Tachyzoites of Toxoplasma gondii attach to and enter all nucleated cells, evading killing by oxidative and non-oxidative mechanisms both during and after cell entry. This proposal outlines our plan for investigation of I.) selected ligands and receptors involved in attachment of tachyzoites to cells and II.) the mechanism by which tachyzoites enter macrophages without triggering a respiratory burst and reside in a vacuole in all cells which neither acidifies nor fuses with lyosomes. Specific plans for I include a.) definition of the role of complement component Clq in cell binding of tachyzoites, b.) characterization of receptors for the extracellular matrix protein laminin on tachyzoites and the role of laminin and host cell receptors in the attachment process, and c.) identification of apical components in the parasite involved in cell binding. Specific plans for II include investigation of a.) the interaction of parasitophorous vacuoles containing T. gondii with other intracellular compartments, b.) the influence of phagocytosis of tachyzoiters via different receptors on vacuole composition and of parasite fate, c.) incorporation and functional activity of components of the phagocyte NADPH oxidase system in the parasitophorous vacuole membrane, d.) functional analysis of acidification of purified parasitophorous vacuoles, e.) immunochemical characterization of the parasitophorous vacuole membrane, and f.) functional analysis of cell components involved in fusion of parasitophorous vacuoles with lysosomes. These studies, for which we have a substantial amount of preliminary data, will investigate important issues in cell attachment and entry by T. gondii, and will address critical issues in the cell biology of intracellular fusion and acidification events. Such experiments should define the molecular mechanisms involved in these events and may suggest strategies for intervention.
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