Tuberculosis is the leading cause of death in the world from a single infectious agent, and is responsible for more than 3 million deaths annually. The high mortality rate in individuals infected with Mycobacterium tuberculosis is due in part to its ability to parasitize macrophages and establish long-term, persistent infection in the host despite cell-mediated immunity. Although the current anti-tubercular drug arsenal is effective in treating individuals suffering from active disease, these drugs are ineffective in treating the 2 billion people that currently suffer from latent tuberculosis, or that are infected with multi-drug resistant strains of M. tuberculosis. One group of transcriptional regulatory determinants that may play a critical role in processes associated with M. tuberculosis latency is the two-component signal transduction systems. These systems mediate adaptation processes and have been shown to contribute to virulence and disease elicitation in other organisms. The goals of this study are to characterize further a two-component system of M. tuberculosis (MprA-MprB) that is required for the establishment and maintenance of persistent infection. In this proposal, we plan to: (i) Identify and characterize the genes regulated by the MprA transcription factor. The genes regulated by MprA will be identified from the M. tuberculosis chromosome using biochemical enrichment and genetic selection techniques, and will be characterized by gene inactivation, promoter expression analysis, and evaluation in model systems for infection. (ii) Analyze the in vivo expression profile of the MprA response regulator, and the genes regulated by MprA. This will be accomplished by expression analysis of these genes using GFP reporter technology, primer extension analysis, molecular beacon technology, and DNA microarray based analysis under physiologically relevant conditions. (iii) Delineate the effects of MprA de-regulation on host-pathogen interactions. This will be accomplished by examining effects of MprA loss or overexpression on M. tuberculosis virulence. Virulence studies will include bacterial survival and cytokine expression analysis as assayed in in vitro tissue culture systems and animal model systems of infection. These studies will also address the effects of MprA de-regulation on Mycobacterium bovis BCG attenuation. The proposal outlined here is expected to improve our understanding of genes required by M. tuberculosis for pathogenesis, and help better define the conditions encountered and responses utilized by M. tuberculosis during the latent stage of infection. We hope that the analysis of two-component systems will aid in the identification of genetic determinants for which novel anti-tubercular drugs can be developed.