Inducible transport systems in the regulation of parasite growth in malaria-infected red blood cells

Elford, B C; Pinches, Robert

doi:10.1042/bst0200790

Cited by 14 publications

(10 citation statements)

References 9 publications

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“…Non-infected RBCs are not able to use exogenous GSH, since they are not supplied with the enzyme γ-glutamyltranspeptidase or a GSH transporter [17]. The infection with P. falciparum alters uptake of several molecules, owing to the induction of new transporters and pores in the plasma membrane of the RBC [47]. Our data support the finding by Ginsburg and Atamna [38] that such induced permeability pathways are responsible for the uptake of the tripeptide GSH.…”

Section: Discussionsupporting

confidence: 85%

Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione

Lüersen

Walter

Müller³

2000

Biochem. J.

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During the erythrocytic cycle, Plasmodium falciparum is highly dependent on an adequate thiol status for its survival. Glutathione reductase as well as de novo synthesis of GSH are responsible for the maintenance of the intracellular GSH level. The first and rate-limiting step of the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). Using L-buthionine-(S, R)-sulphoximine (BSO), a specific inhibitor of the gamma-GCS, we show that the infection with P. falciparum causes drastic changes in the GSH metabolism of red blood cells (RBCs). Infected RBCs lose GSH at a rate 40-fold higher than non-infected RBCs. The de novo synthesis of the tripeptide was found to be essential for parasite survival. GSH depletion by BSO inhibits the development of P. falciparum with an IC(50) of 73 microM. The effect of the drug is abolished by supplementation with GSH or GSH monoethyl ester. Our studies demonstrate that the plasmodicidal effect of the inhibitor BSO does not depend on its specificity towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite GSH in the P. falciparum-infected RBCs. Therefore the depletion of GSH is proposed as a chemotherapeutic strategy for malaria, and gamma-GCS is proposed as a potential drug target.

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Section: Discussionsupporting

confidence: 85%

Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione

Lüersen

Walter

Müller³

2000

Biochem. J.

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“…The GDH-catalyzed reaction has been proposed to be a major source of NADPH in the parasite [19]. The high K m value for ammonia makes the generation of glutamate unlikely [35]. In addition, ammonia, which is toxic for both the parasite and the host, should easily diffuse out of the cells.…”

Section: Discussionmentioning

confidence: 99%

“…Infection of human erythrocytes by P. falciparum causes a 100-fold increase in the uptake of L-glutamine [36]. This exogenous glutamine would be converted into glutamate by the parasite glutamate synthase (GO-GAT, glutamine amide 2-oxoglutarate amidotransferase : Glutamine ϩ 2-oxoglutarateϩNADPHϩH ϩ →2 glutamateϩNADP ϩ ), an enzyme which previously had only been found in bacteria and plants [35]. One may, therefore, speculate that the combined action of GOGAT (which consumes one NADPH, but produces two molecules glutamate) and GDH (which produces one NADPH/glutamate) results in the net generation of NADPH at the expense of exogenous L-glutamine.…”

Section: Discussionmentioning

confidence: 99%

Glutamate dehydrogenase, the marker protein of Plasmodium falciparum

Wagner

Lüdemann

Färber

et al. 1998

European Journal of Biochemistry

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The gene of an NADP ϩ -specific glutamate dehydrogenase was cloned from Plasmodium falciparum, the causative agent of tropical malaria. Southern-blot analysis indicates a single-copy gene. The gene encodes a protein with 470 residues which has 50% of all residues identical with those of the glutamate dehydrogenases from other low eukaryotes and eubacteria. In contrast, the sequence identity with the human enzyme is marginal, which underlines the long evolutionary distance between parasite and host. The gene was overexpressed in Escherichia coli. The kinetic properties of the recombinant enzyme are in good agreement with those of the authentic enzyme. The parasite enzyme is inhibited by D-glutamate and glutarate, but not by chloroquine. Like other coenzyme-specific glutamate dehydrogenases, but in contrast to the dual-specific mammalian enzymes, the P. falciparum enzyme is not affected by GTP and ADP. The physical and chemical properties of the protein are in accordance with the cytosol being the major localization. The gene does not encode a cleavable mitochondrial presequence and the M r of the recombinant protein and the protein isolated from the parasite are indistinguishable on SDS/PAGE. Western-blot analysis of stage-specific parasites shows that glutamate dehydrogenase is present in all intraerythrocytic stages. The signal increased continuously from rings, early trophozoites to late trophozoites and decreased slightly in the segmenter stage. Glutamate dehydrogenase, suggested to be the major source of NADPH in the parasite, is an attractive target molecule for the rational development of new antimalarial drugs.

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“…P. falciparum growth was assessed by measuring the incorporation of the radiolabeled nucleic acid precursor [ 3 H]hypoxanthine (5) in media containing either human pooled serum or heat-inactivated plasma. All assays were started with ring stage-synchronized cultures at 2-4% parasitemia and processed as reported (6)(7)(8). Aliquots of stock solutions of CLT in DMSO were placed in the wells of flatbottomed cell culture plates (Nunc), under sterile conditions, to render final concentrations of 0.1-5 M CLT after the addition of either control or parasitized red cell suspensions in culture medium.…”

Section: Assessment Of Parasite Morphology Stage-specific Developmenmentioning

confidence: 99%

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

Tiffert

Ginsburg

Krugliak

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC50), assessed by parasite incorporation of [ 3 H]hypoxanthine, were between 0.2 and 1.1 M. CLT concentrations of 2 M and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (Ϸ48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 M CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria. In the course of an investigation on the homeostasis of human red cells infected in vitro with the human malaria parasite Plasmodium falciparum, we found that clotrimazole (CLT), an imidazole derivative used as an antifungal agent (1), had a powerful growth-inhibiting effect on the parasite. The vast clinical experience, proven tolerance, and unique lack of acquired fungal resistance to CLT prompted a detailed investigation of its antimalarial effects in cultures of P. falciparum to assess its clinical potential. In this study, we report the in vitro effects of CLT on P. falciparum cultures by assessing the timeand concentration-dependent changes in parasite growth, parasite morphology, stage-specific development, and parasite replication. Materials and MethodsCultures. Five different laboratory strains of P. falciparum [A4 (2), W2, NF54, HB3, and FCR] were cultured in human erythrocytes by standard methods (3) under a low oxygen atmosphere. The culture medium was RPMI 1640, supplemented with 40 mM Hepes, 25 mg͞liter gentamicin sulfate, 10 mM D-glucose, 2 mM glutamine, and either 8.5% (vol͞vol) pooled human serum (A4 clone), or 10% heat-inactivated plasma (strains W2, NF54, HB3, and FCR). Culture media, with or without CLT, were changed daily, unless otherwise indicated. Parasites were synchronized at the ring stage with D-sorbitol (4) for all experiments. Initial parasitemias varied between 2% and 7% for the different e...

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Inducible transport systems in the regulation of parasite growth in malaria-infected red blood cells

Abstract: Paucity of basic biochemical data relating to human malariaFollowing the advent of continuous culture of P. fulczpurum [8, 91, it has been established empirically that adequate exogenous concentrations of both 11glucose and L-glutamine [7, 101 are essential for the Volume 20

Cited by 14 publications

References 9 publications

Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione

Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione

Glutamate dehydrogenase, the marker protein of Plasmodium falciparum

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

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