Toxoplasma gondii is a major opportunistic pathogen during pregnancy and in immunocompromised individuals, including organ transplant and AIDS patients. Toxoplasma is able to invade and survive within virtually any nucleated cell in a wide range of vertebrate hosts. This ability is responsible for the very high prevalence of this parasite which ranges from 25-50% of the human population. Because chronic infections are very long lived, there is a significant risk of reactivation at any time the immune system becomes compromised. Consequently, there is a pressing need for a grater understanding of the unique adaptations for intracellular survival by this parasite. Previous studies have shown that Toxoplasma resides in a unique intracellular vacuole termed the parasitophorous vacuole which forms a protective interface that is key to intracellular survival. In addition to avoidance of lysosomal digestion, segregation of the vacuole from the endocytic system provides protection from immune surveillance. The goals of the project are to define the mechanisms of entry and modification of the host cell that are responsible for formation of the parasitophorous vacuole. The resistance to fusion by Toxoplasma-containing vacuoles will be examined using high-resolution microscopy to determine the interaction of this vacuole with other host vesicular pathways, both endocytic and exocytic. In addition, in vitro biochemical assays will be used to examine the parasitophorous vacuole for inhibitors of vesicular fusion. Pathways for protein and lipid secretion by the parasite will be delineated both in vitro using biochemical approaches in infected host cells. Molecular and cellular biological studies will be used to identify mechanisms for protein targeting within the parasite-containing vacuole and to identify the role of secretory proteins in nutrient uptake and processing. Characterization of the parasitophorous vacuole may provide a means of targeting intracellular parasites for endocytic digestion or for interfering with nutrient uptake by the parasite. The information gained here is directly relevant to understanding antigen processing and presentation for immunological intervention and may also provide a rational approach for targeted delivery of drugs to the parasitophorous vacuole.
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