The proposed project seeks to investigate molecular, genetic, and pharmacological aspects of folate metabolism in the intracellular protozoan parasite Toxoplasma gondii, a common infectious agent which is a particularly insidious opportunistic pathogen of immunodeficient patients. Acute toxoplasmosis is also a leading cause of neurological abnormalities resulting from fetal infection. Aside from its clinical importance, toxoplasma is an appealing organisms for laboratory study: the parasite is convenient to grow in vitro, and lends itself to genetic analysis more readily than do other protozoan parasites. Because T. gondii is incapable of thymidine salvage, the folate pathway is a key target for both clinical therapy and, potentially, the introduction of selectable markers for genetic studies in the laboratory.
The specific aims of this proposal are to study the pharmacology of drugs which block different stages of folate metabolism, isolate various types of mutants altered in their response to these agents, and to clone and characterize the fused gene encoding the parasite dihydrofolate reductase/thymidylate synthase enzyme. The pharmacological studies take advantage of an in vitro assay developed to assess the extent of synergism observed when drugs affecting different steps in the folate pathway are used in combination. These assays will be useful in characterizing drug- resistant mutants, establishing the spectrum of drug sensitivity in laboratory and clinical isolates to T. gondii, and assessing the promise of new therapeutic agents for the treatment of toxoplasmosis. Mutant parasites isolated from clinical cases or selected in the laboratory will be studied to determine the molecular basis of drug resistance in Toxoplasma, and to refine treatment strategies to minimize complications arising from the emergence of resistant strains. Sequence analysis of cDNA clones (already isolated) encoding the parasite DHFR/TS enzyme will be necessary for the analysis of mutant strains, and in combination with genomic clones will provide the basis for engineering vectors for the development of transfection protocols. DHFR/TS sequences will also be of consider able value in elucidating the evolutionary relationship of the parasitic protozoa and the origins of this interesting fused enzyme. Antibodies prepared to the DHFR/TS protein and synthetic polypeptides will be used to purify protein for future structural and enzymatic studies, which will be valuable in the rational design of new parasiticidal drugs. In addition to assisting in the design of new treatment strategies, this study initiates a comprehensive effort on the part of the PI to further the development of Toxoplasma as a useful paradigm for the genetic analysis of parasite cell and molecular biology. In particular, the recently demonstrated ability to microscopically manipulate the parasite oocyst should facilitate the isolation of progeny form genetic crosses, and offers a novel approach to the development of a parasite of a parasite transfection system.
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