Project Summary Toxoplasma gondii is the etiological agent of toxoplasmosis, the second leading cause of foodborne- illness related deaths in the U.S. Pathology during the acute phase of infection is driven by multiple cycles of parasite intracellular replication, resulting host cell lysis and inflammation. Parasites, by definition, depend on their host for resources; nonetheless, the mechanisms by which T. gondii acquires nutrients to sustain its fast- replicative stage are poorly understood. Our group has discovered that T. gondii actively ingests and digests host cytosolic proteins. However, the molecular mechanisms by which macromolecules traverse the parasitophorous vacuole membrane (PVM) that confines the parasite remain unknown. We have identified the dense granule protein GRA14 as a candidate for the recruitment of the host Endosomal Sorting Complexes Required for Transport (ESCRT). GRA14 is a transmembrane protein that contains motifs homologous to the late domain motifs of the HIV Gag protein in its C-terminus that is oriented towards the host cytosol. Late domain motifs mediate HIV-1 budding from the host cell by recruiting components of the ESCRT system. Using an HIV virus-like particle (VLP) release assay, we found that the motif-containing portion of GRA14 is sufficient to substitute for HIV GAG late domain to mediate ESCRT-dependent VLP budding. Furthermore, results from proximity ligation assays suggest that GRA14 abuts the ESCRT protein ALIX in infected host cells. Analysis of GRA14-deficient parasites revealed a marked reduction in ingestion of a host-derived reporter protein compared to WT parasites. Thus, we hypothesize that the parasite uses GRA14 to actively recruit host ESCRT components to its PVM to trigger the formation of vesicles containing cytosolic material that further bud into the parasitophorous vacuole lumen. If correct, this will be a seminal discovery of an intracellular pathogen effectively converting a non-endosomal compartment into replicative niche akin to a multivesicular body (MVB) via exploitation of host ESCRT. Hypothesis: Parasite effectors are necessary for the recruitment of the host endosomal sorting complex required for transport (ESCRT) which facilitates vesicular trafficking of host cytosolic material Aim 1: Elucidate the role of GRA14 in ESCRT recruitment to the PVM Aim 2: Uncover a role for the endosomal sorting required for transport (ESCRT) machinery in internalization of host cytosolic material