(provide by the applicant): Toxoplasma gondii is a widespread Apicomplexan parasite that causes devastating disease in immunocompromised persons and the congenitally infected fetus. The pathology of toxoplasmosis is due to repeated cycles of host cell invasion and lysis by the actively dividing form of T. gondii, the tachyzoite. Despite the importance of invasion to the life cycle of the parasite and the pathology of toxoplasmosis, little is known about the tachyzoite proteins that mediate invasion. Because T. gondii is a haploid, obligate intracellular parasite, forward and reverse genetic approaches to studying invasion are problematic. Small molecules will be used as a means to circumvent this difficulty and identify gene products that play an important role in invasion. A collection of over 14000 structurally diverse small molecules has been screened in a high-throughput invasion assay, and 28 invasion inhibitors have been identified. Unexpectedly, 7 invasion enhancers were also discovered. In secondary assays, several of the bioactive small molecules were shown to affect motility of the parasite and/or secretion from its apical organelles. The goals of the proposal are to: (I) Test the invasion inhibitors and enhancers against several other related and unrelated parasites, to determine whether they target conserved components of the Apicomplexan invasion machinery; (II) Test whether any of the motility inhibitors/enhancers exert their effects through parasite myosin A or the multi-protein complex to which it belongs; (III) Test whether an inhibitor of cGMP phosphodiesterase identified in the screen affects parasite cGMP levels during invasion; (IV) Screen a comprehensive collection of >500 small molecules of known biological function for an effect on invasion; and (V) Use synthetic, biochemical and genetic methods to determine the in vivo targets of 3-5 of the highest priority invasion inhibitors/enhancers, where prioritization is based on the results of I-IV. Target identification will be greatly facilitated by the recently released sequence of the Toxoplasma genome. A strength of the proposal is the level to which biological experiments and synthetic chemistry will be integrated to address the project's goals. This work has the potential to provide important new insights into the proteins and pathways involved in host cell invasion by T. gondii and related parasites, and it may simultaneously identify lead compounds for the development of new and urgently needed anti-parasitic drugs.
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