Shafik SH et al. (2022), Mechanistic basis for multidrug resistance and ...

XB-ART-58873

PLoS Biol 2022 May 04;205:e3001616. doi: 10.1371/journal.pbio.3001616.

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Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT.

Shafik SH , Richards SN , Corry B , Martin RE .

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Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite's susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite's susceptibility to lumefantrine and mefloquine-a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite's digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine's access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.

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Species referenced: Xenopus laevis
Genes referenced: akr1c2

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	Fig 1. Heterologous expression of functional human P-gp and PfMDR1 in Xenopus oocytes. (a) Schematic showing the transport of a [3H]drug via human P-gp or PfMDR1 in the Xenopus oocyte system. (b) The relationship between the quantity of human P-gp cRNA injected into the oocyte and the level of human P-gp–mediated [3H]vinblastine transport measured. (c) The transport of [3H]vinblastine via human P-gp was approximately linear with time for at least 2 hours. (d) The human P-gp–mediated efflux of [3H]vinblastine from oocytes was reduced by known inhibitors of the transporter (nicardipine, PSC8333, vanadate, and verapamil) and was unaffected by amantadine (a drug that does not interact with human P-gp). (e) The field isoforms of PfMDR1 characterized in this study. Residues that differ from the wild-type amino acid sequence are shaded gray. (f) Immunofluorescence microscopy images confirmed that PfMDR1 localized to the oocyte plasma membrane. The expression of 3xHA-tagged PfMDR1NYSND resulted in a fluorescent band external to the pigment layer, indicating that the protein was expressed at the oocyte surface. The band was not present in ne. The length of the scale bar is 50 μm. Images showing the presence of the other HA-tagged PfMDR1 isoforms at the oocyte surface are shown in S4 Fig. (g) Semiquantification of PfMDR1 protein levels in the membranes of oocytes expressing different 3xHA-PfMDR1 isoforms indicated that the 7 isoforms were expressed at similar levels. (h) The transport of [3H]vinblastine via PfMDR1. (i) The efflux of [3H]lumefantrine from oocytes expressing PfMDR1NYSND was reduced by nicardipine, PSC8333, vanadate, and verapamil and was unaffected by amantadine. (j) The relationship between the quantity of PfMDR1NYSND cRNA microinjected into the oocyte and the level of PfMDR1NYSND-mediated [3H]lumefantrine transport measured. (k) The transport of [3H]lumefantrine via PfMDR1NYSND was approximately linear with time for at least 2 hours. (l) Microinjection of additional ATP into the oocyte greatly stimulated [3H]lumefantrine transport via PfMDR1NFCDY. The data are the mean of n = 4 to 9 independent experiments, each yielding similar results and overlaid as individual data points in panels d, g, h, and i, and the error is the SEM. Where not visible, the error bars fall within the symbols. The asterisks denote a significant difference from human P-gp (panel d), 3xHA-PfMDR1NYSND (panel g), or PfMDR1NYSND (panels h and i); ***P < 0.001, ns, not significant (1-way ANOVA). The data underlying this figure is supplied in S3 Data. ne, nonexpressing oocytes; PfMDR1, Plasmodium falciparum multidrug resistance protein 1; P-gp, P-glycoprotein. https://doi.org/10.1371/journal.pbio.3001616.g001
	Fig 2. Field isoforms of PfMDR1 differ significantly in their capacities for antimalarial drug transport. (a–i) Field isoforms of PfMDR1 transported the antimalarial drugs [3H]lumefantrine (a), [3H]mefloquine (b), [3H]chloroquine (c), [3H]quinidine (d), [3H]amodiaquine (e), [3H]piperaquine (f), [3H]dihydroartemisinin (g), methylene blue (h), and quinacrine (i). (j–l) All of the field isoforms of PfMDR1 also transported the human P-gp substrates rhodamine B (j) and [3H]vinblastine (k), but none transported [3H]amantadine (l). The transport of methylene blue, quinacrine, and rhodamine B was detected using the intrinsic fluorescence of these compounds and a fluorescence-based transport assay (see S2 Text). The data are the mean of n = 4 independent experiments (each yielding similar results and overlaid as individual data points), and the error is the SEM. The asterisks denote a significant difference from PfMDR1NYSND; P < 0.05, P < 0.01, **P < 0.001, ns, not significant (1-way ANOVA). The data underlying this figure is supplied in S3 Data. ne, nonexpressing oocytes; PfMDR1, Plasmodium falciparum multidrug resistance protein 1. https://doi.org/10.1371/journal.pbio.3001616.g002
	Fig 3. Characterization of lumefantrine, mefloquine, and chloroquine transport via PfMDR1. (a–c) There were significant differences in the apparent kinetic parameters of lumefantrine (a), mefloquine (b), and chloroquine (c) transport between field isoforms of PfMDR1. The concentration dependence of PfMDR1-mediated drug transport was calculated by subtracting the leakage from ne from that of oocytes expressing a PfMDR1 isoform at each drug concentration. (d–f) The transport of [3H]vinblastine via PfMDR1 was inhibited by lumefantrine (d), [3H]mefloquine (e), and [3H]chloroquine (f). The data are the mean of n = 4 independent experiments (each yielding similar results), and the error is the SEM. The asterisks denote a significant difference from PfMDR1NYSND; P < 0.05, P < 0.01, **P < 0.001, ns nonsignificant (1-way ANOVA). The data underlying this figure is supplied in S3 Data. ne, nonexpressing oocytes; PfMDR1, Plasmodium falciparum multidrug resistance protein 1. https://doi.org/10.1371/journal.pbio.3001616.g003
	Fig 4. Quinine transport via PfMDR1. (a) Field isoforms of PfMDR1 transported [3H]quinine, with PfMDR1NFSDD exhibiting the highest level of quinine transport activity. (b) There were significant differences in the apparent kinetic parameters for quinine transport via field isoforms of PfMDR1. The concentration dependence of PfMDR1-mediated quinine transport was calculated by subtracting the leakage from ne from that of oocytes expressing a PfMDR1 isoform at each drug concentration. The data are the mean of n = 4 independent experiments (each yielding similar results and overlaid as individual data points in panel a), and the error is the SEM. The asterisks denote a significant difference from PfMDR1NYSND; P < 0.01 and *P < 0.001 (1-way ANOVA). The data underlying panels a and b of this figure is supplied in S3 Data. (c) Inward-open homology model of PfMDR1 based on the C. elegans P-gp crystal structure (PDB 4F4C) showing the location of the mutations found in the 5 field PfMDR1 isoforms characterized in this study (orange). A putative binding pose of quinine in the central cavity is shown (pink). (d) A comparison of the inward-open model and outward-open models (based on the crystal structure of human P-gp; PDB 6C0V) as viewed from the side of the protein facing into the DV lumen. The N86Y mutation is part of a cluster of 3 residues forming the extracellular gate of the transporter (the participating residues are shown). These 3 residues are in close proximity in the inward-open state but move apart in the outward-open conformation. (e) Putative quinine binding site in PfMDR1NFSDD. Amino acids interacting with quinine are indicated (apart from L71, I1071, and F1072 that are removed to clearly view the binding pose). Atoms are shaded as follows: carbon in quinine, pink; carbon in the 1042D residue, orange; nitrogen, blue; oxygen, red; hydrogen, white. ne, nonexpressing oocytes; PfMDR1, Plasmodium falciparum multidrug resistance protein 1; P-gp, P-glycoprotein. https://doi.org/10.1371/journal.pbio.3001616.g004
	Fig 5. Correlations between rates of drug transport via PfMDR1 in Xenopus oocytes and in vitro parasite drug resistance indices. The rates of PfMDR1-mediated transport of lumefantrine, mefloquine, chloroquine, and quinine (Figs 2 and 4, and S1 Data) were plotted against the in vitro resistance index for the relevant drug and parasite strain (S3 Table). Where not shown, error bars fall within the symbols. (a) A positive correlation was observed between the rate of lumefantrine transport via PfMDR1 and the in vitro lumefantrine response (Pearson correlation coefficient = 0.76, r2 = 0.58, P = 0.078). (b) A positive correlation was observed between the rate of mefloquine transport via PfMDR1 and the in vitro mefloquine response (Pearson correlation coefficient = 0.75, r2 = 0.56, P = 0.008). The data point for Dd2 is an outlier and its removal significantly improved the correlation (Pearson correlation coefficient = 0.97, r2 = 0.94, P = 0.006). Dd2 parasites typically harbor 2 to 4 copies of PfMDR1 and have been reported to express greater levels of PfMDR1 than the other parasite strains included in the analysis [85,87]. Given that pfmdr1 amplification has been associated with mefloquine resistance [30–32,88,89], it is possible that the overexpression of PfMDR1 in Dd2 parasites imparts a higher level of resistance to this strain (see S3 Text for an extended analysis). (c) An inverse correlation was observed between the rate of chloroquine transport via PfMDR1 and the in vitro chloroquine response. The removal of the 7G8 data point (see S3 Text for an extended analysis) resulted in a stronger correlation between the rate of chloroquine transport via PfMDR1 and the parasite’s chloroquine response in vitro (Pearson correlation coefficient = −0.86, r2 = 0.74, P = 0.054). (d) There was no correlation between the rate of quinine transport via PfMDR1 and the in vitro quinine response (see S4 Text for an extended analysis). The data for the in vitro resistance indices are the mean of n = 2 to 18 published studies, and the data for the PfMDR1-mediated drug transport rates are the mean of n = 4 independent experiments. The error is the SEM except for the in vitro resistance indices that were calculated from 2 studies, in which case the error is the range/2. The data underlying this figure is supplied in S3 Data. PfMDR1, Plasmodium falciparum multidrug resistance protein 1. https://doi.org/10.1371/journal.pbio.3001616.g005
	Fig 6. Transport of lumefantrine and mefloquine via PfCRT in Xenopus oocytes and in situ. (a, d) The transport of [3H]lumefantrine (a) and [3H]mefloquine (d) via PfCRTDd2 and PfCRT7G8 was reduced by verapamil (VP; 100 μM). The asterisks denote a significant difference from the ne (gray asterisks), the PfCRT7G8 control (orange asterisks), or the PfCRTDd2 control (red asterisks). (b, e) The transport of lumefantrine (b) and mefloquine (e) via PfCRTDd2 and PfCRT7G8 was saturable. The asterisks denote a significant difference from PfCRTDd2 (red asterisks). (c, f) The effects of unlabeled lumefantrine (c) and unlabeled mefloquine (f) on [3H]VDPVNF transport via PfCRT3D7, PfCRTDd2, and PfCRT7G8. The asterisks denote a significant difference from PfCRT3D7 (blue asterisks). (g) Lumefantrine and mefloquine (2.5 μM) increased the rate of DV alkalinization in the chloroquine-resistant C67G8 and C4Dd2 parasite lines but not in the chloroquine-sensitive C2GC03 line. Unless labeled ns, P < 0.05 relative to the absence of a test solute. (h) The increase in the rate of DV alkalinization caused by lumefantrine and mefloquine was inhibited by VP (50 μM). The asterisks denote a significant difference from the relevant C2GC03 (blue asterisks), C67G8 (orange asterisks), or C4Dd2 (red asterisks) treatments in the absence of verapamil. (i, j) Lumefantrine (i) and mefloquine (j) increased the rate of DV alkalinization in a concentration-dependent manner. The data are the mean of n = 4 independent experiments, each yielding similar results and overlaid as individual data points in panels a, d, g, and h, and the error is the SEM. Where not visible, the error bars fall within the symbols. P < 0.01, *P < 0.001, ns, not significant (1-way ANOVA). The data underlying this figure is supplied in S3 Data. ne, nonexpressing oocytes; PfCRT, Plasmodium falciparum chloroquine resistance transporter. https://doi.org/10.1371/journal.pbio.3001616.g006
	Fig 7. Roles of PfMDR1 and PfCRT in the parasite’s susceptibility to lumefantrine, mefloquine, and chloroquine. Mechanistic explanations for how polymorphisms in pfmdr1 and pfcrt alter the parasite’s response to lumefantrine and mefloquine (a) and chloroquine (b). Lumefantrine, mefloquine, and chloroquine are weak bases that enter the DV via 2 main routes: (1) simple diffusion of the neutral species across the membrane and subsequent protonation within the acidic DV lumen; and (2) ATP-driven import via PfMDR1. Wild-type PfMDR1 has a high capacity for drug transport and this activity, together with the inability of wild-type PfCRT to efflux lumefantrine, mefloquine, or chloroquine from the DV, causes these drugs to sequester within the DV. Overexpression of PfMDR1 results in a further increase in the rate of drug transport from the cytosol into the DV and thus greater accumulation of lumefantrine, mefloquine, and chloroquine in the DV (as well as concomitant reductions in their cytosolic concentrations). In parasites carrying a mutant PfMDR1 isoform (and/or only one pfmdr1 copy) as well as a mutant isoform of PfCRT, there is a marked reduction in the DV accumulation of lumefantrine, mefloquine, and chloroquine as a result of (1) a decrease in the rate of drug import via PfMDR1; and (2) the PfCRT-mediated efflux of drugs from the DV back into the cytosol. The reduction in the concentration of chloroquine at its primary site of action allows the parasite to evade its killing effects, thereby causing chloroquine resistance. On the other hand, the concomitant increases in the cytosolic drug concentrations render these parasites more sensitive to lumefantrine and mefloquine, indicating that the primary targets of both drugs are located outside of the DV. CQ, chloroquine; DV, digestive vacuole; LM, lumefantrine; MQ, mefloquine; PfCRT, Plasmodium falciparum chloroquine resistance transporter; PfMDR1, Plasmodium falciparum multidrug resistance protein 1. https://doi.org/10.1371/journal.pbio.3001616.g007

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	Fig 1. Heterologous expression of functional human P-gp and PfMDR1 in Xenopus oocytes. (a) Schematic showing the transport of a [3H]drug via human P-gp or PfMDR1 in the Xenopus oocyte system. (b) The relationship between the quantity of human P-gp cRNA injected into the oocyte and the level of human P-gp–mediated [3H]vinblastine transport measured. (c) The transport of [3H]vinblastine via human P-gp was approximately linear with time for at least 2 hours. (d) The human P-gp–mediated efflux of [3H]vinblastine from oocytes was reduced by known inhibitors of the transporter (nicardipine, PSC8333, vanadate, and verapamil) and was unaffected by amantadine (a drug that does not interact with human P-gp). (e) The field isoforms of PfMDR1 characterized in this study. Residues that differ from the wild-type amino acid sequence are shaded gray. (f) Immunofluorescence microscopy images confirmed that PfMDR1 localized to the oocyte plasma membrane. The expression of 3xHA-tagged PfMDR1NYSND resulted in a fluorescent band external to the pigment layer, indicating that the protein was expressed at the oocyte surface. The band was not present in ne. The length of the scale bar is 50 μm. Images showing the presence of the other HA-tagged PfMDR1 isoforms at the oocyte surface are shown in S4 Fig. (g) Semiquantification of PfMDR1 protein levels in the membranes of oocytes expressing different 3xHA-PfMDR1 isoforms indicated that the 7 isoforms were expressed at similar levels. (h) The transport of [3H]vinblastine via PfMDR1. (i) The efflux of [3H]lumefantrine from oocytes expressing PfMDR1NYSND was reduced by nicardipine, PSC8333, vanadate, and verapamil and was unaffected by amantadine. (j) The relationship between the quantity of PfMDR1NYSND cRNA microinjected into the oocyte and the level of PfMDR1NYSND-mediated [3H]lumefantrine transport measured. (k) The transport of [3H]lumefantrine via PfMDR1NYSND was approximately linear with time for at least 2 hours. (l) Microinjection of additional ATP into the oocyte greatly stimulated [3H]lumefantrine transport via PfMDR1NFCDY. The data are the mean of n = 4 to 9 independent experiments, each yielding similar results and overlaid as individual data points in panels d, g, h, and i, and the error is the SEM. Where not visible, the error bars fall within the symbols. The asterisks denote a significant difference from human P-gp (panel d), 3xHA-PfMDR1NYSND (panel g), or PfMDR1NYSND (panels h and i); ***P < 0.001, ns, not significant (1-way ANOVA). The data underlying this figure is supplied in S3 Data. ne, nonexpressing oocytes; PfMDR1, Plasmodium falciparum multidrug resistance protein 1; P-gp, P-glycoprotein. https://doi.org/10.1371/journal.pbio.3001616.g001
	Fig 2. Field isoforms of PfMDR1 differ significantly in their capacities for antimalarial drug transport. (a–i) Field isoforms of PfMDR1 transported the antimalarial drugs [3H]lumefantrine (a), [3H]mefloquine (b), [3H]chloroquine (c), [3H]quinidine (d), [3H]amodiaquine (e), [3H]piperaquine (f), [3H]dihydroartemisinin (g), methylene blue (h), and quinacrine (i). (j–l) All of the field isoforms of PfMDR1 also transported the human P-gp substrates rhodamine B (j) and [3H]vinblastine (k), but none transported [3H]amantadine (l). The transport of methylene blue, quinacrine, and rhodamine B was detected using the intrinsic fluorescence of these compounds and a fluorescence-based transport assay (see S2 Text). The data are the mean of n = 4 independent experiments (each yielding similar results and overlaid as individual data points), and the error is the SEM. The asterisks denote a significant difference from PfMDR1NYSND; P < 0.05, P < 0.01, **P < 0.001, ns, not significant (1-way ANOVA). The data underlying this figure is supplied in S3 Data. ne, nonexpressing oocytes; PfMDR1, Plasmodium falciparum multidrug resistance protein 1. https://doi.org/10.1371/journal.pbio.3001616.g002
	Fig 3. Characterization of lumefantrine, mefloquine, and chloroquine transport via PfMDR1. (a–c) There were significant differences in the apparent kinetic parameters of lumefantrine (a), mefloquine (b), and chloroquine (c) transport between field isoforms of PfMDR1. The concentration dependence of PfMDR1-mediated drug transport was calculated by subtracting the leakage from ne from that of oocytes expressing a PfMDR1 isoform at each drug concentration. (d–f) The transport of [3H]vinblastine via PfMDR1 was inhibited by lumefantrine (d), [3H]mefloquine (e), and [3H]chloroquine (f). The data are the mean of n = 4 independent experiments (each yielding similar results), and the error is the SEM. The asterisks denote a significant difference from PfMDR1NYSND; P < 0.05, P < 0.01, **P < 0.001, ns nonsignificant (1-way ANOVA). The data underlying this figure is supplied in S3 Data. ne, nonexpressing oocytes; PfMDR1, Plasmodium falciparum multidrug resistance protein 1. https://doi.org/10.1371/journal.pbio.3001616.g003
	Fig 4. Quinine transport via PfMDR1. (a) Field isoforms of PfMDR1 transported [3H]quinine, with PfMDR1NFSDD exhibiting the highest level of quinine transport activity. (b) There were significant differences in the apparent kinetic parameters for quinine transport via field isoforms of PfMDR1. The concentration dependence of PfMDR1-mediated quinine transport was calculated by subtracting the leakage from ne from that of oocytes expressing a PfMDR1 isoform at each drug concentration. The data are the mean of n = 4 independent experiments (each yielding similar results and overlaid as individual data points in panel a), and the error is the SEM. The asterisks denote a significant difference from PfMDR1NYSND; P < 0.01 and *P < 0.001 (1-way ANOVA). The data underlying panels a and b of this figure is supplied in S3 Data. (c) Inward-open homology model of PfMDR1 based on the C. elegans P-gp crystal structure (PDB 4F4C) showing the location of the mutations found in the 5 field PfMDR1 isoforms characterized in this study (orange). A putative binding pose of quinine in the central cavity is shown (pink). (d) A comparison of the inward-open model and outward-open models (based on the crystal structure of human P-gp; PDB 6C0V) as viewed from the side of the protein facing into the DV lumen. The N86Y mutation is part of a cluster of 3 residues forming the extracellular gate of the transporter (the participating residues are shown). These 3 residues are in close proximity in the inward-open state but move apart in the outward-open conformation. (e) Putative quinine binding site in PfMDR1NFSDD. Amino acids interacting with quinine are indicated (apart from L71, I1071, and F1072 that are removed to clearly view the binding pose). Atoms are shaded as follows: carbon in quinine, pink; carbon in the 1042D residue, orange; nitrogen, blue; oxygen, red; hydrogen, white. ne, nonexpressing oocytes; PfMDR1, Plasmodium falciparum multidrug resistance protein 1; P-gp, P-glycoprotein. https://doi.org/10.1371/journal.pbio.3001616.g004
	Fig 5. Correlations between rates of drug transport via PfMDR1 in Xenopus oocytes and in vitro parasite drug resistance indices. The rates of PfMDR1-mediated transport of lumefantrine, mefloquine, chloroquine, and quinine (Figs 2 and 4, and S1 Data) were plotted against the in vitro resistance index for the relevant drug and parasite strain (S3 Table). Where not shown, error bars fall within the symbols. (a) A positive correlation was observed between the rate of lumefantrine transport via PfMDR1 and the in vitro lumefantrine response (Pearson correlation coefficient = 0.76, r2 = 0.58, P = 0.078). (b) A positive correlation was observed between the rate of mefloquine transport via PfMDR1 and the in vitro mefloquine response (Pearson correlation coefficient = 0.75, r2 = 0.56, P = 0.008). The data point for Dd2 is an outlier and its removal significantly improved the correlation (Pearson correlation coefficient = 0.97, r2 = 0.94, P = 0.006). Dd2 parasites typically harbor 2 to 4 copies of PfMDR1 and have been reported to express greater levels of PfMDR1 than the other parasite strains included in the analysis [85,87]. Given that pfmdr1 amplification has been associated with mefloquine resistance [30–32,88,89], it is possible that the overexpression of PfMDR1 in Dd2 parasites imparts a higher level of resistance to this strain (see S3 Text for an extended analysis). (c) An inverse correlation was observed between the rate of chloroquine transport via PfMDR1 and the in vitro chloroquine response. The removal of the 7G8 data point (see S3 Text for an extended analysis) resulted in a stronger correlation between the rate of chloroquine transport via PfMDR1 and the parasite’s chloroquine response in vitro (Pearson correlation coefficient = −0.86, r2 = 0.74, P = 0.054). (d) There was no correlation between the rate of quinine transport via PfMDR1 and the in vitro quinine response (see S4 Text for an extended analysis). The data for the in vitro resistance indices are the mean of n = 2 to 18 published studies, and the data for the PfMDR1-mediated drug transport rates are the mean of n = 4 independent experiments. The error is the SEM except for the in vitro resistance indices that were calculated from 2 studies, in which case the error is the range/2. The data underlying this figure is supplied in S3 Data. PfMDR1, Plasmodium falciparum multidrug resistance protein 1. https://doi.org/10.1371/journal.pbio.3001616.g005
	Fig 6. Transport of lumefantrine and mefloquine via PfCRT in Xenopus oocytes and in situ. (a, d) The transport of [3H]lumefantrine (a) and [3H]mefloquine (d) via PfCRTDd2 and PfCRT7G8 was reduced by verapamil (VP; 100 μM). The asterisks denote a significant difference from the ne (gray asterisks), the PfCRT7G8 control (orange asterisks), or the PfCRTDd2 control (red asterisks). (b, e) The transport of lumefantrine (b) and mefloquine (e) via PfCRTDd2 and PfCRT7G8 was saturable. The asterisks denote a significant difference from PfCRTDd2 (red asterisks). (c, f) The effects of unlabeled lumefantrine (c) and unlabeled mefloquine (f) on [3H]VDPVNF transport via PfCRT3D7, PfCRTDd2, and PfCRT7G8. The asterisks denote a significant difference from PfCRT3D7 (blue asterisks). (g) Lumefantrine and mefloquine (2.5 μM) increased the rate of DV alkalinization in the chloroquine-resistant C67G8 and C4Dd2 parasite lines but not in the chloroquine-sensitive C2GC03 line. Unless labeled ns, P < 0.05 relative to the absence of a test solute. (h) The increase in the rate of DV alkalinization caused by lumefantrine and mefloquine was inhibited by VP (50 μM). The asterisks denote a significant difference from the relevant C2GC03 (blue asterisks), C67G8 (orange asterisks), or C4Dd2 (red asterisks) treatments in the absence of verapamil. (i, j) Lumefantrine (i) and mefloquine (j) increased the rate of DV alkalinization in a concentration-dependent manner. The data are the mean of n = 4 independent experiments, each yielding similar results and overlaid as individual data points in panels a, d, g, and h, and the error is the SEM. Where not visible, the error bars fall within the symbols. P < 0.01, *P < 0.001, ns, not significant (1-way ANOVA). The data underlying this figure is supplied in S3 Data. ne, nonexpressing oocytes; PfCRT, Plasmodium falciparum chloroquine resistance transporter. https://doi.org/10.1371/journal.pbio.3001616.g006
	Fig 7. Roles of PfMDR1 and PfCRT in the parasite’s susceptibility to lumefantrine, mefloquine, and chloroquine. Mechanistic explanations for how polymorphisms in pfmdr1 and pfcrt alter the parasite’s response to lumefantrine and mefloquine (a) and chloroquine (b). Lumefantrine, mefloquine, and chloroquine are weak bases that enter the DV via 2 main routes: (1) simple diffusion of the neutral species across the membrane and subsequent protonation within the acidic DV lumen; and (2) ATP-driven import via PfMDR1. Wild-type PfMDR1 has a high capacity for drug transport and this activity, together with the inability of wild-type PfCRT to efflux lumefantrine, mefloquine, or chloroquine from the DV, causes these drugs to sequester within the DV. Overexpression of PfMDR1 results in a further increase in the rate of drug transport from the cytosol into the DV and thus greater accumulation of lumefantrine, mefloquine, and chloroquine in the DV (as well as concomitant reductions in their cytosolic concentrations). In parasites carrying a mutant PfMDR1 isoform (and/or only one pfmdr1 copy) as well as a mutant isoform of PfCRT, there is a marked reduction in the DV accumulation of lumefantrine, mefloquine, and chloroquine as a result of (1) a decrease in the rate of drug import via PfMDR1; and (2) the PfCRT-mediated efflux of drugs from the DV back into the cytosol. The reduction in the concentration of chloroquine at its primary site of action allows the parasite to evade its killing effects, thereby causing chloroquine resistance. On the other hand, the concomitant increases in the cytosolic drug concentrations render these parasites more sensitive to lumefantrine and mefloquine, indicating that the primary targets of both drugs are located outside of the DV. CQ, chloroquine; DV, digestive vacuole; LM, lumefantrine; MQ, mefloquine; PfCRT, Plasmodium falciparum chloroquine resistance transporter; PfMDR1, Plasmodium falciparum multidrug resistance protein 1. https://doi.org/10.1371/journal.pbio.3001616.g007