In vitro generation of novel pyrimethamine resistance mutations in the Toxoplasma gondii dihydrofolate reductase

Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widel y used in the treatment of opportunistic infections caused by the protozoan parasite Toxoplasma gondii. In order to assess the potential role of dhfr sequence polymorphisms in drug treatment failures, we examined the dhfr-ts genes of representative isolates for T. gondii virulence types I, II, and I II, These strains exhibit differences in their sensitivities to pyrimethami ne but no differences in predicted dhfr-ts protein sequences. To assess the potential for pyrimethamine-resistant dhfr mutants to emerge, three drug-s ensitive variants of the T. gondii dhfr-ts gene (the wild-type T.gondii seq uence and two mutants engineered to reflect polymorphisms observed in drug- sensitive Plasmodium falciparum) were subjected to random mutagenesis and t ransfected into either wild-type T. gondi parasites or dhfr-deficient Sacch aromyces cerevisiae under pyrimethamine selection. Three resistance mutatio ns were identified, at amino acid residues 25 (Trp --> Arg), 98 (Leu --> Se r), and 131 (Leu --> His).