Toxoplasma gondii owes much of its success to the battery of secreted effectors that allow it to develop its replicative niche, the parasitophorous vacuole (PV), and evade host immune clearance. Toxoplasma"s dense granule proteins (GRAs) are a class of effectors that carry out these functions in the PV, at the PV membrane (PVM), and in the host cytosol. In general, GRA proteins remain largely under described as they are often dispensable in tissue culture despite being critical to parasite pathogenesis. For example, a small subset of GRAs are responsible for reshaping the PVM into the intravacuolar network (IVN), a tubulovesicular network that is necessary for parasite virulence. While the exact role of the IVN is undefined, genetic phenotypes have implicated that it mediates diverse processes including nutrient uptake and effector export across the PVM. Interestingly, despite having typical traits of integral membrane proteins, the GRAs that build and decorate the IVN are trafficked through the parasite secretory system as soluble, heteromultimeric complexes. This regulation of membrane insertion and switch in solubility suggest that there are chaperoning interactions that keep these GRAs out of surrounding membranes, such as those of the endomembrane system or the parasite plasma membrane, until they reach the IVN. Outside of these observations, how these GRAs contribute to IVN biogenesis, how they are regulated during secretion and trafficking, and what proteins are critical at each of these trafficking steps remains unknown.

To identify regulators of trafficking, I will use immunoprecipitation of soluble versus membrane bound GRAs coupled with mass spectrometry. Taken together, the data from these experiments will provide mechanistic details on Toxoplasma dense granule trafficking in the parasite and PV as well as on IVN biogenesis. Using this information, I aim to develop an in vitro system that reconstitutes the release of chaperoning and insertion into the membrane for the IVN GRAs. I will further use this system to determine structures of both the chaperoned complexes and the GRA oligomers that form the IVN tubules. Finally, I am using structural biology to determine the structures of the folded GRAs to determine how they contribute to successful GRA trafficking and parasite survival.