Abstract
BackgroundAnti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1.MethodsImpacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted.ResultsCo-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact.ConclusionsThese results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum.
Highlights
Anti-malarial drug resistance may be limited by decreased fitness in resistant parasites
Parasites under study Plasmodium falciparum strain NF10, the product of a genetic cross between the Brazilian 7G8 and Ghanaian GB4 strains, was engineered previously to provide lines wild type or mutant at PfMDR1 N86Y and Y184F, loci that have been polymorphic in African parasites [14] (Table 1)
Considering other common polymorphisms relevant to drug resistance, NF10 parasites have wild type sequence at PfMDR1 positions 1034, 1042, and 1246; have one copy of the pfmdr1 gene; and contain the P. falciparum chloroquine resistance transporter (PfCRT) position 72–76 CVIET haplotype and 5 other PfCRT mutations (A220S, Q271E, N326S, I356T, and R371I), a genotype associated with resistance to chloroquine [36]
Summary
Anti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1. With development of resistance to older drugs, standard therapy for falciparum malaria in Africa moved to artemisinin-based combination therapy (ACT) early this century [3]. ACT, including artemether-lumefantrine, artesunate-amodiaquine, dihydroartemisinin-piperaquine, artesunate-mefloquine, and artesunate-pyronaridine, combines a potent and fastacting artemisinin derivative with a slower acting partner drug [4]. Partner drugs play a key role in assuring the efficacy of ACT, by eliminating remaining parasites after the short acting artemisinins are cleared, and by protecting against emergence of artemisinin resistance. Resistance to artemisinins in southeast Asia [5] and varied activity of most partner drugs [6] threatens the efficacy of ACT. Widely used partner drugs have opposing drug sensitivity profiles, with decreased sensitivity to amodiaquine (a close relative of chloroquine) associated with increased sensitivity to lumefantrine and mefloquine [7]
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