Toxoplasma gondii is a serious pathogen of humans and livestock in the U.S.A. and world-wide. In addition to its well-known pathogenesis in the developing fetus, in recent years this protozoan parasite has ncreased its notoriety through the fatal disease it can cause in AIDS patients. Currently, there is no vaccine for Toxoplasma that is designed to impact human health and no drug capable of eliminating the persistent, chronic infection. Disease in AIDS patients is thought to largely result from the reactivation of a chronic infection that persists through the ability of the parasite to differentiate from the actively dividing tachyzoite stage to an encysted bradyzoite stage. The resulting tissue destruction, particularly in the brain and lungs can lead to severe disease or even death. Our goal is to identify and characterize parasite genes (and their respective products) that are critical for the differentiation of tachyzoites to bradyzoites. To identify these genes, we will use a combination of genetic and molecular techniques. For the molecular analysis, we will use microarrays to monitor changes in gene expression over a time-course of differentiation. A novel method developed by us will be used to determine whether these differences are the result of changes in mRNA synthesis or stability. A combination of forward and reverse genetics will then be used to determine the cis-elements mediating these changes. To identify genes with a central role in the differentiation process, we will select mutants that are not able to differentiate from tachyzoite to bradyzoite. This will be done by selecting for parasites that fail to express bradyzoite-specific surface markers. The genes will be identified and the role of their protein products will be determined. In this way, we will learn what pathways are key to differentiation and growth of bradyzoites. This work will lay the foundation for generation of an animal vaccine that produces a self-limiting infection that cannot be transmitted to other animals or humans. Such a vaccine would break the zoonotic cycle of animal to human transmission.
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