The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas'disease, has emerged as a HIV/AIDS-related opportunistic infection. Current anti-T. cruzi agents are highly toxic, and there is no effective treatment for chronic Chagas'disease. Improvement in our understanding of the molecular signaling responsible for its differentiation may lead to effective inhibitors of these processes providing new and novel therapeutic approaches. Differentiation of T. cruzi involves cAMP, but the pathway in which it is acting remains unknown. It is likely, that as in other eukaryotes, cAMP-dependent protein kinase (PKA) is a major signal transduction pathway controlling cell differentiation. The PKA catalytic subunit (TcPKA-C) gene and the PKA regulatory subunit gene (TcPKA-R) of T. cruzi were cloned by our laboratory and data indicates that the PKA catalytic and regulatory subunits are developmentally regulated. These data support the hypothesis that PKA activity is important in T. cruzi stage differentiation. We now plan to study the functions of TcPKA in differentiation. The effects of both overexpression of TcPKA with a constitutively active TcPKAC and blockade of TcPKA with a PKI clone, a dominant negative inhibitory TcPKA-R as well as conditional knockout of TcPKA-C on differentiation and proliferation will be studied in transfected parasites. Mechanisms of intracellular targeting of TcPKA-R in T. cruzi will be examined by deletion analysis of Nterminus of TcPKA-R and by analyzing the post-translational modifications of TcPKA-R with mass spectrometry. To further delineate the components of this pathway, we will identify the TcPKA downstream interacting molecules, using both yeast two-hybrid assays and proteomic approaches.