The long-term goal of this project is to develop a new diagnostic test to detect latent infection with Mycobacterium tuberculosis. The rationale for this study is provided by recent evidence that bacteria express a specific program of new genes in the stationary phase that are crucial for virulence and long-term survival. The specific hypothesis for this proposal is that M. tuberculosis specifies an analogous regulatory system for the expression of gene products required to establish and maintain long-term latent infection, and that these latency-specific proteins comprise new antigens recognized by the host. After identification and characterization, these new antigens wild provide the basis for a new diagnostic test. The specific aims of this proposal are: l) To develop defined culture conditions that lead to stationary phase growth of M. tuberculosis in artificial media and within macrophages. Stationary growth will be induced by nutrient limitation and O2 restriction. To mimic in vivo conditions, organisms will be grown in macrophages under reduced O2 levels. The new proteins synthesized under these conditions will be analyzed by one and two-dimensional gel electrophoresis. 2) To clone and characterize the alternate RNA polymerase sigma factor (rpoS analogue) of M. tuberculosis that controls the expression of stationary phase genes. Sigma factor genes will be cloned in E. coli, sequenced, and the rpoS analogue identified by homology, functional assay, and expression in stationary phase. 3) To define the new proteins produced under control of this sigma factor in vitro and within macrophages. M. tuberculosis will be transformed with the rpoS gene expressed constitutitively and the synthesis of new proteins analyzed on one and two-dimensional gel electrophoresis. 4) To clone selected stationary phase and rpoS- controlled M. tuberculosis antigens in E. coli, to overproduce and purify these antigens for immunologic testing. 5) To characterize the human immune response to M. tuberculosis stationary phase proteins in patients with latent infection. Both antibody and T-cell reactivity to these proteins will be tested and compared to non- infected controls.