F. tularensis Virulence Protein Structure and Function

F. tularensis 毒力蛋白结构和功能

基本信息

批准号：
7314377
负责人：
Karl E Klose
金额：
$ 19.01万
依托单位：
UNIVERSITY OF TEXAS SAN ANTONIO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7314377
关键词：
Affinity Amino Acids Applications Grants Biochemistry Biomolecular Nuclear Magnetic Resonance Categories Cell Death Induction Collaborations Complement Country Data Depth Development Disease Dose Exploratory/Developmental Grant Francisella Francisella tularensis Funding Future Genetic Screening Human Immunoprecipitation Individual Knowledge Laboratories Measures NMR Spectroscopy Nature Organism Pathogenesis Phagolysosome Phagosomes Play Precipitation Preventive Protein Overexpression Proteins Research Risk Roentgen Rays Role Role playing therapy Solutions Structure Subunit Vaccines Tularemia United States National Institutes of Health Vaccines Virulence Virulence Factors X ray diffraction analysis X-Ray Diffraction bacterial genetics insight killings macrophage mortality novel therapeutics programs protein structure function structural biology therapeutic vaccine three dimensional structure tool transmission process

项目摘要

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.

描述（由申请人提供）：土拉弗朗西斯菌被认为是 A 类生物武器，因为它易于传播，感染剂量低，与肺炎土拉热病相关的死亡率高，而且它已在多个国家的生物武器计划中得到深入研究和开发。国家。人们对兔热病的发病机制知之甚少，这主要是由于缺乏对兔热病的研究。 Klose 实验室已鉴定出两种土拉弗拉菌基因产物：IglC 和 IglD，它们对于毒力至关重要。这些蛋白质与任何其他已知蛋白质没有显着的同源性，目前尚不清楚它们的确切功能。然而，它们在逃避吞噬溶酶体融合、吞噬体逃逸和诱导受感染巨噬细胞内的细胞死亡方面发挥作用，这些特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 Hart 实验室之间的合作项目，旨在研究 F. tularensis IglC 和 IglD 的结构和功能。在第一个具体目标中，将确定土拉弗拉菌 IglC 和 IglD 的三维结构。 IglC 和 IglD 蛋白将被过表达、纯化，并使用成熟的单晶 X 射线衍射工具确定其结构，并辅以 NMR 光谱。该目标将由 UTHSCSA 的 Hart 实验室执行，目标 2 的结果将在出现时纳入该目标。在第二个具体目标中，将通过细菌遗传学和生物化学的结合来表征 F. tularensis IglC 和 IglD 的功能。利用 GSK 标记的蛋白质，将确定受感染巨噬细胞内 IglC 和 IglD 的分泌性质。 IglC 和 IglD 功能所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 Klose 实验室执行，目标 1 的结果将在出现时纳入此目标中。这些研究将极大地增加我们对这种潜在生物武器及其发病机制的了解。研究结果将有助于开发新型疗法和亚单位疫苗。人们对土拉弗朗西斯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种土拉弗拉菌基因产物：IglC 和 IglD，它们对于毒力至关重要。这些研究将极大地增加我们对这种生物体及其致病方式的了解。研究结果将有助于开发新型疗法和亚单位疫苗。