Kinase regulation of trafficking at the Toxoplasma intravacuolar network

PROJECT SUMMARY/ABSTRACT Toxoplasma gondii has the remarkable ability to infect virtually any cell type of almost all warm-bloodedanimals and is arguably the most successful parasite on earth, having infected an estimated one-third ofhumans globally. While initial infection typically resolves without complication, the parasite is able to persistfor the life of its host, and can re-emerge in the immunocompromised and immunosuppressed to cause fataldisease. Toxoplasma, like other apicomplexan parasites, must invade a host cell to survive and replicate. Once inside a host cell, the parasite survives and replicates within a specialized organelle called theparasitophorous vacuole. Disruption of the vacuole results in parasite death, and the parasite secretes abattery of proteins into the vacuole to facilitate its biogenesis and regulate trafficking of nutrients andeffector proteins. A principle structure within the parasitophorous vacuole is the intravacuolar network ofmembranous tubules (the IVN), which is thought to act as a major trafficking apparatus. IVN biogenesis isformed by the direct action of oligomeric complexes of parasite proteins and mutants that disrupt the IVNshow reduced virulence in animal models of infection. We have identified a parasite-specific protein kinasethat regulates the membrane association of a subset of the proteins that associate with the parasitophorousvacuolar and IVN membranes, and deletion of this kinase results in vacuoles with aberrant IVN tubulation.While we have identified the kinase substrates and the sites of phosphorylation, the interactions that areregulated by this phosphorylation are unknown. The goal of the proposed studies is to determine theprecise molecular mechanisms by which phosphorylation regulates the inter- and intra-molecularinteractions that drive IVN biogenesis. First, we will determine how the components of the proteincomplexes that drive IVN biogenesis change as the complexes progress through the parasite secretorysystem and insert into the IVN membrane. We will use molecular genetic, cellular, and biochemical methodsto determine the molecular mechanisms by which phosphorylation regulates these protein-proteininteractions to facilitate IVN development. Furthermore, we will use innovative biophysical methods togenerate the first structural models of these critical parasite protein complexes to determine the biophysicalmechanism by which they induce IVN formation.