The goal of this proposal is to develop a strategy to interrupt both acute toxoplasmosis and the reactivation of latent toxoplasmosis based on the absolute necessity of Toxoplasma gondii motility in host cell invasion and the unique mechanism of parasite movement. Active invasion of many host cell types is responsible for the pathology of cerebral and disseminated toxoplasmosis associated with AIDS. A deeper understanding of T. gondii myosin, a likely candidate for the primary motor protein powering gliding movement of myosin, a likely candidate for the primary motor protein powering gliding movement of invasive zoites, may lead to the discovery of drug targets based on inhibition of T. gondii motility. The nucleotide sequence of the T. gondii myosin gene or genes will be deduced from cDNA and genomic libraries by the use of a previously defined 89 bp probe. The predicted protein structure of the full length gene will be compared with known functional domains of heterologous myosin genes. T. gondii myosin genes will be expressed in prokaryote and eukaryote systems in order to analyze biochemical and functional properties of myosin of importance for specific drug design. Antibodies specific for T. gondii myosin within invasive zoites and its function in motility and invasion. Site directed mutagenesis of T. gondii myosin and transformation of T. gondii by altered myosin genes will allow study of the role of myosin in movement and invasiveness, and may provide information on the mechanism of drugs affecting motility. Myosin gene expression will be studied in pathogenic tachyzoite and bradyzoite stages of the asexual cycle, in order to understand the role of myosin in reactivation of bradyzoites in chronic infection. Such reactivation is thought to be the major pathway for initiation of toxoplasmic encephalitis in AIDS. Conditional mutants will be sought that fail to transform to tachyzoites in cell culture, in order to further understand the transition between stages, and to furnish a stable in vitro model for testing pharmacological agents active against bradyzoites.
Showing the most recent 10 out of 55 publications
