Dense granule protein, GRA64 interacts with host cell ESCRT proteins during Toxoplasma gondii infection

The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In attempts to identify novel parasite secreted proteins that influence host cell activity, we identified and characterized a trans-membrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrate that GRA64 appears to interact with certain components of the host Endosomal Sorting Complexes Required for Transport (ESCRT). Genetic disruption of GRA64 does not affect acute Toxoplasma virulence in mice nor encystation as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of Dgra64 tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic Toxoplasma infection.