F. tularensis Virulence Protein Structure and Function

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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.