Project Summary/Abstract Microbial dormancy, defined as a state of reduced metabolic activity, is well recognized in bacterial systems as a mechanism of transient resistance to environmental stresses, including antibiotic treatment. We have recently reported that Trypanosoma cruzi, the agent of Chagas disease, resists long-term exposure to highly cytotoxic compounds due to the ability of a subpopulation of intracelllular amastigotes to spontaneously assume a dormant state. This new understanding of T. cruzi biology has implications for how currently available drugs are delivered and how new compounds capable of reversing dormancy, may be identified. In this application, we propose experiments that will define the timeframe and limits of T. cruzi dormancy in vivo, among different parasite isolates, if different host tissues and with respect to the chronicity of the infections. The immunological implications of dormancy will be investigated and this parasitological and immunological information will be integrated to enhance treatment efficacy using currently available drugs. Using a novel high content imaging screen, we will identify small molecules that prevent or reverse dormancy and/or drive the stage conversion of both dormant and actively replicating amastigotes into trypomastigotes. These compounds will serve as research tools to investigate the molecular pathways involved in dormancy and will be preliminarily investigated as new therapeutic entities for Chagas disease. We believe that the observation of dormancy in T. cruzi and the demonstration of its clear role in drug treatment failure is a game changer for treatment design and drug discovery in T. cruzi. The work in this proposal charts a way forward for therapy for Chagas disease, using new tools to increase our understanding of the complex set of interacting forces that determine treatment outcomes, and integrating this information to better use current drugs and discover of new ones.