The success of Toxoplasma gondii as an intracellular parasite is dependent on its unique ability to invade and survive within virtually any nucleated cell in a wide range of vertebrate hosts. Upwards of 50% of the world's human population is chronically infected with Toxoplasma, although these infections are normally asymptomatic. The emergence of toxoplasmosis as a major opportunistic pathogen in pregnancy and AIDS, has necessitated a greater understanding of the unique adaptations for intracellular survival employed by this parasite. The proposed project provides a comprehensive analysis of the parasitophorous vacuole occupied by Toxoplasma which forms a protective interface that is key to intracellular survival. We have developed in vitro systems to examine the composition and kinetics of vacuole development with respect to models for normal phagocytosis. This comparative approach will be used to identify the biochemical basis of the resistance to fusion by Toxoplasma-containing vacuoles. To explore the active modification of this compartment by the parasite, we have characterized the secretion of two prominent proteins that reside in the mature vacuole (GRA2 and NTPase). Our preliminary findings indicate an important role for intracellular calcium in regulating the secretion of parasite proteins within the vacuole. We will examine this regulation in detail and explore the feasibility of using pharmacological agents that disrupt calcium homeostasis to block secretion and effect intracellular survival. Finally, we will identify new parasite secretory proteins that are targeted to the vacuole membrane to expand our understanding of the unique modifications of this compartment. These findings will provide key information about the composition of the parasitophorous vacuole that may provide a means of targeting intracellular parasites for destruction. The information gained here is also directly relevant to understanding antigen processing and presentation for immunological intervention and may provide a rational approach for targeted delivery of drugs to the parasitophorous vacuole.
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