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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Chloroquine-resistant malaria parasites (Plasmodium falciparum) show an increased leak of H+ ions from their internal digestive vacuole in the presence of chloroquine. This phenomenon has been attributed to the transport of chloroquine, together with H+, out of the digestive vacuole (and hence away from its site of action) via a mutant form of the parasite's chloroquine resistance transporter (PfCRT). Here, using transfectant parasite lines, we show that a range of other antimalarial drugs, as well as various ‘chloroquine resistance reversers’ induce an increased leak of H+ from the digestive vacuole of parasites expressing mutant PfCRT, consistent with these compounds being substrates for mutant forms, but not the wild-type form, of PfCRT. For some compounds there were significant differences observed between parasites having the African/Asian Dd2 form of PfCRT and those with the South American 7G8 form of PfCRT, consistent with there being differences in the transport properties of the two mutant proteins. The finding that chloroquine resistance reversers are substrates for mutant PfCRT has implications for the mechanism of action of this class of compound.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Chloroquine (CQ) acts against the intraerythrocytic stage of the human malaria parasite Plasmodium falciparum, and is thought to exert its toxic effect in the parasite's acidic digestive vacuole (DV). In this organelle, haemoglobin acquired from the host erythrocyte cytosol is digested, generating peptides/amino acids and toxic haem moities (Francis et al., 1997). In its neutral form, CQ is membrane-permeant and can diffuse across the membranes separating the DV interior from the extracellular milieu. However, once inside the acidic DV, CQ becomes protonated (mostly diprotonated), which renders it less membrane-permeant and results in its accumulation to high concentrations (Homewood et al., 1972; Yayon et al., 1984; Ferrari and Cutler, 1991). CQ is thought to exert its antiplasmodial effect by binding to haem and preventing its incorporation into inert haemozoin crystals (Orjih et al., 1994; Bray et al., 1998; 1999; Fitch, 2004).

Chloroquine is now ineffective in most malaria-endemic regions as a result of the emergence and spread of CQ-resistant (CQR) P. falciparum parasites. CQR parasites accumulate significantly less CQ than CQ-sensitive (CQS) parasites (Fitch, 1970; Krogstad et al., 1987). The decreased accumulation has been attributed to mutations in a DV membrane transport protein, the P. falciparum chloroquine resistance transporter (PfCRT) (Fidock et al., 2000), and it has been proposed that the mutant form of the protein transports CQ out of the DV, away from its primary site of accumulation and action (Bray et al., 2005a; Sanchez et al., 2005).

There are multiple lines of evidence that mutations in PfCRT can influence parasite susceptibility to a number of other antimalarial drugs (Cooper et al., 2002; 2007; Mu et al., 2003; Ferdig et al., 2004; Johnson et al., 2004). Direct evidence comes from the pfcrt allelic exchange study performed by Sidhu et al. (2002). Replacing the wild-type pfcrt allele in CQS GC03 parasites with the CQ-resistance-conferring Dd2 or 7G8 alleles was found to render parasites more sensitive to artemisinin (ART), mefloquine (MQ) and quinine (QN) (Sidhu et al., 2002). Further support for an interaction of various antimalarial drugs with PfCRT comes from the recent study by Martin et al. (2009), in which it was shown that the antimalarial drugs amodiaquine (AQ), MQ, quinidine (QD), QN and primaquine (PQ), as well as the CQ resistance reverser verapamil, inhibit the transport of radiolabelled CQ in Xenopus oocytes expressing the Dd2-mutant form of PfCRT (Martin et al., 2009).

We have shown previously that in CQR (but not CQS) parasites the addition of CQ results in an enhanced leak of H+ ions from the DV, consistent with the drug effluxing from the DV of CQR parasites in the protonated form (and/or in symport with H+ ions) (Lehane et al., 2008). In experiments with transfectant parasite lines differing only in their pfcrt allele, the CQ-associated H+ leak from the DV seen in CQR parasites was attributed to CQ-resistance-conferring mutations in PfCRT (Lehane and Kirk, 2008). In this study we have used the same approach, in conjunction with transfectant parasites, to investigate the interaction of different antimalarial drugs and CQ resistance reversers with PfCRT in the parasite. The results provide evidence that a range of drugs are transported by mutant (but not wild-type) PfCRT, with the drug transport properties varying between two different mutant forms of the protein.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Amodiaquine-, quinidine- and quinine-associated H+ leaks from the malaria parasite's DV are conferred by mutations in PfCRT

The weak-base quinoline drugs AQ, MQ, QD and QN, together with the structurally unrelated antimalarial drug ART, were tested for their ability to induce a H+ leak from the DV in the pfcrt transfectant lines generated by Sidhu et al. (2002). In each case, the drug of interest was added to saponin-isolated trophozoite-stage parasites in which the DV had been loaded with fluorescein-dextran. Fluorescence was monitored both before and after the addition of the drug, allowing an assessment of the effects of the drug on fluorescence/pHDV. Two minutes after the addition of drug the DV H+ pump inhibitor concanamycin A (100 nM) was added, with the rate of the consequent DV alkalinization providing a measure of the rate of leakage of H+ from the DV.

Each of the drugs was assessed initially at a concentration of 5 µM and in the presence and absence of verapamil (50 µM). It was shown previously that 50 µM verapamil inhibits completely the CQ-associated H+ leak observed from the DV in CQR strains (Lehane and Kirk, 2008; Lehane et al., 2008). In the case of AQ, it was found that the addition of a 5 µM concentration caused a marked rise in the fluorescence ratio before concanamycin A was added (data not shown), consistent with it causing a rise in pHDV. The concentration of AQ was therefore lowered to 0.625 µM; at this concentration AQ caused a slight increase in the fluorescence ratio in each of the three strains, typically of the order of 15% of the maximum increase seen following the subsequent addition of concanamycin A. An increase of similar magnitude (i.e. ∼15% of that resulting from the addition of concanamycin A) was also seen on addition of 5 µM mefloquine. For all the other drugs shown in Fig. 1, their addition at a concentration of 5 µM had little effect on the fluorescence signal measured at either of the two excitation wavelengths, or on the fluorescence ratio, indicating that they did not cause significant quenching of the fluorescence or have a significant effect on resting pHDV.

image

Figure 1. A, B. Representative fluorometer traces showing the alkalinization of the DV following the addition of concanamycin A (100 nM, at the point indicated by the black triangle) to isolated mature trophozoite-stage CQR C4Dd2 (A) and CQS C2GC03 (B) parasites suspended in the presence of 5 µM QN (dark grey traces), in the presence of 5 µM QN and 50 µM verapamil (‘QN + VP’; light grey traces), in the presence of 50 µM verapamil alone (‘VP’; black traces), and in the absence of QN or verapamil (‘control’; dark grey traces). The compounds were added 2 min before the addition of concanamycin A. C. Averaged data showing the effects of antimalarial drugs on the rate of DV alkalinization measured following the addition of concanamycin A (expressed as the inverse of the half-time for DV alkalinization) in the CQS C2GC03 (grey) strain and in the CQR C4Dd2 (white) and C67G8 (black) strains, in the presence and absence (solvent control) of 50 µM verapamil. The compounds tested were added to suspensions of saponin-isolated trophozoite-stage parasites containing fluorescein-dextran in their DVs 2 min prior to the addition of concanamycin A (100 nM). The concentration of each drug was 5 µM, with the exception of AQ for which the concentration was 0.625 µM. The data (shown + SEM) are averaged from at least three independent experiments for each condition and strain.

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Figure 1A and B shows representative traces, illustrating the effect of one of the drugs tested, QN, on the rate of alkalinization of the DV in C4Dd2 (Fig. 1A) and C2GC03 (Fig. 1B) parasites following the addition of concanamycin A. In CQR C4Dd2 parasites, the presence of QN (5 µM) caused a pronounced increase in the rate of the alkalinization that occurred on addition of concanamycin A, and this increase was reversed by verapamil (Fig. 1A). By contrast, in CQS C2GC03 parasites, the presence of QN had no significant effect on the rate of the concanamycin A-induced alkalinization (Fig. 1B).

Figure 1C summarizes the data obtained with the five different antimalarial drugs tested in the three different strains. As has been reported previously (Lehane and Kirk, 2008), in the absence of drug, the rate of alkalinization of the DV following the addition of concanamycin A was faster in C67G8 parasites than in C2GC03 or C4Dd2 parasites. AQ, QD and QN were found to increase the rate of concanamycin A-induced DV alkalinization (expressed as the inverse of the time taken for half-maximal alkalinization) in the CQR C4Dd2 and C67G8 strains (P < 0.02, paired t-tests), but not in the CQS C2GC03 strain (P > 0.3, paired t-tests). In each case (for both C4Dd2 and C67G8 parasites), the drug-associated increase in the H+ leak was completely inhibited by 50 µM verapamil such that the DV alkalinization rate in the presence of the drug plus verapamil was not significantly different from the rate in the presence of verapamil alone (P > 0.09, paired t-tests).

Mefloquine and ART each caused a small increase in the rate of DV alkalinization in C2GC03 parasites (P ≤ 0.03, paired t-tests); these increases were unaffected by verapamil (P > 0.5, paired t-tests). ART did not affect the rate of DV alkalinization in C4Dd2 or C67G8 parasites (P > 0.4, paired t-tests). MQ caused a slight increase in the rate of DV alkalinization in C4Dd2 parasites (P = 0.001, paired t-test) and a slight decrease in C67G8 parasites (P = 0.01, paired t-test). Verapamil had a slowing effect on DV alkalinization in the presence of MQ in both strains (P < 0.004, paired t-tests).

The data presented in Fig. 1 are consistent with AQ, QD and QN being substrates for the CQ-resistance-conferring Dd2 and 7G8 forms of PfCRT but not the wild-type form, with the increased H+ leak attributable to the drug effluxing from the vacuole in the protonated form and/or in symport with H+ ions. The very small verapamil-insensitive increases in the rates of concanamycin A-induced DV alkalinization caused by MQ and ART in C2GC03 parasites were not investigated further, nor were the slight effects of MQ in C4Dd2 and C67G8 parasites.

The concentration dependence of the H+ leaks associated with AQ, QD and QN in the CQR C4Dd2 and C67G8 strains was investigated in more detail. Figure 2 shows the drug-associated increases in the initial rates of concanamycin A-induced DV alkalinization as a function of the drug concentration. These were obtained by subtracting the initial rate of alkalinization measured in the absence of drug from that measured in the presence of the different concentrations of the drugs. In time course experiments with AQ (0.156 µM), QD (1.25 µM) and QN (1.25 µM), it was found that a 12 min pre-incubation with drug (before concanamycin A addition) was sufficient for the drugs to exert maximal or near-maximal effects on the rate of DV alkalinization in both strains (not shown). A 12 min pre-incubation was therefore used for the concentration dependence experiments shown in Fig. 2.

image

Figure 2. Concentration dependence of the increases in the initial rates of concanamycin A-induced DV alkalinization associated with amodiaquine (A), quinidine (B) and quinine (C) in isolated mature trophozoite-stage CQR C4Dd2 parasites (open circles) and C67G8 parasites (closed circles). The drugs were added to parasite suspensions 12 min before the addition of concanamycin A (100 nM). The data are averaged from three to five independent experiments for each strain and are shown ± SEM (except for A in which, for clarity, only positive error bars are shown for C67G8 and only negative error bars are shown for C4Dd2). Where not shown, error bars fall within the symbols.

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Amodiaquine had a significant effect on the initial rate of DV alkalinization at concentrations ≥ 0.156 µM for C67G8 parasites and ≥ 0.625 µM for C4Dd2 parasites (P < 0.04, paired t-tests). With one exception (0.3125 µM; P = 0.02, unpaired t-test), there was no significant difference between the two CQR strains in the size of the AQ-induced increase in the initial rate of DV alkalinization at any AQ concentration (P > 0.09, unpaired t-tests).

By contrast, there were substantial differences between C4Dd2 parasites and C67G8 parasites in the magnitudes of the QD- and QN-induced increases in the initial rate of DV alkalinization. QD increased the initial rate of DV alkalinization at concentrations ≥ 1.25 µM for C4Dd2 parasites and ≥ 5 µM for C67G8 parasites (P < 0.04, paired t-tests), and the QD-associated initial rates were significantly higher in C4Dd2 parasites than in C67G8 parasites at all concentrations ≥ 1.25 µM (P < 0.04, unpaired t-tests). QN caused a significant increase in the initial rate of DV alkalinization at concentrations ≥ 1.25 µM in C4Dd2 parasites and ≥ 2.5 µM in C67G8 parasites (P < 0.03, paired t-tests). There was a significant difference in the QN-associated initial rates between C4Dd2 parasites and C67G8 parasites at all concentrations ≥ 1.25 µM (P < 0.02, unpaired t-tests), with the rates being higher in C4Dd2 parasites than in C67G8 parasites.

A range of CQ resistance reversers give rise to a H+ leak from the DV in parasites with mutant PfCRT

Most of the CQ resistance reversers identified to date are weak bases that, like CQ and other quinoline drugs, are protonated in the acidic environment of the DV (van Schalkwyk and Egan, 2006). The extent to which such compounds are substrates for mutant PfCRT is unknown. The following, structurally diverse (Table 1), CQ resistance reversers were therefore tested for their effects on the rate of H+ leakage from the DV in parasites expressing the different forms of PfCRT: verapamil (a calcium channel blocker), chlorpheniramine (an antihistamine), desipramine (an antidepressant), promethazine (an antihistamine), fluoxetine (an antidepressant) and PQ (a drug active against Plasmodium vivax liver-stage parasites). CQ was also included for comparison. Each compound was tested at a concentration of 5 µM alone, and (with the exception of verapamil) in the presence of 50 µM verapamil. The compounds were added to the parasites 12 min before the addition of concanamycin A to allow time for their accumulation in the DV. At the 5 µM concentration tested here, desipramine and fluoxetine caused small increases in the fluorescence ratio in both CQR strains, as did promethazine in the C4Dd2 strain (∼15% of the increase seen following the subsequent addition of concanamycin A). The other CQ resistance reversers had little effect on fluorescence/pHDV at the concentrations tested here.

Table 1.  Chemical structures of the chloroquine resistance reversers investigated in this study.
CompoundStructureReference for CQ resistance reversal
Verapamilinline imageKrogstad et al. (1987); Martin et al. (1987)
Chlorpheniramineinline imageBasco and Le Bras (1994)
Desipramineinline imageBitonti et al. (1988)
Promethazineinline imageOduola et al. (1998)
Fluoxetineinline imageGerena et al. (1992)
Primaquineinline imageBray et al. (2005b)

Figure 3A and B shows representative traces, illustrating the effect of one of the CQ resistance reversers, chlorpheniramine, on the rate of alkalinization of the DV in C67G8 (Fig. 3A) and C2GC03 (Fig. 3B) parasites following the addition of concanamycin A. In CQR C67G8 parasites, the presence of chlorpheniramine (5 µM) caused a pronounced increase in the rate of the alkalinization that occurred on addition of concanamycin A, and this increase was reversed by verapamil (Fig. 3A). By contrast, in CQS C2GC03 parasites, the presence of chlorpheniramine had no significant effect on the rate of the concanamycin A-induced alkalinization (Fig. 3B).

image

Figure 3. A, B. Representative fluorometer traces showing the alkalinization of the DV following the addition of concanamycin A (100 nM, at the point indicated by the black triangle) to isolated mature trophozoite-stage CQR C67G8 (A) and CQS C2GC03 (B) parasites suspended in the presence of 5 µM chlorpheniramine (‘CPh’; dark grey traces), in the presence of 5 µM chlorpheniramine and 50 µM verapamil (‘CPh + VP’; light grey traces), in the presence of 50 µM verapamil alone (‘VP’; black traces), and in the absence of chlorpheniramine or verapamil (‘control’; dark grey traces). The compounds were added 12 min before the addition of concanamycin A. C. Averaged data showing the effect of a range of CQ resistance reversers, each at a concentration of 5 µM (verapamil, chlorpheniramine, desipramine, promethazine, fluoxetine and primaquine), and CQ (5 µM) on the rate of DV alkalinization measured following the addition of concanamycin A (expressed as the inverse of the half-time for DV alkalinization) in the CQS C2GC03 strain (grey) and in the CQR C4Dd2 (white) and C67G8 (black) strains, in the presence and absence of 50 µM verapamil. The compounds were added to parasites 12 min prior to the addition of concanamycin A (100 nM). The data (shown + SEM) are averaged from at least three independent experiments for each condition and strain.

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Figure 3C summarizes the data obtained with the various resistance reversers of interest.

In CQS C2GC03 parasites, none of the compounds tested affected the rate of DV alkalinization (P > 0.1, paired t-tests).

In CQR C4Dd2 parasites, verapamil caused a significant increase in the rate of alkalinization, with the increase seen in the presence of 5 µM verapamil being significantly higher than that seen here [and seen previously; (Lehane and Kirk, 2008)] in the presence of 50 µM verapamil. Figure 4 shows the concentration dependence of the effect of verapamil on the rate of concanamycin-A induced DV alkalinization. The DV alkalinization rate in C4Dd2 parasites initially increased with increasing verapamil concentration, reaching a maximum at 5 µM verapamil and decreasing as the concentration was increased further [although still remaining greater than the control at the highest concentration tested (50 µM)].

image

Figure 4. The effect of increasing concentrations of verapamil on the rate of concanamycin A-induced DV alkalinization in CQR C4Dd2 (open circles) and C67G8 (closed circles) parasites. Verapamil was added to parasite suspensions 12 min before the addition of 100 nM concanamycin A. The data (shown mean ± range/2) are averaged from two independent experiments for each strain. Where not shown, error bars fall within the symbols.

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In C4Dd2 parasites, chlorpheniramine, desipramine, promethazine, fluoxetine, PQ and CQ also gave rise to significant increases in the rate of concanamycin A-induced DV alkalinization (P < 0.03, paired t-tests; Fig. 3C). The increase was inhibited completely by 50 µM verapamil in each case, such that there was no significant difference between the verapamil-only condition and any of the ‘compound + verapamil’ conditions (P > 0.4, paired t-tests).

In CQR C67G8 parasites, chlorpheniramine, desipramine, fluoxetine and CQ significantly increased the rate of concanamycin A-induced DV alkalinization (P < 0.04, paired t-tests). This increase was inhibited by verapamil (50 µM). When added to C67G8 parasites on its own, at a lower concentration (5 µM), verapamil slowed DV alkalinization (P = 0.03, paired t-test), with the rate of DV alkalinization decreasing monotonically with increasing verapamil concentration (Fig. 4). This contrasts markedly with the biphasic effect of verapamil on the rate of alkalinization seen in C4Dd2 parasites (Fig. 4).

In the case of chlorpheniramine, fluoxetine and CQ added to C67G8 parasites there was no significant difference between the ‘compound + verapamil (50 µM)’ and verapamil-only condition (P > 0.06, paired t-tests). Verapamil did reduce the desipramine-associated increase in the rate of DV alkalinization (P = 0.0005, paired t-test) in C67G8 parasites, although a significant difference between the desipramine and desipramine + verapamil conditions remained (P = 0.004, paired t-test). The slight increase in the rate of DV alkalinization seen on addition of PQ to C67G8 parasites did not reach statistical significance (P = 0.06, paired t-test). Promethazine caused a slight but significant decrease in the rate of DV alkalinization in this strain (P = 0.03, paired t-test).

Differences in the magnitudes of the PQ- and promethazine-associated H+ leaks between C4Dd2 parasites and C67G8 parasites correlate with differences in their efficacies as CQ resistance reversers

The recent finding that PQ sensitizes CQR parasites to CQ in vitro (Bray et al., 2005b) has generated significant interest (Egan, 2006), as CQ and PQ are already used together to treat malaria caused by P. vivax. Only parasites bearing the Dd2 mutant form of PfCRT or the K1 form (which differs from the Dd2 form only at one residue) were investigated by Bray et al. (2005b). As shown in Fig. 3C, 5 µM PQ gave rise to a more pronounced effect on the rate of DV alkalinization in C4Dd2 parasites compared with those bearing the South American 7G8 form of PfCRT. To investigate this further, we tested the effect of a range of PQ concentrations on the initial rate of DV alkalinization in both strains. Time course experiments with PQ (5 µM) revealed that a 12 min pre-incubation was sufficient for the drug to exert its maximal effect on the rate of concanamycin A-induced DV alkalinization in both strains (not shown); a 12 min incubation was therefore used for the concentration dependence experiments.

The effect of increasing PQ concentrations on the initial rate of PQ-associated DV alkalinization in the C4Dd2 and C67G8 strains is shown in Fig. 5. In C4Dd2 parasites, PQ caused a significant increase in the initial rate of DV alkalinization at all the concentrations tested [≥ 1.25 µM (P < 0.03, paired t-tests)]. In C67G8 parasites, PQ increased the initial rate of DV alkalinization at concentrations ≥ 2.5 µM (P < 0.01, paired t-tests). The initial rate of PQ-associated DV alkalinization was significantly lower in C67G8 parasites than in C4Dd2 parasites at each concentration (P < 0.04, unpaired t-tests).

image

Figure 5. Concentration dependence of the increase in the initial rate of concanamycin A-induced DV alkalinization associated with PQ in isolated mature trophozoite-stage CQR C4Dd2 parasites (open circles) and C67G8 parasites (closed circles). PQ was added to parasite suspensions 12 min before the addition of concanamycin A (100 nM). The data (shown mean ± SEM) are averaged from five independent experiments for C4Dd2 parasites and from six independent experiments for C67G8 parasites.

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The greater magnitude of the PQ-associated H+ leak in C4Dd2 parasites compared with C67G8 parasites is consistent with there being differences between the Dd2 and 7G8 forms of PfCRT in their interaction with the drug. To investigate whether such differences might be manifest in the ability of the drug to modulate PfCRT-mediated CQ resistance we tested the effect of increasing PQ concentrations on the IC50 values for CQ (determined using 48 h [3H]hypoxanthine incorporation assays) in C2GC03, C4Dd2 and C67G8 parasites. The data are shown in Fig. 6A. PQ did not have a significant effect on the CQ IC50 value of C2GC03 parasites at any of the concentrations tested (0.5, 1, 2, 4 and 8 µM; P > 0.07, paired t-tests), but did have a significant effect in C4Dd2 parasites at all the concentrations tested (P < 0.04, paired t-tests), and in C67G8 parasites at concentrations ≥ 2 µM (P < 0.03, paired t-tests). Because the three lines differed in their CQ IC50 values in the absence of PQ (Fig. 6A), PQ concentration was also plotted against the ‘response modification index’ (the CQ IC50 value in the presence of PQ divided by the value in the absence of PQ; Fig. 6A inset), which provides a measure of the extent of CQ resistance reversal. From Fig. 6A it can be seen that PQ reverses CQ resistance more effectively in C4Dd2 parasites than in C67G8 parasites, with the response modification index being significantly different between the two strains at all but the lowest PQ concentration tested (P < 0.05, unpaired t-tests).

image

Figure 6. The effect of increasing concentrations of PQ (A) and promethazine (B) on CQ susceptibility in the C2GC03 (grey), C4Dd2 (white) and C67G8 (black) strains of P. falciparum, expressed in terms of the CQ IC50 value and the CQ response modification index (CQ IC50 with PQ/CQ IC50 without PQ; insets). In both cases, the data are derived from three independent 48 h [3H]hypoxanthine incorporation assays for each strain, and are shown ± SEM. In the main figures (and for the C4Dd2 data in the inset of the lower panel), only positive error bars are shown for C67G8 and only negative error bars are shown for C2GC03 and C4Dd2 for reasons of clarity. Where not shown, error bars fall within the symbols.

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Promethazine, similarly to PQ, gave rise to a large H+ leak in C4Dd2 parasites at a concentration of 5 µM but did not give rise to a detectable H+ leak in C67G8 parasites at this concentration (Fig. 3C). Promethazine significantly reduced the CQ IC50 value in both C4Dd2 and C67G8 parasites at all the concentrations tested (0.5, 1, 2, 4 and 8 µM; P < 0.02, paired t-tests; Fig. 6B), while having no significant effect on the (lower) CQ IC50 value in C2GC03 parasites at any concentration (P > 0.06, paired t-tests). There was a significant difference in the response modification index between C4Dd2 and C67G8 parasites at promethazine concentrations of 0.5 µM (P = 0.0005, unpaired t-test) and 1 µM (P = 0.01, unpaired t-test), with the modification of CQ response being greater in C4Dd2 parasites, but not at higher concentrations (P > 0.08, unpaired t-tests).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this study, a range of antimalarial drugs and CQ resistance reversers, including AQ, QD, QN, chlorpheniramine, desipramine, fluoxetine and PQ were shown to induce an enhanced leak of H+ ions from the DV in C4Dd2 and C67G8 parasites, but not in C2GC03 parasites. The data are consistent with these compounds being substrates for the Dd2 and 7G8 forms of PfCRT, with the enhanced H+ leak attributable to the compounds effluxing from the vacuole in the protonated form and/or in symport with H+ ions.

Verapamil and promethazine induced an enhanced leak of H+ from the DV in C4Dd2 parasites, but not in C67G8 parasites, consistent with these compounds being substrates for the Dd2 form, but not the 7G8 form, of PfCRT. MQ and ART did not enhance the leak of H+ from the DV substantially in any of the strains tested. However, the possibility remains that one or both of these drugs are transported but that this could not be detected using the method used here. The results are consistent with the proposal by Sanchez and colleagues that, based on their observations of CQ uptake stimulation in erythrocytes infected with CQR Dd2 parasites, AQ, PQ, QD and QN (as well as quinacrine, which was not studied here), but not ART or MQ (or halofantrine or pyrimethamine), are substrates for the CQ efflux system (Sanchez et al., 2004).

It was shown previously that the magnitude of the CQ-induced increase in the initial rate of concanamycin A-induced DV alkalinization was similar in C4Dd2 parasites and C67G8 parasites (Lehane and Kirk, 2008). The same result was seen here for AQ (Fig. 2A). However, the magnitudes of the QD and QN effects differed markedly between C4Dd2 parasites and C67G8 parasites, with the effect in C4Dd2 parasites being greater in both cases (Fig. 2B and C). There is reportedly a difference in the expression level of PfCRT protein between C4Dd2 parasites and C67G8 parasites, with C4Dd2 parasites expressing ∼30–40% less protein than C67G8 parasites (Sidhu et al., 2002). This difference in expression level is opposite to what would be required to explain the greater magnitudes of the QD- and QN-induced H+ leaks in C4Dd2 parasites compared with C67G8 parasites. It is therefore likely that one or more of the amino acids that differ between the forms of PfCRT present in the two strains are responsible for the observed differences, and that the Dd2 and 7G8 forms of PfCRT differ in their capacities to transport QD and QN.

In contrast to the situation with CQ, the replacement of wild-type PfCRT with the Dd2 or 7G8 mutant forms does not lead to resistance to AQ, QD or QN (Sidhu et al., 2002). AQ is thought to share the same mechanism of action as CQ (Hawley et al., 1998). It is intriguing then, that an efflux of AQ from the DV upon the introduction of mutant forms of PfCRT does not have a statistically significant effect on parasite susceptibility to this drug {although it should be noted that, although not statistically significant, C4Dd2 and C67G8 parasites did have higher IC50 values for AQ than did C2GC03 parasites [26 nM and 36 nM vs. 18 nM; (Sidhu et al., 2002)]}. One possible explanation is that the rate of diffusion of AQ into the DV exceeds that of CQ and ensures that there is a sufficient amount of the drug present to bind its target (haem) as rapidly as it is generated, even in the presence of an efflux pathway.

Reconciling efflux data with parasite drug sensitivity is also complicated for QN. Sidhu et al. (2002) reported QN IC50 values for C2GC03, C4Dd2 and C67G8 parasites of 171 nM, 93 nM and 72 nM respectively. The data obtained here are consistent with the recent observation of a verapamil-sensitive increase in the rate of [3H]QN loss from erythrocytes infected with C4Dd2 parasites compared with those infected with C2GC03 parasites (Sanchez et al., 2008). QN is known to bind haem and to inhibit its conversion to haemozoin (Chou et al., 1980; Dorn et al., 1998; Hawley et al., 1998), although whether this accounts for its antimalarial properties is not clear (Foley and Tilley, 1998). The fact that QN efflux from the DV, which results in a reduction in QN accumulation (Sanchez et al., 2008), is associated with an increase in QN sensitivity in the GC03 background used to make the pfcrt transfectant lines (Sidhu et al., 2002) suggests that in GC03 the primary target of QN might not reside in the DV.

If QN's primary target lies outside the DV, a reduction in drug accumulation in the DV should only alter the parasite's sensitivity to the drug if, in the absence of the efflux pathway, the DV accumulates QN to such an extent that the drug is depleted from the compartment in which the target resides. Whether there was a depletion of QN from the target site in C2GC03 parasites under the conditions of the parasite proliferation assays performed by Sidhu et al. (2002) is not clear. Another possibility is that QN's target lies in the DV membrane itself. Sanchez and colleagues found that pfmdr1 transfectant parasite lines, which were engineered to express different mutant forms of the DV membrane protein P-glycoprotein homologue 1 (Pgh1) and were found to differ in their susceptibilities to QN (Sidhu et al., 2005), did not differ in the extent to which they accumulated radiolabelled QN (Sanchez et al., 2008). Based on this the authors suggested that Pgh1 could be a target of QN (Sanchez et al., 2008). Likewise, PfCRT itself is thought to have an essential function (Waller et al., 2003) and could be a target for some quinoline drugs.

Data pertaining to the mechanisms of accumulation and action of QD are even scarcer than for QN. Sidhu et al. (2002) reported that there was no significant difference between the QD IC50 value of C2GC03 parasites (27 nM) and the QD IC50 values of either C4Dd2 parasites (38 nM) or C67G8 parasites (22 nM). The fact that QD efflux does not appear to have a significant effect on parasite susceptibility to this drug (at least in the GC03 strain) is consistent with its primary target being outside the DV. The mechanism(s) by which mutations in PfCRT increase parasite susceptibility to MQ and ART also remain to be elucidated. Although not measurable with the method used here, it remains possible that these drugs are transported by some forms of PfCRT.

The finding that a range of CQ resistance reversers give rise to an increased leak of H+ from the DV of parasites expressing mutant PfCRT, consistent with their being transported via the mutant protein, has obvious implications for their mechanism of action. It is likely that at least some such compounds inhibit PfCRT-mediated drug transport via a competitive mechanism.

Chlorpheniramine, desipramine and fluoxetine (each at a concentration of 5 µM) all gave rise to a substantial H+ leak from the DV in both C4Dd2 and C67G8 parasites but not in C2GC03 parasites, consistent with their being substrates of both the Dd2 and 7G8 forms of PfCRT but not the wild-type form.

Primaquine, at a concentration of 5 µM, gave rise to a significant effect on the rate of DV alkalinization in C4Dd2 parasites but a smaller effect that did not reach statistical significance in C67G8 parasites. The subsequent testing of a range of concentrations of PQ revealed that there was a PQ-associated H+ leak from the DV in both strains but that it was greater in C4Dd2 parasites than in C67G8 parasites (Fig. 5). PQ was a more effective reverser of CQ resistance in the former strain (Fig. 6), although this difference between the strains was more subtle than that observed for the PQ-associated H+ leak from the DV. CQ resistance reversal by PQ had only been examined previously in parasites with the Dd2 or K1 forms of PfCRT, and it was proposed that PQ inhibits CQ efflux from the DV by binding to sites in the pore of mutant PfCRT (Bray et al., 2005b). Based on the data presented here, it seems likely that PQ is a competitive inhibitor of CQ efflux via mutant PfCRT. Further studies are required to elucidate whether this is the sole mechanism by which PQ inhibits CQ efflux, or one of multiple modes of inhibition.

Promethazine and verapamil (each at a concentration of 5 µM) gave rise to a large H+ leak from the DV in C4Dd2 parasites but no detectable leak in C67G8 parasites. The unusual biphasic concentration dependence data for verapamil in C4Dd2 parasites, with the verapamil-induced H+ leak increasing with increasing verapamil concentration up to 5 µM, and decreasing with increasing verapamil concentration thereafter, may be indicative of multiple types of interaction of this compound with the Dd2 form of PfCRT; for example, verapamil may be a substrate for the mutant protein at lower concentrations, but inhibit the protein, perhaps via an uncompetitive or non-competitive mechanism, at higher concentrations, a phenomenon that has been demonstrated for other transport proteins (e.g. Barzilay and Cabantchik, 1979). Whether this is the case remains to be determined. The absence of a detectable verapamil-induced (or promethazine-induced) H+ leak from the DV of C67G8 parasites may point to the inhibition by these compounds of CQ transport via the 7G8 form of PfCRT being uncompetitive or non-competitive in nature, although other explanations [e.g. the masking of an induced H+ leak by other effects (e.g. buffering of the DV, or inhibition of a H+ leak pathway)] cannot be excluded.

Verapamil is known to reverse CQ resistance in vitro in both lines, but its effect (at a concentration of 0.8 µM) is greater in C4Dd2 parasites than in C67G8 parasites (Sidhu et al., 2002). A study of field strains also showed that parasites from South America and Papua New Guinea (which have the amino acids SVMNT at positions 72–76 of PfCRT like 7G8) are less responsive to verapamil-mediated CQ resistance reversal than strains from Africa and Southeast Asia (which have CVIET at positions 72–76) (Mehlotra et al., 2001). Promethazine sensitized both C4Dd2 parasites and C67G8 parasites to CQ (Fig. 6), but was more effective at lower concentrations in C4Dd2 parasites.

In summary, the data obtained here are consistent with mutant-PfCRT-mediated efflux of the antimalarial drugs AQ, QD and QN from the DV. Differences were observed between parasites bearing the Dd2 and 7G8 forms of PfCRT with regard to QD and QN efflux. Furthermore, differences were exposed between quinoline antimalarial drugs with regard to the relationship between their efflux from the DV on one hand and parasite susceptibility to the drug on the other. We also present evidence for the efflux of a range of CQ resistance reversers from the DV in parasites with mutant forms of PfCRT. Again, differences were observed between the Dd2 and 7G8 forms of PfCRT for some compounds. This reinforces the importance of including strains with a variety of pfcrt alleles in studies of CQ resistance reversers that are being considered for clinical testing. The data are consistent with competitive inhibition of CQ efflux via mutant PfCRT being one of the mechanisms by which CQ resistance reversers sensitize parasites to CQ.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Parasite strains and culture

The three P. falciparum strains used in this study are pfcrt transfectant strains generated by Sidhu et al. (2002) and generously provided by D. A. Fidock (Columbia University, New York). GC03, a CQS progenitor of a cross between a CQS (HB3) and a CQR (Dd2) strain (Wellems et al., 1990), was used to generate the transfectant lines. The C4Dd2 and C67G8 lines were generated by replacing the wild-type pfcrt allele in GC03 with CQ-resistance-conferring mutant forms of pfcrt from the CQR Dd2 (Southeast Asian) and 7G8 (South American) strains respectively. [The form of PfCRT in the C67G8 strain contains an additional I351M mutation not found in 7G8 parasites (D.A. Fidock, pers. comm.)] The C2GC03 line is a CQS recombinant control line that retained the wild-type pfcrt gene. The parasites were cultured and synchronized as described previously (Allen and Kirk, 2010), and were maintained in the presence of 5 µM blasticidin (Invitrogen, Australia) and 5 nM WR99210 (Jacobus Pharmaceuticals, Princeton, NJ). These selection agents were not present during parasite proliferation assays or fluorometry experiments.

Measurement of the leak of H+ from the DV

Parasite DVs were loaded with the membrane-impermeant pH-sensitive dye fluorescein-dextran (pKa∼6.4; ∼10 × 103Mr; Invitrogen, Australia) to enable changes in pHDV to be monitored. Uninfected erythrocytes were loaded with fluorescein-dextran (55 µM) and inoculated with trophozoite-infected erythrocytes, as described previously (Krogstad et al., 1985; Saliba et al., 2003; Lehane and Kirk, 2008; Lehane et al., 2008). Typically, the trophozoite-infected erythrocytes used for inoculation were separated from uninfected erythrocytes using a Miltenyi Biotec VarioMACS magnet (Paul et al., 1981; Staalsoe et al., 1999) and experiments were performed after one complete asexual cycle (∼48 h). Alternatively, 1–2 ml of packed fluorescein-dextran-loaded erythrocytes was inoculated with an equal volume of a ∼20% parasitaemia, ∼4% haematocrit culture of trophozoite-infected erythrocytes and experiments were performed after two complete cycles. In either case, at the time of experimentation most of the trophozoites were growing within dye-loaded erythrocytes and contained the dye in their DVs as a result of endocytosis of the host erythrocyte cytosol.

Fluorometry experiments were performed as described previously (Lehane et al., 2008). Briefly, fluorescein-dextran-loaded mature trophozoite-stage parasites were functionally isolated from their host erythrocytes by permeabilization of the erythrocyte and parasitophorous vacuole membranes using saponin, under conditions shown not to affect the integrity of the parasite plasma membrane or its ability to maintain large ion gradients (Spillman et al., 2008). The saponin-isolated parasites were then washed several times and resuspended (at a density of ∼107 cells ml−1) in a saline solution (125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM glucose, 25 mM HEPES; pH 7.1) at 37°C. For each trace, a 1 ml aliquot of the suspension was transferred to a cuvette and fluorescence measurements were performed at 37°C using a PerkinElmer Life Sciences LS-50B spectrofluorometer with a dual excitation Fast Filter accessory. The ratio of the fluorescence intensity at 520 nm using two excitation wavelengths (490 nm and 450 nm) was used as an indicator of pHDV. The experiments entailed monitoring the alkalinization of the DV following the addition of the H+ pump inhibitor concanamycin A, in the presence and absence of various antimalarial drugs and CQ resistance reversers (purchased from Sigma, Australia).

Rates of DV alkalinization (following the addition of concanamycin A) are expressed either in terms of the half-time for alkalinization or the initial rate of alkalinization. Alkalinization half-times were used in initial surveys of the effects of a range of reagents on the alkalinization rate, and in those subsequent experiments in which alkalinization rates were compared under conditions in which the rate decreased. In such cases the alkalinization time courses were commonly sigmoidal in appearance (Lehane and Kirk, 2008; Lehane et al., 2008), making it impractical to compare initial alkalinization rates. Half-times for DV alkalinization (t1/2) were determined by fitting the following sigmoidal curve to the data by regression analysis using SigmaPlot 2004 (Systat Software): F = F0 + Fmax/[1 + (t/t1/2)c], where F is the fluorescence ratio, F0 is the initial fluorescence ratio (this was set to the resting fluorescence ratio averaged over the 20 s immediately prior to the opening of the fluorometer chamber to add concanamycin A), Fmax is the maximal change in fluorescence ratio, t is time following the addition of concanamycin A, and c is a fitted constant.

In several sets of experiments, in which quantitative comparisons were made between rates of alkalinization under conditions in which the reagents of interest caused an increased alkalinization rate (Figs 2 and 5), initial rates (rather than half-times) were used. In these cases the first 30–100 s of the concanamycin A-induced alkalinization time course was fitted to the following exponential function: F = F0 + a(1 − e−bt), where F is the fluorescence ratio, F0 is the starting fluorescence ratio, t is time following the addition of concanamycin A, and a and b are fitted constants. Multiplication of a and b yielded the initial rate of alkalinization. To account for differences in signal intensity between experiments, the fluorescence ratio was normalized prior to determining the initial rate by dividing by the maximum change in fluorescence ratio for that trace. Within each experiment, the initial rate of alkalinization for the control trace without drug was subtracted from the initial rates in the presence of drug.

Parasite proliferation assays

Parasite proliferation was measured in 96-well plates over 48 h using the [3H]hypoxanthine incorporation method (Desjardins et al., 1979). [3H]hypoxanthine (0.4 µCi, in a volume of 25 µl low-hypoxanthine medium) was added at the 24 h time point. The parasitaemia (consisting of predominantly ring-stage parasites) and haematocrit were both 1%.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported by Australian National Health and Medical Research Council Grant 418055. We are grateful to David Fidock for the generous gift of the parasite strains used in this study, to Robert Summers for helpful discussion, and to the Canberra Branch of the Australian Red Cross Blood Service for the provision of blood.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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