The goal of the proposed studies is to develop the basis for new chemotherapy that will depend on inhibition of pyrimidine biosynthesis. This pathway is a likely target for chemotherapeutic intervention since some of the parasite enzymes are markedly different from mammalian cells. The investigator presents preliminary evidence that T. gondii is dependent upon its de novo pathway of pyrimidine synthesis. Thus, elimination of the de novo pathway by irreversible genetic deletion of a key enzyme should block replication of the parasite and confirm the investigator's hypothesis that the parasite cannot make effective use of host cell pyrimidines. In the first specific aim, the investigator intends to genetically delete the T. gondii gene for carbamoyl phosphate synthetase (CPSII), the first committed step of pyrimidine synthesis in order to produce an auxotroph that will be completely dependent on salvage of uracil. A defined uracil auxotroph will confirm the validity of the de novo pyrimidine synthesis as a chemotherapeutic target. The effect of selected inhibitors of de novo pyrimidine synthesis on the intracellular growth of T. gondii will be studied in cell culture. Inhibitors of intracellular replication will represent useful lead compounds for further development. Uracil auxotrophs will be tested for in vivo growth and virulence in a mouse model. In the second specific aim, the investigator will expand the parasite's salvage repertoire by expression of cytosine deaminase and thymidine kinase in T. gondii. This will allow the use of these salvage enzymes for positive and negative selection. The linker region of dihydrofolate reductase-thymidylate synthetase (DHFR-TS) will be developed as a locus that can be expanded to express genes of interest in T. gondii.
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