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. 2016 May 18:7:11553.
doi: 10.1038/ncomms11553.

Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies

M Isabel Veiga  1   2 , Satish K Dhingra  1 , Philipp P Henrich  1 , Judith Straimer  1 , Nina Gnädig  1 , Anne-Catrin Uhlemann  3 , Rowena E Martin  4 , Adele M Lehane  1   4 , David A Fidock  1   3
Affiliations

Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies

M Isabel Veiga et al. Nat Commun. .

Abstract

Antimalarial chemotherapy, globally reliant on artemisinin-based combination therapies (ACTs), is threatened by the spread of drug resistance in Plasmodium falciparum parasites. Here we use zinc-finger nucleases to genetically modify the multidrug resistance-1 transporter PfMDR1 at amino acids 86 and 184, and demonstrate that the widely prevalent N86Y mutation augments resistance to the ACT partner drug amodiaquine and the former first-line agent chloroquine. In contrast, N86Y increases parasite susceptibility to the partner drugs lumefantrine and mefloquine, and the active artemisinin metabolite dihydroartemisinin. The PfMDR1 N86 plus Y184F isoform moderately reduces piperaquine potency in strains expressing an Asian/African variant of the chloroquine resistance transporter PfCRT. Mutations in both digestive vacuole-resident transporters are thought to differentially regulate ACT drug interactions with host haem, a product of parasite-mediated haemoglobin degradation. Global mapping of these mutations illustrates where the different ACTs could be selectively deployed to optimize treatment based on regional differences in PfMDR1 haplotypes.

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Figures

Figure 1
Figure 1. Chemical structures of antimalarials used in ACTs.
Clinically used artemisinin derivatives are shown on the top left. DHA, dihydroartemisinin.
Figure 2
Figure 2. Geographical distribution of PfMDR1 haplotypes at residues 86 and 184 and pfmdr1 copy number variations.
Data were compiled using the Sanger Pf3k data set (www.malariagen.net/apps/pf3k/release_3/index.html) and comprised 2,512 genomes. Pie charts represent proportions of PfMDR1 haplotypes (numerical data per country in Supplementary Table 1). Country colourings show average pfmdr1 amplification compared with the 3D7 reference genome with single-copy pfmdr1. Vertical bars in Southeast Asia show the proportions of haplotypes represented among pfmdr1 copy number variants. Vertical bars colouring: White: no pfmdr1 amplification; blue: pfmdr1 amplification plus NY haplotype; red: pfmdr1 amplification plus NF haplotype. Countries where PfMDR1 individual polymorphism data are available via WWARN (www.wwarn.org/tracking-resistance/molecular-surveyor-pfmdr1-pfcrt) are indicated in grey (data not evaluated herein due to the lack of combined N86 and Y184 status in the full data set).
Figure 3
Figure 3. Schematic of the ZFN-based pfmdr1 editing strategy.
The pmdr1 plasmid expresses the 2A-linked pfmdr1-specific ZFN pair from the calmodulin promoter and the human dhfr selectable marker. These ZFNs create a double-stranded break that can be repaired using the plasmid-borne homologous donor sequence that extends 0.6 kb upstream and 1.8 kb downstream of the target site (yellow thunderbolt). The four pmdr1 transfection plasmids carry three silent mutations at the binding sites to prevent ZFNs cleaving the plasmids or the edited sequence. These plasmids encode the four haplotypes at residues 86 and 184. Shown is the example of transfecting a N86/Y184 parasite with the pmdr1NY plasmid (Table 2). Chromatograms show sequence analysis of genomic and recombinant DNA samples. Mutations at the ZFN binding site (red arrows) and the N86Y and Y184F change of codons are indicated.
Figure 4
Figure 4. In vitro IC50 response of pfmdr1-modified and parental lines.
IC50 values (nM) were determined by incubating parasites for 72 h across a range of drug concentrations. Parasite growth was determined by measuring parasitaemia using flow cytometry with cells stained with Mito Tracker Deep Red and SYBR Green. Mean±s.e.m. IC50 values are presented for lumefantrine (LMF), mefloquine (MFQ), dihydroartemisinin (DHA) and piperaquine (PPQ). Five to ten assays were performed for each drug (Supplementary Tables 3 and 4). Statistical evaluations comparing mutant pfmdr1-modified lines against recombinant control lines of the same genetic backgrounds (NF10mdr1-YF-1 and KC5mdr1-YF-1 for NF10 and KC5 lines, respectively) were performed using two-tailed Mann–Whitney U-tests. *P<0.05, **P<0.01 and ***P<0.001.
Figure 5
Figure 5. In vitro IC50 response of pfmdr1-modified and parental lines.
IC50 values (nM) are presented for CQ, md-CQ, md-ADQ and QN. Five to ten assays were performed for each drug (Supplementary Tables 3 and 4). Statistical evaluations comparing mutant pfmdr1-modified lines against recombinant control lines of the same genetic backgrounds were performed using two-tailed Mann–Whitney U-tests, as described for Fig. 4. *P<0.05, **P<0.01 and ***P<0.001.
Figure 6
Figure 6. Proposed roles for PfMDR1 and PfCRT in the P. falciparum response to CQ and LMF.
(a) CQ (a weak base) and the hydrophobic drug LMF are thought to enter the DV via simple diffusion across the membrane. Within the DV, CQ is thought to trigger parasite killing by binding to reactive haem moieties and preventing their biomineralization into chemically inert haemozoin crystals. LMF also shows moderate binding to haem; however, its main site of action is suspected to be outside of the DV. It is thought that PfCRT facilitates the efflux of drugs from the DV, and that PfMDR1 transports drugs into the DV. (b–e) Point mutations in these transporters are known to differentially impact parasite susceptibility to CQ and LMF. The N86Y mutation is thought to ablate PfMDR1-mediated CQ transport activity, but the resulting reduction in the rate of CQ influx is insufficient to confer CQ resistance in parasites that carry wild-type PfCRT (designated as K76) (b,c). CQ resistance-conferring isoforms of PfCRT (designated K76T) mediate the efflux of CQ from the DV. The reduced rate of CQ influx in PfMDR1 N86Y parasites may further decrease CQ concentrations in the DV and thus augment CQ resistance. However, this effect is only evident in parasites expressing a high-capacity transporter of CQ (for example, GB4 PfCRT). This is perhaps because the loss of CQ influx via PfMDR1 causes a substantial decrease in the time taken to reach sub-lethal levels of CQ in the DV only when the drug is being extruded from the DV via K76T PfCRT at a relatively high rate. (e) LMF might also be a substrate of PfMDR1 N86 (b,d) and the introduction of N86Y (c,e) might likewise reduce the capacity of PfMDR1 to import LMF into the DV. In this scenario, the heightened susceptibility of CQ-resistant N86Y PfMDR1 parasites to LMF could be due to the increased accumulation of LMF in the cytosol (where its main antimalarial target may be located). The relative sizes of the CQ and LMF text in these panels reflect the predicted distribution of the drugs between the DV and cytosol.
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