Structural Studies of the MPT64 Family of Mycobacterial Virulence Factors

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DESCRIPTION (provided by applicant): The MPT64 protein and its homologs form a highly conserved family of secreted proteins found within the pathogenic Mycobacteria genus. The founding member of this family from Mycobacterium tuberculosis (MPT64, protein Rv1980c) is expressed only when the Mycobacteria cells are actively dividing. By virtue of this relatively unique expression profile, Rv1980c is currently under phase III clinical trials in South Africa and Peru to evaluate its potential to replace tuberculin, or purified protein derivative, as a benchmark diagnostic for active Tuberculosis infection. Despite numerous NIH-sponsored initiatives supporting Tuberculosis research and the potential importance of Rv1980c to the improved diagnosis of the single most widespread bacterial infectious disease in the world, traditional genetic and biochemical approaches have failed to provide any explanation as to the biological role(s) of Rv1980c or any other members of this family of proteins. To address this need, we propose the use of state of the art methods in structural biology to provide a better understanding of the biological roles of the MPT64 family through the following specific aims: Specific Aim 1: To determine an ensemble of high-resolution solution structures for Rv1980c by heteronuclear NMR spectroscopy. Specific Aim 2: To investigate the areas of structural similarity and divergence within the MPT64 family by solving the high-resolution crystal structure of MAP0087, 1of 2 family members found in the genome of Mycobacterium avium subsp. paratuberculosis (Map). Specific Aim 3: To use heteronuclear NMR to compare the structural and dynamic properties of wild type, disulfide-linked Rv1980c with a non-linked mutant protein to provide insights into the mechanism of Rv1980c skin barrier transit. The results of Aims 1 and 2 will be correlated and used assign likely biological roles to MPT64 family members by virtue of structural homology to proteins of known function; this will further our understanding of these proteins in the complex pathogenesis of M. tuberculosis and related organisms. The results of Aim 3 will provide mechanistic insight into Rv1980c skin barrier transit and may suggest approaches to improving this promising diagnostic agent. The studies proposed here have the potential to impact both the treatment and diagnosis of widespread Mycobacterial disease, and therefore lie within the mission of the agency.