Toxoplasma gondii is an obligate intracellular parasite which resides in a unique membrane bounded vacuole inside host cells. Like all intracellular protozoan parasites, T. gondii must salvage purines from the host Cell in order to survive and replicate The vacuolar membrane surrounding the parasite inside host cells is freely permeable to small molecules, providing the parasite with ready access to mM concentrations of ATP from the host Cell cytosol. One of the most prevalent proteins produced by the parasite (1-2% of total parasite protein) is a nucleoside triphosphate hydrolase (NTPase) which sequentially cleaves all nucleoside triphosphates to the di-phosphate and the non-phosphate form. The NTPase is secreted by the parasite, a distinctly unusual process for an ATPase, and accumulates in the vacuolar space and at the vacuolar membrane. At this site, the enzyme could participate in purine salvage by supplying AMP to a membrane bound parasite 5'-nucleotidase, with subsequent generation of high levels of adenosine. Three tandomly repeated genomic copies of the NTPase have been identified, two of which are expressed in the virulent RH strain, and are over 97% identical at the nucleotide and amino acid level. The ATPase activity of the isoforms is strikingly different and only the less active isoform is expressed in two non- virulent, slow-growing isolates, raising the possibility that NTPase expression is related to replicative potential inside cells. The NTPase will be investigated in depth as a potential therapeutic target and as a paradigm for targets in the vacuolar space and membrane amenable to novel strategies for intervention. Experiments are proposed to further characterize expression of NTPase isoforms in different parasite strains, to delete and replete NTP isoforms by genetic means, to study the regulation and stage specificity of expression, and to look for homologues in other coccidian parasites. The structure and function of the NTPase will be studied by determining the subunit composition, the enzyme kinetics and the subcellular distribution of the various isoforms, by delineating residues involved in nucleotide binding and dithiol- mediated regulation of activity, by identifying inhibitors of functional activity, and by evaluating the role of the enzyme in nucleoside salvage and other functions within the vacuolar space.
