The protozoan parasite Toxoplasma gondii is a ubiquitous human pathogen which has emerged as a leading opportunistic infection associated with AIDS. Clinical management of toxoplasmosis has traditionally relied on antifolates, but complications associated with the chronic therapy needed for immunodeficient patients have left us with no adequate treatment for this devastating disease. This proposal seeks to employ newly developed genetic and pharmacological tools to investigate drug sensitivity and resistance in Toxoplasma, focusing particularly on folate metabolism and the parasite's bifunctional dihydrofolate reductase-thymidylate synthase enzyme (DHFR-TS). Goals of this research include the development of improved treatment strategies for acute toxoplasmosis. Sequences and probes derived from the T. gondii DHFR-TS gene have been employed to examine the predicted structure of the enzyme in wild-type parasites, and to develop functional vectors for transient and stable molecular transformation of the parasite. Clinical cases of antifolate- resistant toxoplasmosis and similar mutants isolated in the lab will be screened for differences in sensitivity to combined pyrimethamine/sulfonamide treatment (in addition to sensitivity to pyrimethamine or sulfa alone), and examined for possible DHFR-TS mutations or altered gene expression. Mechanisms of DHFR-independent resistance will be identified by genetic means. DHFR-TS enzyme function will be assessed in transgenic parasites bearing mutations derived from four sources: naturally occurring allelic variation, mutations identified from drug- resistant laboratory strains and clinical isolates, modeling studies on the T. gondii enzyme, and point mutations thought to be associated with antifolate resistance in field isolates of the related parasite Plasmodium falciparum (malaria). Findings from this research will be combined with structure/function studies on the recombinant DHFR-TS enzyme, to assist in the design of novel antifolates with improved activity against the parasite. Because of the tremendous power of genetic techniques for the identification and analysis of drug targets, available transformation schemes will be modified to permit: (1) Targeted gene disruption and replacement, used in this context to examine mutations at the DHFR-TS locus; (2) Insertional mutagenesis and marker rescue, used to clone potential targets for therapeutic intervention and negative selectable markers for gene replacement studies; (3) Molecular cloning by complementation, focusing on DHFR-independent antifolate resistance genes which appear to be particularly important for pyrimethamine resistance in Toxoplasma; and (4) Optimal expression and overexpression of recombinant protein. In addition to their value for the analysis of folate metabolism and drug resistance, these tools should be broadly applicable to other studies on the biology and biochemistry of Toxoplasma.
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