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Toxoplasma gondii is a widespread protozoan parasite that is capable of causing congenital disease in developing infants and severe complications in immunocompromised patients. Nearly a third of the human population is chronically infected by T. gondii.<P> Current therapies are unable to cure chronic infection and intolerance, due to toxicity, often results from long-term treatment. Our ability to effectively treat T. gondii infection requires the identification of new therapeutic targets and the development of a rational drug design strategy. T. gondii is highly effective at parasitizing a broad range of warm-blooded hosts and can infect nearly any nucleated cell type. During the invasion process T. gondii injects a heterogeneous mixture of proteins from secretory organelles, known as rhoptries, into the host cytoplasm. The result is that many of the well known innate mechanisms employed by hosts to combat infectious parasites become unresponsive or inoperative.<P> Host pathways involved in apoptosis, metabolite sequestering, and the immune response become effectively co-opted allowing T. gondii to complete its intracellular life cycle unimpeded. Proteomic analysis of the rhoptry organelles identified the presence of a highly expanded family of serine/threonine (S/T) kinases (ROP kinases) and genetic mapping studies of virulence genes implicated several of these as critical virulence factors. <P>Approximately 20 active ROP kinases have been identified in the T. gondii genome and phylogenetic, structural, and functional analyses have indicated that they differ significantly from any of the major families of S/T kinases found in humans. Several polymorphic ROP kinases have since been shown to directly modulate parasite virulence yet, surprisingly, none were essential for parasite viability.<P> The goal of this application is to provide a comprehensive analysis of the ROP kinase family by systematically determining their role in parasite viability and virulence. We will first define the essential members of the kinase family and characterize the phenotypic effects of protein knockdown on the intracellular life cycle in vitro as well as virulence in the mouse model. The role of the essential ROP kinases in host cell modulation will be assessed through microarray based comparison of transcriptional changes occurring after infection. The isolation of native core ROP kinase complexes and the trapping of host substrates will complete the analysis by providing insight into additional factors which may play a role in regulating kinase function or determining substrate specificity. In summary this project will integrate various phenotypic analyses, host cell transcriptional profiling, and proteomic characterization of ROP kinase complexes to yield a composite view of the role that essential ROP kinases play in critical host-pathogen interactions. More importantly the resulting analyses may identify a host of new potential therapeutic targets.
Public Health Relevance: Nearly one third of the world's human population and over 60 million people in the U.S. are estimated to be infected by the protozoan parasite Toxoplasma gondii. Although normally not life-threatening, this parasite can cause serious health problems and even death in people with weakened immune systems or in the developing fetus. Current drug therapies are unable to cure infection and long-term treatments often become toxic to the patient; the goal of this project is to identify essential parasite proteins responsible for establishing infection that have the capacity to serve as new potential therapeutic targets.