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Plasmodium falciparum: Thiol status and growth in normal and glucose-6-phosphate dehydrogenase deficient human erythrocytes
EXPERIMENTALPARASITOLOGY57, 239-247 (1984)
Plasmodium falciparum: Thiol Status and Growth in Normal and Glucose-6-Phosphate Dehydrogenase Deficient Human Erythrocytes JACQUELINE
MILLER,
JACOB GOLENSER,
DAN
T. SPIRA
Department of Parasitology, The Kuvin Centre for the Study of Infectious and Tropical Diseases, Hebrew University-Hadassah Medical School, Jerusalem, Israel AND NECHAMA
S. KOSOWER
Department of Human Genetics, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel (Accepted for publication
5 January 1984)
MILLER, J., GOLENSER, J., SPIRA, D. T., AND KOSOWER, N. S. 1984. Plasmodium falciparum: Thiol status and growth in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes. Experimental Parasitology 57, 239-247. The relationship of the thiol status of the human erythrocyte to the in vitro growth of Plasmodium falciparum in normal and in glucose-6-phosphate dehydrogenase (G6PD)-deficient red cells was investigated. Pretreatment with the thiol-oxidizing agent diamide led to inhibition of growth of P. falciparum in G6PD-deficient cells, but did not affect parasite growth in normal cells. Diamidetreated normal erythrocytes quickly regenerated intracellular glutathione (GSH) and regained normal membrane thiol status, whereas G6PD-deficient cells did not. Parasite invasion and intracellular development were affected under conditions in which intracellular GSH was oxidized to glutathione disulfide and membrane intrachain and interchain disulfides were produced. An altered thiol status in the G6PD-deficient erythrocytes could underlie the selective advantage of G6PD deficiency in the presence of malaria. INDEX DESCRIPTORS: Plasmodium falciparum; Protozoa, parasitic; Malaria, human; Erythrocyte; Thiol status; Glucose-6-phosphate dehydrogenase (G6PD) deficiency; Diamide .
INTRODUCTION
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with increased morbidity and mortality in humans (Beutler 1983). Nevertheless, this inherited abnormality has attained a high frequency, most notably in many areas where endemic malaria due to Plasmodium falciparum existed. The suggestion that G6PD deficiency may confer a selective advantage in the presence of P. fulciparum (Allison 1960; Motulsky 1960) has been supported by some population studies (Luzzatto et al. 1969; Siniscalco et al. 1966; Luzzatto 1979; Guggenmoos-Holzmann et al. 1981),
though questioned by others (Martin et al. 1979; Bernstein et al. 1980). Hemolysis in G6PD-deficient individuals may be precipitated by certain drugs, infections, or fava bean ingestion (Beutler 1983). Under these conditions, erythrocyte glutathione (GSH) is irreversibly oxidized to the disulfide (GSSG) due to the failure of NADPH regeneration in the deficient cells. In addition, oxidative membrane damage, with or without intracellular hemoglobin denaturation, is found in vivo in G6PD-deficient individuals with active hemolysis (Johnson et al. 1979). GSSG has previously been shown to inhibit protein synthesis (Kosower et al. 239 0014-4894184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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1972; London et al. 1976; Ernst et al. 1978). It was proposed that an increased erythrocyte GSSG, by lowering the rate of parasite proliferation, provides a molecular basis for the selective advantage of G6PD-deficient cells in the presence of malaria (Kosower and Kosower 1970). Enhanced oxidative damage to the infected G6PD-deficient cell has also been considered as a cause for diminished parasitemia (Eckman and Eaton 1979). This is in agreement with the observed in vivo killing of malaria parasites by free radical-generating agents (Clark and Hunt 1983; Allison and Eugui 1982). In vitro culture of the erythrocytic forms of P. fulciparum (Trager and Jensen 1976) has made possible the direct study of whether or not G6PD-deficient cells are more resistant to the parasite. Diminished parasite multiplication was observed in G6PD-deficient cells when exposed to free radical-generating sources (Friedman 1979). Golenser et al. (1983) have shown that G6PD-deficient erythrocytes treated with isouramil, a component of fava bean extract which gives rise to radical species, do not support parasite growth. Inhibition of parasite growth was also observed in some G6PD-deficient cells without overt exposure to oxidative stress (Luzzatto et al 1983; Roth et al. 1983). The alterations in the cells responsible for the inhibitory effects are not known. Oxidation of cellular GSH and defined oxidative changes in membrane thiols without free radical damage to hemoglobin and membrane can be induced experimentally by the use of the thiol-oxidizing agent diamide (Kosower et al. 1969; Kosower et al. 1978). The alterations induced by diamide in G6PD-deficient cells (Kosower et al. 1982) are similar to those found in vivo in some G6PD-deficient individuals (Johnson et al. 1979). Thus, the in vitro intraerythrocytic growth of P. fulcipurum can be studied in detail in relation to the thiol status of the red cell. Here we show that the multiplication of
ET AL.
is inhibited in diamidetreated, G6PD-deficient erythrocytes. Parasite growth is markedly affected under conditions in which cellular GSH has been oxidized, and membrane disulfides have been present. P. fulcipurum
MATERIALS
AND METHODS
Blood, collected in acid-citrate-dextrose (ACD), was obtained from normal individuals and from healthy males with G6PD deficiency, Mediterranean type, and kept at 4C. Experiments were started within 24 hr after obtaining the blood samples. Blood was centrifuged, and plasma and buffy coat were removed. Erythrocytes were washed twice with phosphate-buffered saline, pH 7.2 (PBS), and resuspended in the same buffer to give a 20% cell suspension. Equal volumes of sterile solutions of diamide in PBS (solutions sterilized with a 45pm filter) were added to cell suspensions with rapid mixing (final diamide concentrations, 0.25- 1.O m&f). Control cell suspensions were mixed with PBS alone. The ceil suspensions were incubated at 37C for 30 min with gentle shaking, washed with RPMI-1640 medium to remove remaining diamide, and suspended in growth medium (RPMI-1640, containing 10% human serum). Plasmodium falciparum (strain FCR3TC, obtained from J. B. Jensen) was cultured according to the method of Trager and Jensen (1976), with some modifications, as described previously (Golenser et al. 1983), and with the addition of 10 mM glucose and 40 PM S-fluorocytosine. Cultures were synchronized by employing a combination of the methods of Jensen (1978) and Lambros and Vanderberg (1979), as previously described (Golenser er al. 1983). Suspensions enriched in trophozoites and schizonts were obtained by the gelatin sedimentation technique of Jensen (1978). A fraction with parasitized erythrocytes enriched with trophozoites and schizonts (the inoculum) was added to a 2.5% erythrocyte suspension (recipient cell population), so that the initial parasitemia was 0.5-l% of the cell suspension, unless otherwise stated, and contamination by the inoculum cells was less than 1.5% of the recipient cell population. The cell suspensions were dispensed into a 96-well microplate (100 pi/well) and incubated at 37C, using the candle jar method of ‘Bager and Jensen (1976). The supematant was replaced every day by fresh growth medium. Samples in triplicate were removed at time intervals and Giemsa-stained smears were prepared for parasite counts and for morphological examination. For analysis of thiols and membrane proteins, nonparasitized control and diamide-treated erythrocytes were incubated as 5% cell suspensions in growth medium in 35-mm Petri dishes (2.0 ml/dish), using the
Plasmodium
fakiparum:
GROWTH
IN
candle jar method (‘Rager and Jensen 1976). The supematant was changed daily, as done for the parasitized cells. Samples were removed at intervals, and the cells were washed in PBS. Aliquots of cell suspension in PBS were labeled with monobromobimane (mBBr), as previously described (Kosower et al. 1979). Erythrocyte ghosts were prepared according to the method of Steck and Kant (1974). Ghosts were solubilized and analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis, according to established methods (Fairbanks et al. 1971; Liu et al. 1977), without addition of dithiothreitol. After electrophoresis, gels were fmed, photographed under ultraviolet (360 nm) illumination, and stained with Coomssie blue and photographed again, as described previously (Kosower et al. 1981).
For GSSG determination, cells were lysed in 5 mM phosphate buffer, pH 8.0. Hemolysates (0.5 r&f hemoglobin) were incubated for 15 min at 37C in the presence and absence of 1.0 mM NADPH and 0.05 unit GSSG reductase (Sigma, type III, 145 units/mg protein)/ml of hemolysate. Samples were then analyzed for GSH. Analyses of GSH, hemoglobin, and membrane thiols were carried out on nonlabeled aliquots, using 5,5-dithiobis(2nitrobenzoic acid) (Ellman 1959) or on mBBr-labeled aliquots (Kosower et al. 1981). RESULTS
To study the multiplication of Plasmodium falciparum in normal and G6PD-deticient erythrocytes, the erythrocytes were pretreated with various concentrations of diamide, washed to remove any remaining reagent, and then infected with P. falciparum. Figure 1 shows the course of the parasitemia in erythrocytes from four normal and four G6PD-deficient individuals. In normal cells, the rate of multiplication of parasites in cells pretreated with diamide was similar to that of control cells. In G6PD-deficient erythrocytes, the multiplication of parasites in control cells was similar to that in normal cells. A significant inhibition in parasite multiplication was observed in G6PD-deficient cells pretreated with diamide. In experiments carried out on erythrocytes obtained from other G6PD-deficient males, slight individual differences were noted in the magnitude of the effects, but the overall results were the same. The inhibition of parasite growth in
NORMAL
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DAYS IN
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1. The effect of diamide on the growth of Plusmodium falciparum in erythrocytes from 4 normal humans (J. G., A. T., I. K., and D. K.) and from 4 G6PD-deficient humans (M. Z., D. N., E. M., and E. T.). Erythrocytes (10% suspensions) were incubated for 30 min with or without 1.0 mM diamide, washed, and resuspended in growth medium, and then inoculated with parasitized erythrocytes. FIG.
G6PD-deficient erythrocytes was dose dependent and ranged from partial inhibition in samples pretreated with 0.25-0.5 mM diamide to a complete inhibition of growth in samples treated with 1.0 mM diamide (Fig. 2). The morphology of the parasites in control deficient cells was similar to that of parasites in normal cells, though some desynchronization was noted in several of the control deficient samples within 2-3 days of culture. Some of the parasites in the diamide-treated deficient cells were morphologically abnormal. The behavior of the parasites during the initial 24 hr of culture was followed by examining samples every 5-6 hr after the addition of the inoculum of trophozoites and schizonts to the recipient cell suspensions. Results are shown in Fig. 3. In control samples of both normal (Fig.3A) and G6PD-deficient eythrocytes (Fig.3B), ring forms appeared concomitantly with the decrease in the number of mature parasites, starting at about 6 hr after inoculum addition (Figs. 3A and B). These results demonstrate that the merozoites, derived from schizonts in the
242
MILLER 0 0 A 0
ET AL. I
LOmM OIAMIDE 0.5mM DIAMIDE 0.25mM DIAMID PBS
160 -
I
I
A. NORMAL
I
I
,
I
l-
B.GGPD(-) P-
h +
0
I
2
3
4
CULTURE
FIG. 2. Growth of Plasmodium falciparum in normal and in G6PD-deficient human erythrocytes pretreated with various concentrations of diamide. Erythrocytes (10% suspensions) were treated for 30 min with diamide in concentrations of 0.25, A; 0.5, 0; and 1.0 mM, 0. Control cells suspended in PBS, 0.
inoculum, had invaded the recipient cells and developed into ring forms. P. falciparum added to diamide-treated normal cells behaved similarly to the control cells, with ring forms appearing at a similar or a slightly lower rate in diamidetreated cells as compared to that in the control cells (Fig. 3A). In marked contrast, very few ring forms appeared in the diamide-treated, G6PD-deficient cells at the time when many of the mature forms had disappeared (Fig. 3B). These results indicate that the merozoites which were released from the donor cells had either not invaded the diamide-treated deficient cells or did not develop into recognizable ring forms in these cells. To study cellular and membrane thiols, unparasitized control and diamide-treated erythrocytes were used. At the beginning of culture, less GSH was present in the control G6PD-deficient cells than in the control normal cells. GSH declined in both types of cells over several days in culture (Fig. 4A). Pretreatment of the red cells with diamide initially resulted in the oxidation of
i-
10 -
0.
’ 6
’ ’ 12 18 HOURS
I I ’ 3 6 12 IN CULTURE
I it3
L 24
FIG. 3. The dium falciparum
development of ring forms of Plasmoin normal and in G6PD-deficient human erythrocytes pretreated with diamide. Erythrocytes (10% suspensions) were incubated for 30 min with or without 1.0 m&f diamide, then washed and resuspended in growth medium. Cell suspensions were then inoculated with parasitized erythrocytes (mature forms) to an initial parasitemia of 5% and cultured. Samples were examined every 5-6 hr during the first 24 hr of culture. (A) Normal cells: 0, control; 0, diamide treated. (B) G6PD-deficient cells: A, control; A, diamide treated.
GSH to GSSG in both normal and G6PDdeficient erythrocytes. In normal erythrocytes, GSH is rapidly regenerated in the presence of glucose, so that a short incubation in the growth medium led to almost complete regeneration of the cellular GSH. In contrast, GSH was not regenerated in the G6PD-deficient cells, so that the diamide-treated deficient cells had no or very little GSH throughout the culture period (Fig. 4A). Initially, the oxidized GSH was present in these cells as GSSG. No GSSG was found in the deficient cells after culture for 24-48 hr. Membrane SH group content was similar in both normal and deficient control cells at the beginning of the culture, with no significant change during the subsequent incubation (Fig. 4B). In diamide-treated normal erythrocytes, the level of mem-
Plasmodium
fUkipUrUi?Z:
GROWTH
IN NORMAL
AND G6PD-DEFICIENT
G6PDd. normal
GGPDd. normal -
‘. g
2-
‘.
A.‘ds--A
.
-2
;
_
L
+
-
+
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DIAMIDE
-
+
-
+
5 I
.
0123401234 DAYS
IN CULTURE
FIG. 4. Glutathione (GSH) and membrane SH groups in normal and in G6PD-deficient human erythrocytes treated with diamide. Erythrocytes (10% suspensions) were incubated for 30 min with or without 1.O mM diamide, washed, resuspended in growth medium, and incubated under the same conditions used for culture of Plasmodium falciparum parasitized cells. (A) GSH, (B) membrane SH groups. 0, normal cells, control; 0, normal cells, diamide treated; A, G6PD-deficient cells, control; A, G6PD-deficient, diamide treated.
brane SH groups was similar to that of normal control cells throughout the culture period. In the diamide-treated G6PD-deficient cells, membrane thiol groups were diminished (Fig. 4B). Profiles of membrane proteins of erythrocytes at the time of inoculum addition are shown in Fig. 5. The distribution of the fluorescence and the stained protein bands in the diamide-treated normal cells was similar to that of the control cells. In contrast, membrane proteins from diamide-treated, G6PD-deficient cells exhibited alterations in the distribution of both the label and the stained protein bands; a significant diminution in labeling was noted in many of the bands. The profile of the stained proteins showed a diminution in some of the bands and the appearance of high-molecularweight proteins. An altered pattern of membrane proteins was also found in diamidetreated deficient cells after several days in culture (not shown).
FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of human erythrocyte membrane proteins isolated from normal and from G6PDdeficient cells incubated for 30 min with or without 1.O m&f diamide. Erythrocytes were then washed in PBS, labeled with monobromobimane, and processed. Left, fluorescence; right, Coomassie blue staining. H.M.W., high-molecular-weight proteins.
Human hemoglobin SH groups are not reactive towards diamide (Kosower er al. 1982). In both normal and G6PD-deficient, diamide-treated erythrocytes, the hemoglobin was not denatured and thiols were unaltered throughout the incubation period. DISCUSSION
The growth of Plasmodium falciparum in relation to the thiol status of the human host’s erythrocytes was examined. We found a significant inhibition of the parasite growth in G6PD-deficient cells following thiol oxidation by diamide. Pretreatment of normal erythrocytes with diamide did not affect the parasite growth. The inhibition of parasite growth in diamide-treated, G6PD-deficient cells may be due to effects on one or more stages in the parasite life cycle, namely, the attachment, internalization, and/or the intracellular development of the parasites. The reduced number of newly formed rings observed in the diamide-treated deficient cells following the initial release of merozoites
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from the inoculum indicates that entry of the parasite into that cell is inhibited. The subsequent deficiency in mature parasites and the appearance of abnormal forms suggest that there is also an inhibition of intracellular parasite development. Treatment of G6PD-deficient erythrocytes (and of glucose-deprived normal cells) with the thiol-oxidizing agent diamide (Kosower et al. 1969) results in the oxidation of GSH to GSSG. When cellular GSH is low, membrane protein thiols are oxidized, with the formation of intrachain and interchain disulfides (Kosower et al. 1981). The normal erythrocyte can, when metabolically active (i.e., in the presence of glucose), recover the original membrane thiol status along with the reduction of GSSG. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired (Kosower et al. 1982). In the experiments described here, the diamide-treated erythrocytes were washed and resuspended in RPM1 medium containing glucose. The normal cell quickly repaired most of the diamide-induced oxidative damage, so that cellular GSH was regenerated to about 80% of control level, membrane disulfides were reduced, and the original, control membrane protein profile was recovered at the time of parasite addition or shortly thereafter. In contrast, no GSH was regenerated in the deficient cell, nor was the membrane oxidative damage repaired at the time of parasite innoculation or thereafter. Invasion of the cell by the parasite involves attachment to the membrane, followed by invagination of the cell membrane and internalization of the parasite, with the completion of endocytosis by the fusion of the erythrocyte membrane at the point of parasite entry (Aikawa et al. 1978; Aikawa et al. 1981; Pasvol and Wilson 1982). A decrease in the density of intramembranous particles (IMP) in the area of invagination (McLaren et al. 1979; Aikawa et al. 1981),
ET AL.
arising from a redistribution of membrane components, has been found. The lateral mobility of IMP, usually restricted by the erythrocyte, may be made possible by a proteolytic release of the restrictive linkage between the IMP and the cytoskelton. Little information is available on parasite invasion of cells in which membrane proteins are modified. Ovalocytes which have an altered cytoskeleton are resistant to parasite invasion (Kidson et al. 1981). Antibodies to spectrin, which cause spectrin crosslinking, prevent the parasite invasion (Pasvol and Wilson 1982; Olson and Kilejian 1982). Miller et al. (1977) and Perkins (1981) have shown that enzymatic treatment of erythrocyte membranes prevents the invasion of the erythrocyte by certain plasmodia. The alterations in membrane thiol status shown here may inhibit parasite invasion at one or more stages. For example, the high-molecular-weight aggregates and the intrachain disultides formed in the membranes of G6PD-deficient cells may interfere with parasite attachment to the cell and subsequent invagination and internalization by diminishing cell deformability and by preventing the formation of IMP-free areas in the membrane. The changes in the membrane could also affect intracellular development of the invading merozoites by altering the transport of various substances necessary for the parasite growth (Cabantchik et al. 1983). Hemoglobin within the human erythrocyte is not altered by diamide treatment, and, as a substrate for growing parasites (Sherman 1979), can come from either normal or G6PD-deficient erythrocytes. Intracellular development of the parasite may, however, be altered as a result of lack of GSH, or the presence of GSSG. Inhibition of P. falciparum growth was found in samples of all G6PD-deficient erythrocytes when parasites were added to the cells shortly after diamide treatment. In some cases, especially those treated with lower concentrations of diamide, the
P/asmodium
fa&arum:
GROWTH
IN NORMAL
number of parasitized erythrocytes began to rise slightly after severaldays in culture. Theseresults indicate that some of the cellular alterations responsiblefor growth inhibition have been reversed. One significant change observed in the diamidetreated deficient cells incubated in culture for 2 days was the elimination of GSSG from the cells. The results indicate that GSSG may play a role in the inhibition of P. falciparum growth. GSSG inhibits the initiation of protein synthesis(Kosower et al. 1972;London et al. 1976;Ernst er al. 1978),and has beenproposedas a possible factor in the relative resistance of the G6PD-deficienterythrocyte to Plasmodium falciparum (Kosower and Kosower 1970). The fitness of the G6PD-deficient phenotype is consideredto be only slightly decreasedand, therefore, only a small increment in the resistanceto malaria would be sufficient to achieve a balanced polymorphism in populations in endemic areas. Thus, an initial, partial inhibition and desynchronizationin the parasitegrowth due to thiol-oxidation in G6PD-deficienterythrocytes would be sufficient to explain the overall relative resistanceto malaria. ACKNOWLEDGMENTS This study was supported by grants from The Lester Aronberg Foundation; from the Chief Scientist’s office, Ministry of Health, Israel; and from the fund for basic research administered by the Israel Academy of Sciences and Humanities.
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Impermeable Ghosts and Inside-out Vesiclesfrom Human Erythrocyte Membranes. In “Methods in Enzymology” (S. Fleischer and L. Packer, eds.), Vol. 31, pp. 172-180. Academic Press,New York, San Francisco,London.
J. A.
1974.
Prepartion
of
673-675.
malar193,
Plasmodium falciparum: Thiol Status and Growth in Normal and Glucose-6-Phosphate Dehydrogenase Deficient Human Erythrocytes JACQUELINE
MILLER,
JACOB GOLENSER,
DAN
T. SPIRA
Department of Parasitology, The Kuvin Centre for the Study of Infectious and Tropical Diseases, Hebrew University-Hadassah Medical School, Jerusalem, Israel AND NECHAMA
S. KOSOWER
Department of Human Genetics, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel (Accepted for publication
5 January 1984)
MILLER, J., GOLENSER, J., SPIRA, D. T., AND KOSOWER, N. S. 1984. Plasmodium falciparum: Thiol status and growth in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes. Experimental Parasitology 57, 239-247. The relationship of the thiol status of the human erythrocyte to the in vitro growth of Plasmodium falciparum in normal and in glucose-6-phosphate dehydrogenase (G6PD)-deficient red cells was investigated. Pretreatment with the thiol-oxidizing agent diamide led to inhibition of growth of P. falciparum in G6PD-deficient cells, but did not affect parasite growth in normal cells. Diamidetreated normal erythrocytes quickly regenerated intracellular glutathione (GSH) and regained normal membrane thiol status, whereas G6PD-deficient cells did not. Parasite invasion and intracellular development were affected under conditions in which intracellular GSH was oxidized to glutathione disulfide and membrane intrachain and interchain disulfides were produced. An altered thiol status in the G6PD-deficient erythrocytes could underlie the selective advantage of G6PD deficiency in the presence of malaria. INDEX DESCRIPTORS: Plasmodium falciparum; Protozoa, parasitic; Malaria, human; Erythrocyte; Thiol status; Glucose-6-phosphate dehydrogenase (G6PD) deficiency; Diamide .
INTRODUCTION
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with increased morbidity and mortality in humans (Beutler 1983). Nevertheless, this inherited abnormality has attained a high frequency, most notably in many areas where endemic malaria due to Plasmodium falciparum existed. The suggestion that G6PD deficiency may confer a selective advantage in the presence of P. fulciparum (Allison 1960; Motulsky 1960) has been supported by some population studies (Luzzatto et al. 1969; Siniscalco et al. 1966; Luzzatto 1979; Guggenmoos-Holzmann et al. 1981),
though questioned by others (Martin et al. 1979; Bernstein et al. 1980). Hemolysis in G6PD-deficient individuals may be precipitated by certain drugs, infections, or fava bean ingestion (Beutler 1983). Under these conditions, erythrocyte glutathione (GSH) is irreversibly oxidized to the disulfide (GSSG) due to the failure of NADPH regeneration in the deficient cells. In addition, oxidative membrane damage, with or without intracellular hemoglobin denaturation, is found in vivo in G6PD-deficient individuals with active hemolysis (Johnson et al. 1979). GSSG has previously been shown to inhibit protein synthesis (Kosower et al. 239 0014-4894184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
240
MILLER
1972; London et al. 1976; Ernst et al. 1978). It was proposed that an increased erythrocyte GSSG, by lowering the rate of parasite proliferation, provides a molecular basis for the selective advantage of G6PD-deficient cells in the presence of malaria (Kosower and Kosower 1970). Enhanced oxidative damage to the infected G6PD-deficient cell has also been considered as a cause for diminished parasitemia (Eckman and Eaton 1979). This is in agreement with the observed in vivo killing of malaria parasites by free radical-generating agents (Clark and Hunt 1983; Allison and Eugui 1982). In vitro culture of the erythrocytic forms of P. fulciparum (Trager and Jensen 1976) has made possible the direct study of whether or not G6PD-deficient cells are more resistant to the parasite. Diminished parasite multiplication was observed in G6PD-deficient cells when exposed to free radical-generating sources (Friedman 1979). Golenser et al. (1983) have shown that G6PD-deficient erythrocytes treated with isouramil, a component of fava bean extract which gives rise to radical species, do not support parasite growth. Inhibition of parasite growth was also observed in some G6PD-deficient cells without overt exposure to oxidative stress (Luzzatto et al 1983; Roth et al. 1983). The alterations in the cells responsible for the inhibitory effects are not known. Oxidation of cellular GSH and defined oxidative changes in membrane thiols without free radical damage to hemoglobin and membrane can be induced experimentally by the use of the thiol-oxidizing agent diamide (Kosower et al. 1969; Kosower et al. 1978). The alterations induced by diamide in G6PD-deficient cells (Kosower et al. 1982) are similar to those found in vivo in some G6PD-deficient individuals (Johnson et al. 1979). Thus, the in vitro intraerythrocytic growth of P. fulcipurum can be studied in detail in relation to the thiol status of the red cell. Here we show that the multiplication of
ET AL.
is inhibited in diamidetreated, G6PD-deficient erythrocytes. Parasite growth is markedly affected under conditions in which cellular GSH has been oxidized, and membrane disulfides have been present. P. fulcipurum
MATERIALS
AND METHODS
Blood, collected in acid-citrate-dextrose (ACD), was obtained from normal individuals and from healthy males with G6PD deficiency, Mediterranean type, and kept at 4C. Experiments were started within 24 hr after obtaining the blood samples. Blood was centrifuged, and plasma and buffy coat were removed. Erythrocytes were washed twice with phosphate-buffered saline, pH 7.2 (PBS), and resuspended in the same buffer to give a 20% cell suspension. Equal volumes of sterile solutions of diamide in PBS (solutions sterilized with a 45pm filter) were added to cell suspensions with rapid mixing (final diamide concentrations, 0.25- 1.O m&f). Control cell suspensions were mixed with PBS alone. The ceil suspensions were incubated at 37C for 30 min with gentle shaking, washed with RPMI-1640 medium to remove remaining diamide, and suspended in growth medium (RPMI-1640, containing 10% human serum). Plasmodium falciparum (strain FCR3TC, obtained from J. B. Jensen) was cultured according to the method of Trager and Jensen (1976), with some modifications, as described previously (Golenser et al. 1983), and with the addition of 10 mM glucose and 40 PM S-fluorocytosine. Cultures were synchronized by employing a combination of the methods of Jensen (1978) and Lambros and Vanderberg (1979), as previously described (Golenser er al. 1983). Suspensions enriched in trophozoites and schizonts were obtained by the gelatin sedimentation technique of Jensen (1978). A fraction with parasitized erythrocytes enriched with trophozoites and schizonts (the inoculum) was added to a 2.5% erythrocyte suspension (recipient cell population), so that the initial parasitemia was 0.5-l% of the cell suspension, unless otherwise stated, and contamination by the inoculum cells was less than 1.5% of the recipient cell population. The cell suspensions were dispensed into a 96-well microplate (100 pi/well) and incubated at 37C, using the candle jar method of ‘Bager and Jensen (1976). The supematant was replaced every day by fresh growth medium. Samples in triplicate were removed at time intervals and Giemsa-stained smears were prepared for parasite counts and for morphological examination. For analysis of thiols and membrane proteins, nonparasitized control and diamide-treated erythrocytes were incubated as 5% cell suspensions in growth medium in 35-mm Petri dishes (2.0 ml/dish), using the
Plasmodium
fakiparum:
GROWTH
IN
candle jar method (‘Rager and Jensen 1976). The supematant was changed daily, as done for the parasitized cells. Samples were removed at intervals, and the cells were washed in PBS. Aliquots of cell suspension in PBS were labeled with monobromobimane (mBBr), as previously described (Kosower et al. 1979). Erythrocyte ghosts were prepared according to the method of Steck and Kant (1974). Ghosts were solubilized and analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis, according to established methods (Fairbanks et al. 1971; Liu et al. 1977), without addition of dithiothreitol. After electrophoresis, gels were fmed, photographed under ultraviolet (360 nm) illumination, and stained with Coomssie blue and photographed again, as described previously (Kosower et al. 1981).
For GSSG determination, cells were lysed in 5 mM phosphate buffer, pH 8.0. Hemolysates (0.5 r&f hemoglobin) were incubated for 15 min at 37C in the presence and absence of 1.0 mM NADPH and 0.05 unit GSSG reductase (Sigma, type III, 145 units/mg protein)/ml of hemolysate. Samples were then analyzed for GSH. Analyses of GSH, hemoglobin, and membrane thiols were carried out on nonlabeled aliquots, using 5,5-dithiobis(2nitrobenzoic acid) (Ellman 1959) or on mBBr-labeled aliquots (Kosower et al. 1981). RESULTS
To study the multiplication of Plasmodium falciparum in normal and G6PD-deticient erythrocytes, the erythrocytes were pretreated with various concentrations of diamide, washed to remove any remaining reagent, and then infected with P. falciparum. Figure 1 shows the course of the parasitemia in erythrocytes from four normal and four G6PD-deficient individuals. In normal cells, the rate of multiplication of parasites in cells pretreated with diamide was similar to that of control cells. In G6PD-deficient erythrocytes, the multiplication of parasites in control cells was similar to that in normal cells. A significant inhibition in parasite multiplication was observed in G6PD-deficient cells pretreated with diamide. In experiments carried out on erythrocytes obtained from other G6PD-deficient males, slight individual differences were noted in the magnitude of the effects, but the overall results were the same. The inhibition of parasite growth in
NORMAL
AND
G6PD-DEFICIENT
DAYS IN
CELLS
241
CULTURE
1. The effect of diamide on the growth of Plusmodium falciparum in erythrocytes from 4 normal humans (J. G., A. T., I. K., and D. K.) and from 4 G6PD-deficient humans (M. Z., D. N., E. M., and E. T.). Erythrocytes (10% suspensions) were incubated for 30 min with or without 1.0 mM diamide, washed, and resuspended in growth medium, and then inoculated with parasitized erythrocytes. FIG.
G6PD-deficient erythrocytes was dose dependent and ranged from partial inhibition in samples pretreated with 0.25-0.5 mM diamide to a complete inhibition of growth in samples treated with 1.0 mM diamide (Fig. 2). The morphology of the parasites in control deficient cells was similar to that of parasites in normal cells, though some desynchronization was noted in several of the control deficient samples within 2-3 days of culture. Some of the parasites in the diamide-treated deficient cells were morphologically abnormal. The behavior of the parasites during the initial 24 hr of culture was followed by examining samples every 5-6 hr after the addition of the inoculum of trophozoites and schizonts to the recipient cell suspensions. Results are shown in Fig. 3. In control samples of both normal (Fig.3A) and G6PD-deficient eythrocytes (Fig.3B), ring forms appeared concomitantly with the decrease in the number of mature parasites, starting at about 6 hr after inoculum addition (Figs. 3A and B). These results demonstrate that the merozoites, derived from schizonts in the
242
MILLER 0 0 A 0
ET AL. I
LOmM OIAMIDE 0.5mM DIAMIDE 0.25mM DIAMID PBS
160 -
I
I
A. NORMAL
I
I
,
I
l-
B.GGPD(-) P-
h +
0
I
2
3
4
CULTURE
FIG. 2. Growth of Plasmodium falciparum in normal and in G6PD-deficient human erythrocytes pretreated with various concentrations of diamide. Erythrocytes (10% suspensions) were treated for 30 min with diamide in concentrations of 0.25, A; 0.5, 0; and 1.0 mM, 0. Control cells suspended in PBS, 0.
inoculum, had invaded the recipient cells and developed into ring forms. P. falciparum added to diamide-treated normal cells behaved similarly to the control cells, with ring forms appearing at a similar or a slightly lower rate in diamidetreated cells as compared to that in the control cells (Fig. 3A). In marked contrast, very few ring forms appeared in the diamide-treated, G6PD-deficient cells at the time when many of the mature forms had disappeared (Fig. 3B). These results indicate that the merozoites which were released from the donor cells had either not invaded the diamide-treated deficient cells or did not develop into recognizable ring forms in these cells. To study cellular and membrane thiols, unparasitized control and diamide-treated erythrocytes were used. At the beginning of culture, less GSH was present in the control G6PD-deficient cells than in the control normal cells. GSH declined in both types of cells over several days in culture (Fig. 4A). Pretreatment of the red cells with diamide initially resulted in the oxidation of
i-
10 -
0.
’ 6
’ ’ 12 18 HOURS
I I ’ 3 6 12 IN CULTURE
I it3
L 24
FIG. 3. The dium falciparum
development of ring forms of Plasmoin normal and in G6PD-deficient human erythrocytes pretreated with diamide. Erythrocytes (10% suspensions) were incubated for 30 min with or without 1.0 m&f diamide, then washed and resuspended in growth medium. Cell suspensions were then inoculated with parasitized erythrocytes (mature forms) to an initial parasitemia of 5% and cultured. Samples were examined every 5-6 hr during the first 24 hr of culture. (A) Normal cells: 0, control; 0, diamide treated. (B) G6PD-deficient cells: A, control; A, diamide treated.
GSH to GSSG in both normal and G6PDdeficient erythrocytes. In normal erythrocytes, GSH is rapidly regenerated in the presence of glucose, so that a short incubation in the growth medium led to almost complete regeneration of the cellular GSH. In contrast, GSH was not regenerated in the G6PD-deficient cells, so that the diamide-treated deficient cells had no or very little GSH throughout the culture period (Fig. 4A). Initially, the oxidized GSH was present in these cells as GSSG. No GSSG was found in the deficient cells after culture for 24-48 hr. Membrane SH group content was similar in both normal and deficient control cells at the beginning of the culture, with no significant change during the subsequent incubation (Fig. 4B). In diamide-treated normal erythrocytes, the level of mem-
Plasmodium
fUkipUrUi?Z:
GROWTH
IN NORMAL
AND G6PD-DEFICIENT
G6PDd. normal
GGPDd. normal -
‘. g
2-
‘.
A.‘ds--A
.
-2
;
_
L
+
-
+
243
CELLS
DIAMIDE
-
+
-
+
5 I
.
0123401234 DAYS
IN CULTURE
FIG. 4. Glutathione (GSH) and membrane SH groups in normal and in G6PD-deficient human erythrocytes treated with diamide. Erythrocytes (10% suspensions) were incubated for 30 min with or without 1.O mM diamide, washed, resuspended in growth medium, and incubated under the same conditions used for culture of Plasmodium falciparum parasitized cells. (A) GSH, (B) membrane SH groups. 0, normal cells, control; 0, normal cells, diamide treated; A, G6PD-deficient cells, control; A, G6PD-deficient, diamide treated.
brane SH groups was similar to that of normal control cells throughout the culture period. In the diamide-treated G6PD-deficient cells, membrane thiol groups were diminished (Fig. 4B). Profiles of membrane proteins of erythrocytes at the time of inoculum addition are shown in Fig. 5. The distribution of the fluorescence and the stained protein bands in the diamide-treated normal cells was similar to that of the control cells. In contrast, membrane proteins from diamide-treated, G6PD-deficient cells exhibited alterations in the distribution of both the label and the stained protein bands; a significant diminution in labeling was noted in many of the bands. The profile of the stained proteins showed a diminution in some of the bands and the appearance of high-molecularweight proteins. An altered pattern of membrane proteins was also found in diamidetreated deficient cells after several days in culture (not shown).
FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of human erythrocyte membrane proteins isolated from normal and from G6PDdeficient cells incubated for 30 min with or without 1.O m&f diamide. Erythrocytes were then washed in PBS, labeled with monobromobimane, and processed. Left, fluorescence; right, Coomassie blue staining. H.M.W., high-molecular-weight proteins.
Human hemoglobin SH groups are not reactive towards diamide (Kosower er al. 1982). In both normal and G6PD-deficient, diamide-treated erythrocytes, the hemoglobin was not denatured and thiols were unaltered throughout the incubation period. DISCUSSION
The growth of Plasmodium falciparum in relation to the thiol status of the human host’s erythrocytes was examined. We found a significant inhibition of the parasite growth in G6PD-deficient cells following thiol oxidation by diamide. Pretreatment of normal erythrocytes with diamide did not affect the parasite growth. The inhibition of parasite growth in diamide-treated, G6PD-deficient cells may be due to effects on one or more stages in the parasite life cycle, namely, the attachment, internalization, and/or the intracellular development of the parasites. The reduced number of newly formed rings observed in the diamide-treated deficient cells following the initial release of merozoites
244
MILLER
from the inoculum indicates that entry of the parasite into that cell is inhibited. The subsequent deficiency in mature parasites and the appearance of abnormal forms suggest that there is also an inhibition of intracellular parasite development. Treatment of G6PD-deficient erythrocytes (and of glucose-deprived normal cells) with the thiol-oxidizing agent diamide (Kosower et al. 1969) results in the oxidation of GSH to GSSG. When cellular GSH is low, membrane protein thiols are oxidized, with the formation of intrachain and interchain disulfides (Kosower et al. 1981). The normal erythrocyte can, when metabolically active (i.e., in the presence of glucose), recover the original membrane thiol status along with the reduction of GSSG. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired (Kosower et al. 1982). In the experiments described here, the diamide-treated erythrocytes were washed and resuspended in RPM1 medium containing glucose. The normal cell quickly repaired most of the diamide-induced oxidative damage, so that cellular GSH was regenerated to about 80% of control level, membrane disulfides were reduced, and the original, control membrane protein profile was recovered at the time of parasite addition or shortly thereafter. In contrast, no GSH was regenerated in the deficient cell, nor was the membrane oxidative damage repaired at the time of parasite innoculation or thereafter. Invasion of the cell by the parasite involves attachment to the membrane, followed by invagination of the cell membrane and internalization of the parasite, with the completion of endocytosis by the fusion of the erythrocyte membrane at the point of parasite entry (Aikawa et al. 1978; Aikawa et al. 1981; Pasvol and Wilson 1982). A decrease in the density of intramembranous particles (IMP) in the area of invagination (McLaren et al. 1979; Aikawa et al. 1981),
ET AL.
arising from a redistribution of membrane components, has been found. The lateral mobility of IMP, usually restricted by the erythrocyte, may be made possible by a proteolytic release of the restrictive linkage between the IMP and the cytoskelton. Little information is available on parasite invasion of cells in which membrane proteins are modified. Ovalocytes which have an altered cytoskeleton are resistant to parasite invasion (Kidson et al. 1981). Antibodies to spectrin, which cause spectrin crosslinking, prevent the parasite invasion (Pasvol and Wilson 1982; Olson and Kilejian 1982). Miller et al. (1977) and Perkins (1981) have shown that enzymatic treatment of erythrocyte membranes prevents the invasion of the erythrocyte by certain plasmodia. The alterations in membrane thiol status shown here may inhibit parasite invasion at one or more stages. For example, the high-molecular-weight aggregates and the intrachain disultides formed in the membranes of G6PD-deficient cells may interfere with parasite attachment to the cell and subsequent invagination and internalization by diminishing cell deformability and by preventing the formation of IMP-free areas in the membrane. The changes in the membrane could also affect intracellular development of the invading merozoites by altering the transport of various substances necessary for the parasite growth (Cabantchik et al. 1983). Hemoglobin within the human erythrocyte is not altered by diamide treatment, and, as a substrate for growing parasites (Sherman 1979), can come from either normal or G6PD-deficient erythrocytes. Intracellular development of the parasite may, however, be altered as a result of lack of GSH, or the presence of GSSG. Inhibition of P. falciparum growth was found in samples of all G6PD-deficient erythrocytes when parasites were added to the cells shortly after diamide treatment. In some cases, especially those treated with lower concentrations of diamide, the
P/asmodium
fa&arum:
GROWTH
IN NORMAL
number of parasitized erythrocytes began to rise slightly after severaldays in culture. Theseresults indicate that some of the cellular alterations responsiblefor growth inhibition have been reversed. One significant change observed in the diamidetreated deficient cells incubated in culture for 2 days was the elimination of GSSG from the cells. The results indicate that GSSG may play a role in the inhibition of P. falciparum growth. GSSG inhibits the initiation of protein synthesis(Kosower et al. 1972;London et al. 1976;Ernst er al. 1978),and has beenproposedas a possible factor in the relative resistance of the G6PD-deficienterythrocyte to Plasmodium falciparum (Kosower and Kosower 1970). The fitness of the G6PD-deficient phenotype is consideredto be only slightly decreasedand, therefore, only a small increment in the resistanceto malaria would be sufficient to achieve a balanced polymorphism in populations in endemic areas. Thus, an initial, partial inhibition and desynchronizationin the parasitegrowth due to thiol-oxidation in G6PD-deficienterythrocytes would be sufficient to explain the overall relative resistanceto malaria. ACKNOWLEDGMENTS This study was supported by grants from The Lester Aronberg Foundation; from the Chief Scientist’s office, Ministry of Health, Israel; and from the fund for basic research administered by the Israel Academy of Sciences and Humanities.
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