Tuberculosis (TB) is a persistent lung infection that has plagued mankind for centuries and ranks as one of the most serious threats to world health today. The 2-3 million deaths attributed yearly to the disease, as well as the emergence of strains resistant to all of the available chemotherapeutic agents, urgently call for the development of new therapies to treat TB. Furthermore, the threat of drug-resistant TB as a bioterrorism agent has led to it's listing as a NIAID Category C Priority Pathogen for biodefense research. The primary objective of the proposed research is to understand the mechanisms by which this pathogen manipulates its human host to evade killing by the immune system. We showed previously that M. tuberculosis utilizes the ESX-1 protein secretion system to export virulence factors that disarm host macrophages. We found that EspR is a key regulator of ESX-1 that is required for secretion and virulence in mice. EspR activates transcription of an operon that includes three ESX-1 components, whose expression in turn promotes secretion of ESX-1 substrates. Surprisingly, efflux of the DNA-binding regulator itself eventually results in reduced transcription, and thus reduced ESX-1 secretion. Our results reveal a direct negative feedback loop that titrates the activity of a secretion system essential for virulence of a major human pathogen. We hypothesize that such a regulatory scheme provides a timing mechanism that allows for a pulse of virulence factor secretion early after infection, followed by inhibition of secretion. This is an appealing hypothesis as many of the ESX-1 virulence factors are also major antigens recognized by the adaptive immune response. Thus, these studies will test the hypothesis that M. tuberculosis activates this pathway early during infection to deliver virulence factors, but then inactivates the pathway in order to hide from the immune system. Results from these studies may reveal ways to engineer better vaccines to fight TB.