Toxoplasma gondii is an opportunistic protozoan parasite that causes central nervous disease in AIDS patients. T. gondii is also a Category B bioterrorism agent. Infection with T. gondii results in dissemination throughout the infected host via the tachyzoite form. After dissemination, the rapidly proliferating T. gondii tachyzoites differentiate into slowly replicating bradyzoites that remain latent within cysts for the life of the host. Clinically apparent toxoplasmosis in AIDS patients is thought to occur due to reactivation of latent bradyzoites as immune function wanes. Bradyzoite differentiation is stress mediated, and our data suggest similarities to cAMP regulated stress-induced differentiation in other eukaryotic pathogens. Manipulation of cyclic nucleotide signaling pathways affects the frequency of bradyzoite formation. To examine the role of parasite signaling pathways, we have cloned and characterized the T. gondii cAMP dependent kinase, PKA, the major effector of cAMP signaling. Transfection studies have revealed that the catalytic subunits of the cAMP dependent kinase, PKA 1 and PKA2, differ in localization and in their effect on parasite proliferation. We hypothesize that PKA 1 and PKA2 have opposing regulatory functions on metabolism and cell cycle regulation during tachyzoite-bradyzoite transition. We plan to disrupt PKA genes to further characterize their roles in parasite proliferation and bradyzoite differentiation. In an independent line of experiments, yeast 2 hybrid screens will identify proteins that interact with PKA 1 and PKA2. Finally, we will use 7-. gondii cDNA microarrays to examine global gene expression patterns in response to conditions known to induce bradyzoite formation and conditions that affect cAMP signaling. Gene expression patterns will be compared to parasites with mutations or deletions of PKA subunits. These studies will further elucidate cAMP signaling pathways in T. gondii and determine the role of cAMP signaling pathways during T. gondii differentiation into latent bradyzoites.
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