The Malaria Parasite Supplies Glutathione to its Host Cell — Investigation of Glutathione Transport and Metabolism in Human Erythrocytes Infected with Plasmodium Falciparum

Abstract

Malaria-infected red blood cells are under a substantial oxidative stress. Glutathione metabolism may play an important role in antioxidant defense in these cells, as it does in other eukaryotes. In this work, we have determined the levels of reduced and oxidized glutathione (GSH and GSSG, respectively) and their distributions in the parasite, and in the host-cell compartments of human erythrocytes infected with the malaria parasite Plasmodium falciparum. In intact trophozoite-infected erythrocytes, [GSH] is low and [GSSG] is high, compared with the levels in normal erythrocytes. Normal erythrocytes and the parasite compartment display high GSH/GSSG ratios of 321.6 and 284.5, respectively, indicating adequate antioxidant defense. This ratio drops to 26.7 in the host-cell compartment, indicating a forceful oxidant challenge, the low ratios resulting from an increase in GSSG and a decline in GSH concentrations. On the other hand, the concentrations of GSH and GSSG in the parasite compartment remain physiological and comparable to their concentrations in normal red blood cells. This results from de novo glutathione synthesis and its recycling, assisted by the intensive activity of the hexose monophosphate shunt in the parasite. A large efflux of GSSG from infected cells has been observed, its rate being similar from free parasites and from intact infected cells. This result suggests that de novo synthesis by the parasite is the dominating process in infected cells. GSSG efflux from the intact infected cell is more than 60-fold higher than the rate observed in normal erythrocytes, and is mediated by permeability pathways that the parasite induces in the erythrocyte's membrane. The main route for GSSG efflux through the cytoplasmic membrane of the parasite seems to be due to a specific transport system and occurs against a concentration gradient. 1)-Glutamylcysteine [Glu(-Cys)] and GSH can penetrate through the pathways from the extracellular space into the host cytosol, but not into that of the parasite. This implies that the parasite membrane is impermeable to these peptides, and that the host cannot supply GSH to the parasite as suggested previously. Exogenous Glu(-Cys) is not converted into GSH in the host cell, arguing that GSH synthetase may not be functional. Compartment analysis of Mg" in infected erythrocytes revealed that the host compartment exhibits a low concentration of Mg2+ (0.5 mM) in comparison with the parasite compartment (4 mM) and the normal erythrocytes (1.5-3 mM). The drop in [Mg"] results in cessation of Glu(-Cys) synthesis, and hence of GSH synthesis in the host-cell compartment. The decrease in [Mg"] can affect other Mg2+-ATP-dependent functions, such as Na' and Ca2+ active efflux. The present investigation confirms that the host-cell compartment is oxidatively distressed, whereas the parasite is efficiently equipped with anti-oxidant means that protect the parasite from the oxidative injury. The parasite has a huge capacity for de nova synthesis of GSH and for the reduction of GSSG. Part o...

Clotrimazole Binds to Heme and Enhances Heme-dependent Hemolysis

“…As free heme is highly toxic to the malarial parasite, the parasite has developed a means of detoxifying heme through polymerization to non-toxic, insoluble hemozoin (8) or by degradation with GSH (9 -11), which is found at millimolar concentrations in red blood cells and parasite compartments (12,13). About 30 -50% of free heme is detoxified by polymerization at the trophozoite stage (10,14,15), and the remainder is detoxified by GSH-dependent degradation.…”

“…Thus, the axial-ligated CLT completely inhibited GSH-dependent heme degradation. The concentration of GSH in red blood cells and in the parasite compartment reaches millimolar levels (12,13). Nevertheless, the high equilibrium association constant of CLT (6.54 ϫ 10 8 M Ϫ2 ) should allow the heme-CLT complex to exist in the erythrocyte and malaria parasite.…”

The Thioredoxin System of the Malaria Parasite Plasmodium falciparum

“…Since Plasmodium, like tumor cells, is particularly susceptible to oxidative challenge, the glutathione system and the thioredoxin system ( Fig. 1) represent potential targets for antiparasitic and antitumor drug design (2)(3)(4)(5)(6)(7).…”

“…Unlike the monothiol tripeptide glutathione, the dithiol thioredoxin (Trx; 12 kDa) has a macromolecular structure and is reduced by thioredoxin reductase (TrxR (EC 1.6.4.5); NADPH ϩ H ϩ ϩ TrxS 2 3 NADP ϩ ϩ Trx(SH) 2 ). High molecular weight TrxRs as they occur in malarial parasites and in mammalian cells are structurally and mechanistically closely related to glutathione reductase but differ significantly from low molecular weight bacterial TrxRs (6, 9 -11).…”

“…Redox Potential of PfTrx-The reversibility of the reaction NADPH ϩ TrxS 2 ϩ H ϩ º Trx(SH) 2 ϩ NADP ϩ was employed for determining the equilibrium constant (18) using the absorbance change at 340 nm as a measure of NADPH oxidation with concomitant production of an equimolar amount of Trx(SH) 2 , the resulting change in absorbance at 340 nm was converted into NADPH concentration change by using a millimolar absorbance coefficient of 6.22 mM Ϫ1 cm Ϫ1 . These experiments yielded an equilibrium constant of 110 Ϯ 10 at pH 7.4 which corresponds to a difference in redox potential of 60.2 Ϯ 2 mV.…”