们的结构。这一目标将由UTHSCSA的HART实验室执行，目标2的结果将在出现时纳入这一目标。在第二个具体目标中，将通过细菌遗传学和生物化学相结合的方法来表征图拉氏F菌IglC和IglD的功能。利用GSK标记的蛋白质，将在感染的巨噬细胞中确定IglC和IglD的分泌性质。IglC和IglD功能所需的氨基酸残基将通过基因筛查进行鉴定。这些蛋白与其他细菌和/或宿主蛋白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。 这些研究将极大地增加我们对这种潜在的生物武器及其发病机制的了解。这一结果将有助于开发新的疗法和亚单位疫苗。 关于图拉方济氏菌是如何致病的，人们知之甚少，这阻碍了针对这种潜在生物武器的疗法和疫苗的开发。这些研究的重点是图拉氏福氏杆菌的两个基因产物，IglC和IglD，它们是毒力所必需的。这些研究将极大地增加我们对这种有机体及其如何导致疾病的了解。这一结果将有助于开发新的疗法和亚单位疫苗。

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.