Fitness effects of DHFR-TS mutations associated with pyrimethamine resistance in apicomplexan parasites

Pyrimethamine resistance in the malaria parasite Plasmodium falciparum is characterized by specific point mutations in the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously explored the effect of these mutations by engineering homologous alleles of Toxoplasma gondii DHFR-TS, which can readily be expressed as recombinant protein for enzymatic studies, or as allelic replacements in transgenic parasites. In order to directly assess the costs of pyrimethamine-resistance in vivo, we have carried out competition studies between mixtures of T. gondii tachyzoites harbouring wild-type or mutant DHFR-TS alleles, both in tissue culture and in mice. Arg59+Asn108 mutants (using the P. falciparum numbering system) exhibit no significant fitness defects in vitro, but a fitness defect of 1.8% per generation in mice. Arg59+Ser223 mutants exhibit fitness defects of >2.8% per generation both in vitro and in vivo, which may explain why this highly pyrimethamine-resistant allele has not been observed in the field. It is important to note that long-term propagation of parasites in vitro or in vivo can produce adaptations affecting fitness by >3.7% per generation, necessitating careful attention to background in head-to-head competition studies. A sensitive PCR-based assay permits different growth rates to be assessed even in the absence of a drug resistance marker that can be scored by plaque assay.