More than two million people die each year from tuberculosis and one third of the world' s population is believed infected with Mycobacterium tuberculosis, the bacterium responsible for this disease. To enable development of new drugs and vaccines for tuberculosis, a thorough understanding of M. tuberculosis physiology and pathogenesis is required. Protein secretion pathways play an important role in bacterial pathogenesis. The long-term objective of this research is to define the protein export systems of mycobacteria and the role they play in M. tuberculosis pathogenesis. Mycobacteria are unusual in possessing two SecA homologues (SecA1 and SecA2). SecA is highly conserved throughout bacteria and is a central component of the general Sec-dependent transport pathway, which exports proteins containing amino-terminal Sec signal sequences. In mycobacteria, SecA1 is the essential "housekeeping" SecA while SecA2 is a non-essential accessory secretion factor. A deletion of the secA2 gene in M. tuberculosis attenuated the virulence of the organism in mice. This suggests that SecA2 exports virulence factors of M. tuberculosis. The investigators identified two antioxidants of M. tuberculosis as being secreted by a SecA2-dependent export pathway. This has led to the hypothesis that SecA2 is part of a virulence mechanism of M. tuberculosis to evade the oxidative attack of the host. Both of these antioxidants lack Sec signal sequences and the mechanistic basis of their export is unknown. The proposed research will characterize the roles of SecA2 in pathogenesis and in protein export. The specific aims of this proposal are the following: (1) Investigate the role of SecA2 and secreted antioxidants in M. tuberculosis pathogenesis by testing a AsecA2 mutant of M. tuberculosis in both macrophages and mice and in its ability to resist oxidative attack, (2) Identify proteins exported by SecA2, and (3) Characterize the basis of SecA2 function in protein export by identifying other proteins that act with SecA2. This study will expand our understanding of the proteins that contribute to M. tuberculosis virulence and the protein secretion pathways of M. tuberculosis. It may also help clarify the role of reactive oxygen species in the host response to M. tuberculosis. This research has the potential of facilitating development of new anti-tuberculosis strategies.