Relationship of chloroquine-induced redistribution of a neutral aminopeptidase to hemoglobin accumulation in malaria parasites

To study the relationship between neutral aminopeptidase activity and hemoglobin accumulation in malaria parasites, we treated mice infected with Plasmodium berghei NYU-2 with chloroquine intraperitoneally in doses ranging from 0.3 to 3 micromol per 25 g mouse. Preparations of infected erythrocytes (normalized to represent 1000 parasites per 1000 erythrocytes) hydrolyzed 1200 nmol of leucine-p-nitroanilide per minute per milliliter of packed erythrocytes, which was 10x more than that of uninfected preparations. The activity in infected preparations was distinguished by resistance to ferriprotoporphyrin IX and puromycin and susceptibility to inhibition by ethanol and Tris. Chloroquine treatment caused the activity in unwashed membrane ghosts of infected preparations to decrease by 50% despite an increase in total activity. Concomitantly, hemoglobin in washed membrane ghosts increased. Electron microscopy revealed that the hemoglobin was retained in endocytic vesicles. Chloroquine-induced redistribution of a neutral aminopeptidase may be the cause of hemoglobin accumulation in endocytic vesicles of malaria parasites.     

Plasmodium falciparum: in vitro drug interaction between chloroquine and enantiomers of amlodipine

Both enantiomers of amlodipine, whose calcium antagonist action resides almost exclusively in the R(-) enantiomer, reversed chloroquine resistance in Plasmodium falciparum in vitro. R(-) enantiomer was slightly more effective than the S(+) enantiomer in potentiating chloroquine action against chloroquine-resistant strains of parasites. No potentiating effect was observed in chloroquine-sensitive parasites. Both enantiomers entered rapidly into parasitized erythrocytes to the same extent. Reversal of chloroquine resistance by the enantiomers of amlodipine was related to dose-dependent increase in the accumulation of chloroquine inside the erythrocytes parasitized by resistant parasites. These results suggest that the potentiating effect on chloroquine is independent of calcium metabolism of malaria parasites.     

Ferriprotoporphyrin IX and cell lysis: a protective role for hydrogen peroxide

Two potentially lytic substances, ferriprotoporphyrin IX (FP) and hydrogen peroxide, may coexist and partially detoxify each other in sickle cells and in erythrocytes infected with malaria parasites. Since hydrogen peroxide can decompose FP, its effect on hemolysis induced by FP and by the complex of FP with chloroquine was investigated. Human erythrocytes suspended at a concentration of 0.5% in a 50 microM solution of FP underwent approximately 42% hemolysis during the course of 2 hours. Twenty-five micromolar chloroquine potentiated hemolysis to 99%, and preincubation of 50 microM FP with 25 microM hydrogen peroxide for 5 minutes reduced hemolysis to 4%. Mixing either FP or hydrogen peroxide first with chloroquine abolished the effect of hydrogen peroxide. Detoxification of FP by hydrogen peroxide may be an important protective mechanism in certain hemolytic anemias, and inhibition of detoxification could account for the effectiveness of chloroquine in malaria.     

The malarial pigment in rat infected erythrocytes and its interaction with chloroquine. A M?ssbauer effect study

M?ssbauer studies of rat erythrocytes infected by Plasmodium berghei malaria parasites, using 57Fe-enriched rat red blood cells, were carried out in order to determine the physical parameters which characterize the malarial pigment iron and to test the effect of the widely used antimalaria drug, chloroquine, on these parameters. The iron in the malarial pigment which is derived from hemoglobin digestion by the intracellular parasite was found to be trivalent, high spin, with M?ssbauer parameters which are significantly different from those of any known iron porphyrin containing compound. No difference was found between the parameters obtained in erythrocytes infected by drug-sensitive and drug-resistant strains of P. berghei, both before and after the treatment with chloroquine. The iron compound consists of microaggregates, about 30 A in diameter. These are somewhat larger in chloroquine-resistant strains and tend to increase in size in chloroquine-sensitive strains upon treatment with the drug. M?ssbauer spectra of erythrocytes infected by a chloroquine-resistant strain revealed pigment iron in relative amounts invariable of those found in chloroquine-sensitive strains, demonstrating that drug-resistant parasites indeed digest hemoglobin.     

Chloroquine-induced masking of a lipid that promotes ferriprotoporphyrin IX dimerization in malaria

Mice infected with the NYU-2 strain of Plasmodium berghei were used to study the effect of chloroquine on masking of a lipid that promotes ferriprotoporphyrin IX dimerization. More than 40% of this lipid was masked and unable to promote dimerization in membrane ghosts from erythrocytes of untreated, infected mice. Thus, preparations of membrane ghosts dimerized 57 +/- 6 nmol of ferriprotoporphyrin IX during a 2-h incubation, whereas the lipids extracted from these preparations dimerized 101 +/- 11 nmol of ferriprotoporphyrin IX (means +/- S.D. for four experiments). Exposure of membrane ghosts to sonication or cold significantly increased the extent of masking. In addition, chloroquine treatment of infected mice increased the extent of masking to approximately 90%. The lipid could be unmasked by extracting it into acetone or by aging erythrocyte membrane ghosts from untreated or chloroquine-treated, infected mice for 24 h at pH 7.4 and 25 degrees C. These findings indicate that masking and unmasking of a lipid is central to the regulation of ferriprotoporphyrin IX dimerization in malaria parasites. They also indicate that chloroquine impairs the function of this regulatory process.     

The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite's 6-phosphogluconate dehydrogenase

Ferriprotoporphyrin IX (FPIX) is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleaside monophosphate kinase and pyrimidine 5'-nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-alpha, phoshoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6-phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.     

Persistent cognitive and motor deficits after successful antimalarial treatment in murine cerebral malaria

Human cerebral malaria causes neurological and behavioral deficits which persist long after resolution of infection and clearance of parasites with antimalarial drugs. Previously, we demonstrated that during active infection, mice with cerebral malaria demonstrated negative behavioral outcomes. Here we used a chloroquine treatment model of cerebral malaria to determine whether these abnormal outcomes would be persistent in the mouse model. C57BL/6 mice were infected with Plasmodium berghei ANKA, and treated for ten days. After cessation of chloroquine, a comprehensive assessment of cognitive and motor function demonstrated persistence of abnormal behavioral outcomes, 10 days after successful eradication of parasites. Furthermore, these deficits were still evident forty days after cessation of chloroquine, indicating persistence long after successful treatment, a hallmark feature of human cerebral malaria. Thus, cognitive tests similar to those used in these mouse studies could facilitate the development of adjunctive therapies that can ameliorate adverse neurological outcomes in human cerebral malaria.     

Ferriprotoporphyrin IX binding substances and the mode of action of chloroquine against malaria

Bovine serum albumin and preparations of cell sap from malaria parasites and normal erythrocytes were tested for ability to protect cellular membranes against the toxicity of ferriprotoporphyrin IX (FP) and a chloroquine-FP complex. Suspensions of $\text{Plasmodium berghei}$ (approximately 7 × 10⁶ parasites per ml, isolated from saponin-lysed, infected erythrocytes) were used as a test system. Toxicity was monitored by measuring changes in turbidity of these suspensions at 700 nm. Parasite cell sap (0.56 mg protein per ml) and albumin (1 mg per ml) completely prevented the toxicity of 40 μM FP. Erythrocyte cell sap (8.6 mg of hemoglobin per ml) provided only partial protection from 40 μM FP. Neither the cell sap preparations nor albumin eliminated the toxicity of a chloroquine-FP complex formed from 20 μM chloroquine and 40 μM FP. These observations suggest that the cell sap preparations contain FP binding substances and that the mode of action of chloroquine may be to shunt FP away from a nontoxic complex with these substances and into a toxic chloroquine-FP complex.     

Inhibition of hemozoin formation in Plasmodium falciparum trophozoite extracts by heme analogs: possible implication in the resistance to malaria conferred by the beta-thalassemia trait.

BACKGROUND: Human falciparum malaria, caused by the intracellular protozoa Plasmodium falciparum, results in 1-2 million deaths per year. P. falciparum digests host erythrocyte hemoglobin within its food vacuole, resulting in the release of potentially toxic free heme. A parasite-specific heme polymerization activity detoxifies the free heme by cross-linking the heme monomers to form hemozoin or malaria pigment. This biochemical process is the target of the widely successful antimalarial drug chloroquine, which is rapidly losing its effectiveness due to the spread of chloroquine resistance. We have shown that chloroquine resistance is not due to changes in the overall catalytic activity of heme polymerization or its chloroquine sensitivity. Therefore, the heme polymerization activity remains a potential target for novel antimalarials. In this study, we investigated the ability of heme analogs to inhibit heme polymerization and parasite growth in erythrocytes. MATERIALS AND METHODS: Incorporation of radioactive hemin substrate into an insoluble hemozoin pellet was used to determine heme polymerization. Incorporation of radioactive hypoxanthine into the nucleic acid of dividing parasites was used to determine the effects of heme analogs on parasite growth. Microscopic and biochemical measurements were made to determine the extent of heme analog entry into infected erythrocytes. RESULTS: The heme analogs tin protoporphyrin IX (SnPP), zinc protoporphyrin IX (ZnPP), and zinc deuteroporphyrin IX, 2,4 bisglycol (ZnBG) inhibited polymerization at micromolar concentrations (ZnPP << SnPP < ZnBG). However, they did not inhibit parasite growth since they failed to gain access to the site of polymerization, the parasite's food vacuole. Finally, we observed high ZnPP levels in erythrocytes from two patients with beta-thalassemia trait, which may inhibit heme polymerization. CONCLUSIONS: The heme analogs tested were able to inhibit hemozoin formation in Plasmodium falciparum trophozite extracts. The increased ZnPP levels found in thalassemic erythrocytes suggest that these may contribute, at least in part, to the observed antimalarial protection conferred by the beta-thalassemia trait. This finding may lead to the development of new forms of antimalarial therapy.     

Ferriprotoporphyrin IX, phospholipids, and the antimalarial actions of quinoline drugs

Two subclasses of quinoline antimalarial drugs are used clinically. Both act on the endolysosomal system of malaria parasites, but in different ways. Treatment with 4-aminoquinoline drugs, such as chloroquine, causes morphologic changes and hemoglobin accumulation in endocytic vesicles. Treatment with quinoline-4-methanol drugs, such as quinine and mefloquine, also causes morphologic changes, but does not cause hemoglobin accumulation. In addition, chloroquine causes undimerized ferriprotoporphyrin IX (ferric heme) to accumulate whereas quinine and mefloquine do not. On the contrary, treatment with quinine or mefloquine prevents and reverses chloroquine-induced accumulation of hemoglobin and undimerized ferriprotoporphyrin IX. This difference is of particular interest since there is convincing evidence that undimerized ferriprotoporphyrin IX in malaria parasites would interact with and serve as a target for chloroquine. According to the ferriprotoporphyrin IX interaction hypothesis, chloroquine would bind to undimerized ferriprotoporphyrin IX, delay its detoxification, cause it to accumulate, and allow it to exert its intrinsic biological toxicities. The ferriprotoporphyrin IX interaction hypothesis appears to explain the antimalarial action of chloroquine, but a drug target in addition to ferriprotoporphyrin IX is suggested by the antimalarial actions of quinine and mefloquine. This article summarizes current knowledge of the role of ferriprotoporphyrin IX in the antimalarial actions of quinoline drugs and evaluates the currently available evidence in support of phospholipids as a second target for quinine, mefloquine and, possibly, the chloroquine-ferriprotoporphyrin IX complex.     

Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.     

Plasmodium berghei NK65: studies on the effect of treatment duration and inoculum size on recrudescence

Recrudescence of Plasmodium berghei NK65 infection was studied to examine factors affecting recrudescence. Treatment with a high dose of chloroquine did not prevent recrudescence, but an extended duration of treatment suppressed the frequency of recrudescence. Infection with a larger number of parasites also resulted in more frequent recrudescences. Recrudescent parasites were as sensitive to chloroquine as those before treatment. Splenectomized mice were administered carbon particles, infected, and treated with chloroquine. Recrudescence was significantly more frequent in these mice than in mice given a sham operation and PBS. The results do not suggest that merozoite stages escape the effect of chloroquine by 'hiding' in phagocytes, but that latent parasites such as dormant ring stages may cause recrudescence.     

Plasmodium berghei: Uptake and distribution of chloroquine in infected mouse erythrocytes

The capacity of mouse erythrocytes infected with Plasmodium berghei to accumulate chloroquine is developed with maturation of the parasites. This is shown by direct comparison of the early and mature stages, which are separated by density difference. After drug accumulation, infected cells were fractionated by saponin lysis or nitrogen decompression to study the drug distribution. Effectiveness of isolating intact parasites and host components was checked by SDS-polyacrylamide gel electrophoresis and by low leakage of parasite-specific lactate dehydrogenase used as a marker enzyme. At low external drug concentration (~10^?7M), chloroquine is principally accumulated in the parasites. However, at higher drug concentrations (~10^?5and ~10^?3M), the proportion of the drug found in the host cytosol fraction is increased. A small but significant proportion of the drug (<20%) is associated with the host cell membrane. The pellet fraction of the freed parasites, further fractionated by freeze-thaw lysis, contains a major proportion of the drug at low external concentrations. However, the pellet fraction obtained from prolonged sonication of the parasites, which contains the bulk of hemozoin pigment, carries only a small proportion of the drug. This indicates that parasite membrane components may bind most of the drug. As external chloroquine concentration is increased, the proportion of drug in the parasite supernatant increases, some or most of which is probably bound by soluble hemecontaining compounds. However, the presence of chloroquine in the parasite does not affect the partition of heme in particulate and soluble forms.     

Free-flow electrophoresis for the separation of malaria infected and uninfected mouse erythrocytes and for the isolation of free parasites (Plasmodium vinckei): a new rapid technique for the liberation of malaria parasites from their host cells.

After aggregation of erythrocytes from malaria infected mice, the parasites (Plasmodium vinckei) could be set free using gentle mechanical forces. The mixture of freed parasites, infected and non-infected erythrocytes, and membraneous material was separated by free-flow electrophoresis. The free parasites produced were very pure and infectious. Morphological and enzymatic data on the separated fractions are presented. Free-flow electrophoresis also allowed the separation of infected and uninfected erythrocytes.     

Epitope mapping and topographic analysis of VAR2CSA DBL3X involved in P. falciparum placental sequestration

Pregnancy-associated malaria is a major health problem, which mainly affects primigravidae living in malaria endemic areas. The syndrome is precipitated by accumulation of infected erythrocytes in placental tissue through an interaction between chondroitin sulphate A on syncytiotrophoblasts and a parasite-encoded protein on the surface of infected erythrocytes, believed to be VAR2CSA. VAR2CSA is a polymorphic protein of approximately 3,000 amino acids forming six Duffy-binding-like (DBL) domains. For vaccine development it is important to define the antigenic targets for protective antibodies and to characterize the consequences of sequence variation. In this study, we used a combination of in silico tools, peptide arrays, and structural modeling to show that sequence variation mainly occurs in regions under strong diversifying selection, predicted to form flexible loops. These regions are the main targets of naturally acquired immunoglobulin gamma and accessible for antibodies reacting with native VAR2CSA on infected erythrocytes. Interestingly, surface reactive anti-VAR2CSA antibodies also target a conserved DBL3X region predicted to form an alpha-helix. Finally, we could identify DBL3X sequence motifs that were more likely to occur in parasites isolated from primi- and multigravidae, respectively. These findings strengthen the vaccine candidacy of VAR2CSA and will be important for choosing epitopes and variants of DBL3X to be included in a vaccine protecting women against pregnancy-associated malaria.     

Quantitative pH measurements in Plasmodium falciparum-infected erythrocytes using pHluorin

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of action of several antimalarial drugs, such as chloroquine, which accumulate in this organelle due to their properties as amphiphilic weak bases that inhibit haem detoxification. It has been suggested that changes in the pH of the digestive vacuole, affecting either drug partitioning or haem solubility and/or biomineralization rates, would correlate with reduced intracellular chloroquine accumulation and, hence, would determine the chloroquine-resistance phenotype. The techniques previously used to quantify digestive vacuolar pH mainly relied on lysed or isolated parasites, with unpredictable consequences on internal pH homeostasis. In this study, we have investigated the baseline steady-state pH of the cytoplasm and digestive vacuole of a chloroquine-sensitive (HB3) and a chloroquine-resistant (Dd2) parasite using a pH-sensitive green fluorescent protein, termed pHluorin. This non-invasive technique allows for in vivo pH measurements in intact P. falciparum-infected erythrocytes under physiological conditions. The data suggest that the pH of the cytoplasm is approximately 7.15 +/- 0.07 and that of the digestive vacuole approximately 5.18 +/- 0.05. No significant differences in baseline pH values were recorded for the chloroquine-sensitive and chloroquine-resistant parasites.     

The plasmodial surface anion channel is functionally conserved in divergent malaria parasites

The plasmodial surface anion channel (PSAC), a novel ion channel induced on human erythrocytes infected with Plasmodium falciparum, mediates increased permeability to nutrients and presumably supports intracellular parasite growth. Isotope flux studies indicate that other malaria parasites also increase the permeability of their host erythrocytes, but the precise mechanisms are unknown. Channels similar to PSAC or alternative mechanisms, such as the upregulation of endogenous host transporters, might fulfill parasite nutrient demands. Here we evaluated these possibilities with rhesus monkey erythrocytes infected with Plasmodium knowlesi, a parasite phylogenetically distant from P. falciparum. Tracer flux and osmotic fragility studies revealed dramatically increased permeabilities paralleling changes seen after P. falciparum infection. Patch-clamp of P. knowlesi-infected rhesus erythrocytes revealed an anion channel with striking similarities to PSAC: its conductance, voltage-dependent gating, pharmacology, selectivity, and copy number per infected cell were nearly identical. Our findings implicate a family of unusual anion channels highly conserved on erythrocytes infected with various malaria parasites. Together with PSAC's exposed location on the host cell surface and its central role in transport changes after infection, this conservation supports development of antimalarial drugs against the PSAC family.     

Enhanced uptake and metabolism of riboflavin in erythrocytes infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [14C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5-8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0-3 h at 37 degrees C in phosphate buffered saline containing MgCl2, glucose, and [14C]riboflavin (2.5-7.5 microM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.     

Falciparum malaria in naturally infected human patients: II. Ultrastructural alterations to erythrocytes infected with asexual forms

Ultrastructural alterations of human erythrocytes infected with asexual forms of Plasmodium falciparum were studied in naturally infected Saudi patients. These included surface knobs and nodules as well as invaginations associated with cytopasmic vesicles observed in erythrocytes infected with asexual forms of the parasites. Such nodules and surface invaginations have been previously described only in erythrocytes infected with P. ovale and P. vivax, respectively. Within the cytoplasm of infected erythrocytes were membrane-bound clefts, similar to those that appear to be a common characteristic in all red cells infected with malaria parasites. Vacuolations were often seen in the peripheral cytoplasm and may represent hemolyzed areas. Collapsed cells with an internal-lucent interior and surrounded by an irregularly folded membrane may represent completely hemolyzed erythrocytes. © 1993 Wiley-Liss, Inc.     

Chloroquine as intercalator: a hypothesis revived

The mode of action of chloroquine is still controversial. Proposed mechanisms of action include (1) DNA intercalation, (2) lysosome accumulation and (3) binding to ferriprotoporphyrin IX. Recent data suggest that intercalation into parasite DNA can occur at physiological concentrations of the drug. Furthermore, structure-activity relationship studies are most consistent with the intercalation mechanism. Regardless of which mechanism is correct, the selective toxicity of chloroquine for malaria parasites is probably due to permease-mediated uptake.