title: Fitness effects of DHFR-TS mutations associated with pyrimethamine resistance in apicomplexan parasites.
abstract: 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.
authors: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.
authors: Leah M
authors: LM
authors: Fohl
authors: David S
authors: DS
authors: Roos
mesh_terms: D000818:Animals; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics*; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011739:Pyrimethamine / Q000494:pharmacology*; D012641:Selection, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism; D014122:Toxoplasma / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
chemical_list: D005493:Folic Acid Antagonists; D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D011739:Pyrimethamine