The Role of Toxoplasma Rhoptries in Host Cell Infection

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DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes severe central nervous system disorders of immunocompromised (AIDS/transplant/lymphoma) individuals and birth defects to congenitally infected neonates worldwide. This parasite also serves as a model system for the study of intracellular Apicomplexan parasites that cause a variety of diseases of global medical and veterinary importance including Plasmodium falciparum, the causative agent of malaria. These parasites actively invade into and reside within a unique parasitophorous vacuole (PV) in the cytoplasm of the host. Invasion and establishment of the PV are mediated in part by the action of the rhoptries, secretory organelles unique to Apicomplexans. The precise role of the rhoptries in this process in not known, largely because only a few rhoptry proteins have been identified to date. To address the function of the rhoptries, we have purified rhoptries and analyzed their contents using proteomic and monoclonal antibody approaches. We have identified the major proteins constituents of the rhoptries, one of which is a protein phosphatase 2C-like (TgPP2L) protein that is targeted to the nucleus of the host cell during infection, indicating a novel role for the rhoptries in host-parasite interaction. We will focus on the TgPP2L, protein and two novel rhoptry proteins identified in our proteomic analysis to determine their roles in invasion. Specifically, we will 1) characterize TgPP2L with respect to its host nuclear localization and directly assess its phosphatase activity; 2) determine the function of TgPP2L by analyzing TgPP2L knockout parasites in vitro and in vivo and by the expression of TgPP2L in human cells; and, 3) determine the functions of the novel rhoptry proteins ROP14 and RON3 by determining their ultimate destination following secretion, identifying interaction partners, and disruption of their genes. The studies proposed here offer important insight into the means by which Apicomplexan parasites establish an environment for intracellular survival and provide an understanding of unique aspects of host pathogen interactions.