A BIOCHEMICAL AND GENETIC MODEL FOR PARASITE RESISTANCE TO ANTIFOLATES - TOXOPLASMA-GONDII PROVIDES INSIGHTS INTO PYRIMETHAMINE AND CYCLOGUANIL RESISTANCE IN PLASMODIUM-FALCIPARUM

We have exploited the experimental accessibility of the protozoan para site Toxoplasma gondii and its similarity to Plasmodium falciparum to investigate the influence of specific dihydrofolate reductase polymorp hisms known from field isolates of drug-resistant malaria. By engineer ing appropriate recombinant shuttle vectors, it is feasible to examine mutations by transient or stable transformation of T. gondii parasite s, in bacterial and yeast complementation assays, and through biochemi cal analysis of purified enzyme, A series of mutant alleles that mirro r P. falciparum variants reveals that the key mutation Asn-108 (Asn-83 in T. gondii) probably confers resistance to pyrimethamine by affecti ng critical interactions in the ternary complex, Mutations such as Arg -59 (T. gondii 36) have limited effect in isolation, but in combinatio n with other mutations they enhance the competitive ability of folate by increasing the speed of product turnover. Val-16 (T. gondii 10) con fers low level resistance to cycloguanil but hypersensitivity to pyrim ethamine, This mutation precludes Asn-108, probably because compressio n of the folate binding pocket introduced by this combination is incom patible with enzyme function. These studies permit detailed biochemica l, kinetic, and structural analysis of drug resistance mutations and r econstruction of the probable phylogeny of antifolate resistance in ma laria.