TARGETING ESSENTIAL ROP KINASES IN TOXOPLASMA

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DESCRIPTION (provided by applicant): Toxoplasma gondii is a widespread protozoan parasite of wild, domestic, and companion animals that also commonly infects humans. Severe infections are normally only found in immunocompromised patients, including HIV infection, cancer chemotherapy, organ transplant, or infants infected in utero. Additionally, emerging studies indicate that severe ocular toxoplasmosis can also occur in healthy adults, and that chronic infection is an underlying risk factor for some forms of psychiatric disease. Toxoplasma isolates differ dramatically in their virulence in animal models and also in human infections. Recent findings reveal that the major virulence determinants of this parasite are secretory proteins derived from the rhoptries (ROPs), which are injected to the host cell at the time of invasion. Many ROPs contain a conserved serine / threonine kinase domain and are predicted to function as kinases, potentially altering host signaling pathways by phosphorylation of specific targets. In preliminary studies, we have developed a systematic gene disruption strategy to target all of the known active ROP kinases in the genome. Disruption of ROP kinases will be used to define their roles during growth in vitro vs. in vivo in the mouse model. Separately, we have designed a single-step insertional strategy to generate conditional expression alleles of essential ROP kinases. Knockdown of individual ROPs will be used to explore the functional pathways controlled by these kinases. We will also employ biochemical assays and mass spectrometry to identify the substrate specificity of ROP kinases, there by providing specific substrates for development of inhibitor assays. Expression of recombinant proteins will be used to develop in vitro activity assays and to screen focused kinase inhibitor libraries to indentify potent inhibitors. The goal of these studies to provide genetic validation of essential kinases in T. gondii and to characterize chemical scaffolds that inhibitor ROP kinases for future development. These studies will help define the roles of an important class of parasite virulence factors and may lead to improved therapeutic intervention against toxoplasmosis. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is an important food borne pathogen of humans that causes disease in those with weakened immune systems. Our studies are designed to identify essential parasite virulence factors and validate them as potential drug targets. These studies may eventually lead to improved therapeutic intervention against severe infections caused by these parasites.