PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX

It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.     

Identification of the acidic compartment of Plasmodium falciparum-infected human erythrocytes as the target of the antimalarial drug chloroquine.

Chloroquine (CQ), the most widely used antimalarial drug, is an acidotropic agent (De Duve, 1983) which accumulates to high levels in malaria-infected erythrocytes. A possible site of accumulation of the drug, the parasite's food vacuole, has been implicated in the mode of action of CQ. We have defined the various compartments of Plasmodium falciparum-parasitized human erythrocytes in terms of their pH and capacity to accumulate bases. The host cell and the parasite cytosols were differentially labeled in situ with pH-sensitive fluorescein, and the parasite food vacuole was revealed by targeting fluoresceinated dextran via endocytosis. The pH of the various compartments obtained from fluorescence excitation spectra were 6.9 for the cytosol of normal and infected erythrocytes and 5.2 for the parasite food vacuole. Determination of CQ and methylamine accumulation in infected erythrocytes, in conjunction with morphometric determination of the relative sizes of the various cellular compartments, provided an independent assessment of the vacuolar pH, yielding a value of 5.0-5.2. Perturbation of the proton gradient, either by lowering extracellular pH or by alkalinization of the food vacuole with NH4Cl or monensin, resulted in a concomitant and reversible decrease in accumulation of the probe. We conclude that drug accumulation in malaria-infected erythrocytes can be fully accounted for by the steady-state proton gradients across the barriers delineating the various cellular compartments and the acidotropic properties of the drug.     

Plasmodium chabaudi: association of reversal of chloroquine resistance with increased accumulation of chloroquine in resistant parasites.

The effects of tricyclic antidepressants, desipramine and imipramine, and phenothiazines, chlorpromazine and trifluoperazine, on chloroquine (CQ)-resistant and CQ-sensitive lines of P. chabaudi were examined in vivo. In mice that received daily injections of these drugs the growth of CQ-resistant and CQ-sensitive parasites was unaffected or affected very slightly, if at all. A combination of CQ and each drug suppressed the growth of CQ-resistant parasites in a dose-dependent manner. In addition, in CQ-sensitive parasites each drug also increased the susceptibility to CQ. Measurements of CQ levels by high-performance liquid chromatography showed that CQ accumulated in sensitive parasites to more than twice the level in resistant parasites at 2 to 4 hr after an injection of CQ. Verapamil and desipramine substantially increased CQ levels in both CQ-resistant and CQ-sensitive parasites. These results suggest that not only Ca2+ antagonists but tricyclic antidepressants reverse CQ resistance in CQ-resistant parasites and enhance the inhibitory effect in sensitive parasites by increasing CQ levels in those parasites. The effects of Ca2+ antagonists, tricyclic antidepressants, and phenothiazines on a pyrimethamine-resistant line of P. chabaudi were also studied. None of the Ca2+ antagonists (verapamil, nicardipine, and diltiazem) affected the growth of the parasite in combination with 20 mg/kg pyrimethamine. Tricyclic antidepressants and phenothiazines suppressed pyrimethamine-resistant parasites to some extent. However, the extent of this suppression was less pronounced as compared with that of suppression of CQ resistance by the same drugs.     

Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance

The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.     

Antiretroviral protease inhibitors potentiate chloroquine antimalarial activity in malaria parasites by regulating intracellular glutathione metabolism

Antiretroviral protease inhibitors significantly potentiated the sensitivity of chloroquine-resistant malaria parasites to the antimalarial drug in vitro and in vivo. Ritonavir was found to be potent in potentiating CQ antimalarial activities in both -resistant and -sensitive lines. The mechanism by which the APIs modulate the CQ resistance in malaria parasites was further investigated. CQ-resistant parasites showed increased intracellular glutathione levels in comparison with the CQ-sensitive parasites. Treatment with APIs significantly reduced the levels of GSH and glutathione S-transferase activities in CQ-resistant parasites. Ritonavir also decreased glutathione reductase activities and glutathione peroxidase activities in CQ-resistant parasite line. Taken together, these results demonstrate that parasite GSH and GST may play an important role in CQ resistance and APIs are able to enhance the sensitivity of CQ-resistant malaria parasite to the drug by influencing the levels of GSH and the activities of the related enzymes.     

Identification of distinct accumulation sites of 4-aminoquinoline in chloroquine sensitive and resistant Plasmodium berghei strains

We report the synthesis of an analogue of chloroquine (CQA) which can be used as a probe to visualize accumulation of 4-aminoquinoline by electron microscopy. A mouse monoclonal antibody against CQA was raised and used for immunodetection by the protein-A gold method on ultrathin cryosections, of CQA treated parasites. We demonstrate that in a P. berghei chloroquine(CQ)-sensitive strain (N strain) the chloroquine analogue used accumulates in the endocytic vacuoles where hemoglobin (Hb) degradation is occurring. In contrast, in a P. berghei CQ-resistant strain (RC strain) the probe was found scattered all over the cytoplasm of the parasite. This result suggests that endocytic vacuoles of the parasite could constitute the site of antimalarial action of CQ.     

Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine

Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.     

Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine.

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.     

Validation of a chloroquine-induced cell death mechanism for clinical use against malaria

An alternative antimalarial pathway of an ‘outdated'' drug, chloroquine (CQ), may facilitate its return to the shrinking list of effective antimalarials. Conventionally, CQ is believed to interfere with hemozoin formation at nanomolar concentrations, but resistant parasites are able to efflux this drug from the digestive vacuole (DV). However, we show that the DV membrane of both resistant and sensitive laboratory and field parasites is compromised after exposure to micromolar concentrations of CQ, leading to an extrusion of DV proteases. Furthermore, only a short period of exposure is required to compromise the viability of late-stage parasites. To study the feasibility of this strategy, mice malaria models were used to demonstrate that high doses of CQ also triggered DV permeabilization in vivo and reduced reinvasion efficiency. We suggest that a time-release oral formulation of CQ may sustain elevated blood CQ levels sufficiently to clear even CQ-resistant parasites.Along with improvements in vector control, surveillance/diagnosis and treatment accessibility, the development of new drugs to counteract the problem of drug resistance remains integral to the eradication agenda.¹ Efforts to develop novel antimalarials have been promising,^{2, 3} and drugs designed specifically to reverse drug resistance are also being uncovered.⁴ However, novel chemical entities are expensive to test and take considerable time before they can be deployed. In comparison, alternative strategies to fully exploit the existing arsenal of antimalarials (largely already affordable and accessible) are likely to be relatively expedient and cost-effective.We had previously demonstrated the existence of a novel parasite programmed cell death (PCD) mechanism that was induced by high concentrations of chloroquine (CQ) and shown that clan CA cysteine proteases were key mediators of the pathway.⁵ We had also observed that the permeabilization of the parasite digestive vacuole (DV) was an important upstream trigger of this pathway and that other lysosomotropic compounds that are not parasite-specific could similarly destabilize the DV to initiate parasite PCD.⁶ We hypothesize that by altering the dosing regimen or formulation of CQ, it might be possible to reinstate CQ into antimalarial chemotherapy by making use of this novel mechanism.⁷In this present study, we begin by showing evidence that CQ treatment is able to result in the extrusion of DV proteases into the parasite cytoplasm. Second, we validate the existence of this PCD pathway in multiple laboratory strains and field isolates to suggest its clinical relevance and universality. Third, we investigate the minimum concentration and duration required for CQ to trigger PCD to determine if the pharmacokinetics of the current CQ regimen might be suitable for initiating PCD. Finally, we make use of two murine malaria models to demonstrate that a short exposure to high levels of CQ is able to induce parasite DV permeabilization in vivo and that this procedure reduces parasite viability.     

In vivo imaging of retrogradely transported synaptic vesicle proteins in Caenorhabditis elegans neurons

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. We have developed a novel in vivo method to exclusively mark and dynamically track retrogradely moving compartments carrying specific endogenous synaptic vesicle proteins in the Caenorhabditis elegans model. Our method is based on the uptake of a fluorescently labeled anti-green fluorescent protein (GFP) antibody delivered in an animal expressing the synaptic vesicle protein synaptobrevin-1::GFP in neurons. We show that this method largely labels retrogradely moving compartments. Very little labeling is observed upon blocking vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. Further, we observe that the retrograde compartment carrying synaptobrevin contains synaptotagmin but lacks the endosomal marker RAB-5. This labeling method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle and can also be extended to other model systems.     

Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments

Recently we demonstrated that low-pH compartments can be visualized with the electron microscope using a basic congener of dinitrophenol, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), which concentrates in acidic compartments and can be detected by immunocytochemistry with a monoclonal anti-dinitrophenol antibody. We now report that DAMP also accumulates in cisternae and vesicles associated with the trans face of the Golgi apparatus. DAMP rapidly leaves this compartment when cells are incubated with the ionophore monensin, which indicates that accumulation is due to the acidic pH in this compartment. Using indirect protein A-gold immunocytochemistry, we localized fibronectin, a major secretory protein in fibroblasts, to the trans Golgi vesicles that took up DAMP. Therefore, the trans cisternae of the Golgi apparatus and forming secretory vesicles have an acidic pH.     

Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 2. Altered vacuolar volume regulation in drug resistant malaria

In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], we reported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initial application to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparum malarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of the parasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previous reports have suggested that mutant PfCRT protein, which causes chloroquine resistance (CQR) in P. falciparum, also causes increased acidification of the DV. Since pH and volume regulation are often linked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisition of SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterative deconvolution using experimental point spread functions, we quantify the volume of the DV for live, intraerythrocytic HB3 (CQS), Dd2 (CQR via drug selection), GCO3 (CQS), and GCO3/C3(Dd2) (CQR via transfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. We find that relative to both CQS strains, both CQR strains show significantly increased DV volume as the organelle forms upon entry into the trophozoite stage of development and that this persists until the trophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelle forms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATP depletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, these data suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation.     

Treatment of Erythrocytes with the 2-Cys Peroxiredoxin Inhibitor,Conoidin A,Prevents the Growth of Plasmodium falciparum and Enhances Parasite Sensitivity to Chloroquine

The human erythrocyte contains an abundance of the thiol-dependant peroxidase Peroxiredoxin-2 (Prx2), which protects the cell from the pro-oxidant environment it encounters during its 120 days of life in the blood stream. In malarial infections, the Plasmodium parasite invades red cells and imports Prx2 during intraerythrocytic development, presumably to supplement in its own degradation of peroxides generated during cell metabolism, especially hemoglobin (Hb) digestion. Here we demonstrate that an irreversible Prx2 inhibitor, Conoidin A (2,3-bis(bromomethyl)-1,4-dioxide-quinoxaline; BBMQ), has potent cytocidal activity against cultured P. falciparum. Parasite growth was also inhibited in red cells that were treated with BBMQ and then washed prior to parasite infection. These cells remained susceptible to merozoite invasion, but failed to support normal intraerythrocytic development. In addition the potency of chloroquine (CQ), an antimalarial drug that prevents the detoxification of Hb-derived heme, was significantly enhanced in the presence of BBMQ. CQ IC₅₀ values decreased an order of magnitude when parasites were either co-incubated with BBMQ, or introduced into BBMQ-pretreated cells; these effects were equivalent for both drug-resistant and drug-sensitive parasite lines. Together these results indicate that treatment of red cells with BBMQ renders them incapable of supporting parasite growth and increases parasite sensitivity to CQ. We also propose that molecules such as BBMQ that target host cell proteins may constitute a novel host-directed therapeutic approach for treating malaria.     

Inhibition of hemoglobin degradation in Plasmodium falciparum by chloroquine and ammonium chloride

The effect of chloroquine (CQ) and NH4Cl on hemoglobin degradation within Plasmodium falciparum was studied by SDS gel electrophoresis. CQ inhibited hemoglobin digestion such that accumulation of hemoglobin in the parasites occurred. Quantitative analysis indicated that the content of hemoglobin in isolated CQ-treated parasites was increased from 2.39 +/- 0.47 micrograms hemoglobin (Hb)/mg protein to 8.34 +/- 0.77 micrograms Hb/mg protein (P less than 0.001) within 45 min and further to 18.7 +/- 1.23 micrograms Hb/mg protein for 2 hr compared with the untreated parasites. These results suggest that the inhibition of hemoglobin degradation in malarial parasites might be the primary target of CQ antimalarial action. The CQ-like effect on the hemoglobin digestion in P. falciparum was observed with another lysosomotropic weak base, NH4Cl, suggesting that the CQ effect on hemoglobin degradation and its antimalarial action, as well as the effect of NH4Cl, are related to their properties of lysosomotropic weak base.     

Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles

The direct identification of the intracellular site where proinsulin is proteolytically processed into insulin has been achieved by immunocytochemistry using an insulin-specific monoclonal antibody. Insulin immunoreactivity is absent from the Golgi stack of pancreatic B-cells and first becomes detectable in clathrin-coated secretory vesicles released from the trans Golgi pole. Clathrin-coated secretory vesicles transform into mature noncoated secretory granules which contain the highest concentration of insulin immunoreactive sites. Maturation of clathrin-coated secretory vesicles is accompanied by a progressive acidification of the vesicular milieu, as evidenced by a cytochemical probe that accumulates in acidic compartments whereupon it can be revealed by immunocytochemistry. Thus packaging of the prohormone in secretory vesicles, and acidification of this compartment, are critical steps in the proper proteolytic maturation of insulin.     

Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria

We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.     

Mutations in transmembrane domains 1, 4 and 9 of the Plasmodium falciparum chloroquine resistance transporter alter susceptibility to chloroquine, quinine and quinidine

Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) can result in verapamil-reversible CQ resistance and altered susceptibility to other antimalarials. PfCRT contains 10 membrane-spanning domains and is found in the digestive vacuole (DV) membrane of intraerythrocytic parasites. The mechanism by which PfCRT mediates CQ resistance is unclear although it is associated with decreased accumulation of drug within the DV. On the permissive background of the P. falciparum 106/1(K76) parasite line, we used single-step drug selection to generate isogenic clones containing unique pfcrt point mutations that resulted in amino acid changes in PfCRT transmembrane domains 1 (C72R, K76N, K76I and K76T) and 9 (Q352K, Q352R). The resulting changes of charge and hydropathy affected quantitative CQ susceptibility and accumulation as well as the stereospecific responses to quinine and quinidine. These results, together with a previously described S163R mutation in transmembrane domain 4, indicate that transmembrane segments 1, 4 and 9 of PfCRT provide important structural components of a substrate recognition and translocation domain. Charge-affecting mutations within these segments may affect the ability of PfCRT to bind different quinoline drugs and determine their net accumulation in the DV.     

A cytochemical ultrastructural study of the lysosomal system of different species of malaria parasites

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.     

Vacuolar H+ Pumping ATPases in Luminal Acidic Organelles and Extracellular Compartments: Common Rotational Mechanism and Diverse Physiological Roles

Cytoplasmic organelles with an acidic luminal pH include vacuoles, coated vesicles, lysosomes, the Golgi apparatus, and synaptic vesicles. Acidic compartments are also known outside specialized cells such as osteoclasts. The unique acidic pH is formed by V-ATPase (Vacuolar type ATPase), other ion transporters, and the buffering action of proteins inside the organelles. V-ATPase hydrolyzes ATP and transports protons inside an organelle or extracellular compartment. We have summarized recent progress on mouse V-ATPases and their varying localizations together with their mechanism emphasizing similarities with F-type ATPases.