Molecular and Biochemical Parasitology, 38 (1990) 105-1i2 Elsevier

105

MOLBIO 01247

Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodium falciparum-infected red cells Patricia A. Maguire and Irwin W. Sherman Department of Biology, Universityof California, RiversMe, CA, U.S.A. (Received 2 March 1989; accepted 16 August 1989)

The membrane lipid composition and [all]cholesterol exchange rate were studied in both normal human erythrocytes and those infected with the human malaria Plasmodiumfalciparum. The host cell membrane was separated from parasite membranes using the Affigel (731) bead method. The purity of the membrane preparation was very high, as judged by SDS-PAGE, and in several instances was estimated to be >98% as determined by the activity of the parasite membrane-specificenzyme, choline phosphotransferase. No difference was found in the content of phosphatidylethanolamine and only small changes were observed for phosphatidylcholine and phosphatidylserine. The sphingomyelin content in red cell membranes of both trophozoite- and schizont-infectcd cells was up to 47% less than that of uninfectedcells, and the cholesterol/phosphollpidratio was decreased 55%. Trophozoiteand schizont-infectedcells exchanged 29 and 33% less cholesterol, respectively, than uninfected cells. These changes in lipid composition and cholesterol exchange could have a marked effect on the function of the red cell membrane of malaria-infected cells and may be responsible, in part, for the increased fluidity and permeability of P. falciparum-infected erythrocytes. Key words: Plasmodiumfalcipcrum; Malaria; Cholesterol; Phospholipid; Sphingomyelin

Introduction The erythrocytic stages of malaria contain no lipid reserves, and all of the lipids of the intracellular parasite are membrane-associated. In general, malaria-infected red cells show an increase in the total a m o u n t of lipid, and a decrease in the cholesterol/phospholipid ratio compared to uninfected red cells. Since malaria parasites appear to be incapable of synthesizing fatty acids and cholesterol de novo and are limited in their capacity for fatty acid saturation and desaturation as well as chain elongation and

Correspondence address: Patricia A. Maguire, Department of Biology, University of California, Riverside, CA 92521, U.S.A. Abbreviations: TIC, thin layer chromatography;SDS-PAGE, sodium dodecyl sulfate-polyaerylamide gel electrophoresis; MDA, malonyldialdehyde; ACE, acetylcholinesterase; CPT, cholinephosphotransferase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin;PS, phosphatidylserine.

shortening reactions, the erythrocytic stages of Plasmodium satisfy their requirements for these lipid precursors by relying on dynamic exchanges with the blood plasma, an activity akin to that of the red cell in which the parasite grows and reproduces [1-4]. It has been reported that both Plasmodium falciparum and Plasmodium knowlesi-infected red cells exchange cholesterol at a rate that is very similar to that of the uninfected red cell [5]. However, based on the data available in the literature, the cholesterol content of the malaria-infected red cell is quite variable: increased in P. knowlesi [5], unchanged in P. falciparum [5], and decreased in Plasmodium lophurae and Plasmodium gallinaceum-infected cells [6,7]. Sources of this variability could be species differences, differing host cell preferences by the different species of parasites, varying levels of parasitemia, a comparison of different developmental stages of the parasite, and the incomplete separation of host cell membranes from the abundant array of parasite membranes.

0166.6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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In view of the high degree of variability of the lipid composition of the malaria-infected red cell, but especially the paucity of information concerning the lipid composition of the erythrocyte membrane in red cells infected with the human malaria P. falciparum, we have developed a '~ighly reproducible Affigel bead technique which permits the isolation cf host cell membranes having a high degree of purity and in amounts sufficient for use with conventional methods of lipid analysis. The bead-isolated membranes have been studied for their cholesterol and phospholipid content, as well as the type and amount of phospholipid present. In addition, we have measured the exchange rate of cholesterol in P. falciparuminfected red cells and compared this with that of uninfected red cells. Materials and Methods

Materials. Beads of Affigel 731 and all reagents for SDS-PAGE were obtained from BioRad Laboratories (Richmond. CA). Cytidine 5'diphosphate [methyl-l~C]choline (50--60 mCi mmol -l) and [7(n)-3Hleholesteroi (5-15 Ci mmol -l) were purchased from Amersham (Arlington Heights, IL). Thin-layer chromatography (TLC) plates were EM Science Silica gel 60 (Darmstadt, F.R.G.). Phospholipid and cholesterol standards for chromatography were ob'";"~'~-,,,,~.,~ from Sigma ~t.'°" Louis, MO). Human serum was purchased from Worldwide Biological (Cincinnati, OH). Parasites. P. falciparum (Gambian, FCR-3 strain) was maintained in continuous culture following the methods of Trager and Jensen [8], and parasites were synchronized at the ring stage by sorbitol lysL of mature forms [9]. Trophozoite- and schizont-infected cells were obtained by the gel. atin flotation method of Pasvol et al. [10].

Membrane isolation. Polyethyleneimine-coated polyacrylamide beads (Affigel 731) were prepared as described by Gruenberg and Sherman [11[. Briefly, the beads were hydrated in deionized water and washed twice (by gravity sedimentation) with 2 M ammonium chloride, twice with deionized water, and twice with 250 mM sucrose,

24 mM sodium phosphate, pH 7.1 (low-ionicstrength buffer). The beads were washed immediately before use to ensure maximal binding of cells. Unused beads were washed once with deionized water and stored at 4°C in 0,5 M sodium chloride, 0.05% (w/v) sodium azide. After gelatin concentration, trophozoite- and schizontinfected cells were washed twice with low-ionicstrength buffer and mixed gently with a 50% (v/v) suspension of washed Affigel beads. Usually, 8--12 ml of bead suspension was used for each 1 ml of packed cells. The bead suspension was gently agitated for 10 rain, followed by 2 washes with low ionic strcngth buffer to remove unbound cells. Bead-bound cells were iysed in 10 mM sodium phosphate, pH 7.1, by rapid vortex mixing for 30 s, followed by 2 min of sonication in a bath sonicator (Bransonic 12) [12]. Typically, 4 to 5 cycles of lysis were required to lyse completely all beadbound cells. The extent of contamination of red cell membranes by parasite membranes was determined in the following way: both whole infected cells (of high parasitemia) and the corresponding Affigel-bound membranes were assayed for acetylcholinesterase (ACE) [13], a marker for the red cell membrane, and cholinephosphotransferase (CPT) [14], a marker for the parasite membranes. The enzyme activity per mg protein [15] was used to compare the specific activity of whole cells with that of Affigel bead-isolated membranes. The control and infected bead-isolated membranes were also solubilized as described previously [11] and the proteins separated by SDS-PAGE (7.5% acrylamide) [16] and stained with Coomassie Blue.

Lipid extraction and thin-layer chromatography. Lipids were extracted from Affigel-bound membranes by the method of Bligh and Dyer [17]. Extracts were flash-evaporated and subjected to two-dimensional TLC; the first dimension was developed with chloroform/methanol/58% ammonium hydroxide/water (60:25:2.5:2.5) and the second dimension involved the use of chloroform/acetone/methanol/acetic acid/water (3:4:1: 1:0.5) [18]. Lipids were visualized with iodine vapor and identified by comparison to pure standards. Phospholipids were quantitated by a modification [19] of the method of Fiske and Subbarow

107

[20] and cholesterol was assayed using the method of Zlatkis et al. [21].

Cholesterol exchange. The rate of cholesterol exchange in intact normal and infected cells was measured using the method of Jain and Shohet [221. Briefly, washed normal, trophozoite-infected or schizont-infected cells (50% paras(tern(a) were incubated with [3H]cholesteroMabeicd serum (10 txCi per 10 ml of heat-inactivated serum) at 37°C for 1-5 h (final hematocrit 33%). At each time point, 250 ILl of cells was removed, washed twice with phosphate-buffered saline (145 mM sodium chloride, 5 mM sodium phosphate, pH 7.4), twice with low-ionic strength buffer, and then the cells were bound to Affigel beads as described above. Following cell lysis and lipid extraction, the extract was divided into two aliquots. One aliquot was used for the cholesterol determination and the other was used for liquid scintillation counting. The results are expressed as percent of the total exchange (dpm (Ixg cholesterol) -l) of the labeled serum. Results

In order to assess accurately the lipid composition of the erythrocyte membrane from P. rid_ ciparum-infected cells, it is critical to obtain membranes that are relatively free of contamination by parasite iipids. This was accomplished by binding malaria-infected cells to Affigel beads,

followed by shearing. This technique leaves the host membrane firmly attached to the beads, and the parasite membranes and soluble proteins remain free in solution. The degree of contaminants of such isolated membranes, as determined by the relative specific activities of ACE and CPT in the whole infected cells and bead-bound membranes (Table I), was determined to be 1.75 _+ 1.34% (N = 4). The degree of membrane purity was confirmed by SDS-PAGE (Fig. 1). The pattern of protein bands from membranes obtained from the Affigel-bead bound schizont-infected cells (lane 1) was nearly identical to that of the uninfected red cell membrane isolated in the same manner (lane 2). Of all the bands, only one band, of approximately 150 kDa, appeared to be associated with the infected cell membrane preparation (see ref. 4 for review of parasite-specific proteins). Because bead-bound host cell membranes are so firmly attached to the solid substratum of the bead, it was possible to extract the membrane lipids directly from the beads without further separation. The extracted lipids were then chromatographed, and the phospholipid and cholesterol contents determined. The phospholipid composition of the red blood cell membrane from normak trophozoiie-infected, and schizont-infected cells (at >70% parasitemia) purified on Affigel beads is shown in Fig. 2. No differences were observed in phosphatidylethanolamine (PE) content. Small differ-

TABLE 1 Purity of Afligel-bound membranes ACE

CPT

CPT/ACE

r; Contamination

Whole infected Bead-bound

7.30 114.17

11.635 (t. 117

0.1187(I 0.(11110

1.18

Whole infected Bead-bound

6.(12 64.48

(I.796 (I. 125

I). 1322 I).0019

1.47

Whole infected Bead-bound

7.64 94.03

(~.353 11.160

(I.11462 (I.IJ(ll 7

3.69

Whole infected Bead-bound

4.77 109.52

1t.529 0.08(I

(). 11t19 0.(1[107

I).66

Whole infected cells (approx. 5(lea paras(tern(a) and the corresponding Afligel bead-bound membranes were assayed for A C E [13] (nm¢~l rain ~ (mg protein) i) and CPT [14] (nmol (rag protein) ~).

108

1

2

~-200

40.

-93

--i

30.

~

25.

~ 20, o_ o

10. 5

-66

-45

Fig. 1. Proteins from Affigel-bead bound normal (lane 2) and schizont-infected (lane 1) human red cells (at >70% parasitemia) were solubilized as described previously [11], separated on a 7.5% acryiamide gel and stained with Coomassie Blue.

ences were observed in the amount of phosphatidylcholine (PC) and phosphatidylserine (PS). However, the most striking differences were observed in the sphingomyelin content (a 36--47% decrease) and in the cholesterol/phospholipid ratio (a 55% decrease). These changes in lipid composition were due to the parasite, as cells incubated in the same medium, but not infected, showed lipid profiles indistinguishable from those of normal red cell membranes. All changes occurred during the first 24 h of parasite development, and no further changes were observed during the last 24 h of maturation, It was of concern that the lipid changes observed could be the result of the membrane isolation technique rather than a consequence of

NORMAL CONTROL

I~

TROPHOZOITE SCHIZONT • 1.200 D

,.,'-i

35.

O

-116

m

• 1.000

E O -r-

llil1inli lliii l,i! PC

PE

SM

PS

0.800 O "-r

-0.600 ~ . O

.o.4oo ,.=, FO3 W

-0.200

0.000

-r C)

CHOL/PL

Fig. 2. Lipids from Affigel bead-isolated membranes from normal, control (normal red cells bound to Affigel bead~, lysed and treated with infected cell lysate, see Results), trophozoite-infected and schizont-infected human red cells (at >70% parasitemia) were extracted, chromatographed, and analyzed as described in Materials and Methods. Results are expressed as mole% of the total phospholipids or cholesterol to phospholipid molar ratio. Values are means - SEM for 9 (normal), 3 (control), or 3 (infected) samples. Sta'tistical differences were determined by Student's t-test. PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; PS, phosphatidylserine; CHOL/PL, cholesterol/phospholipid molar ratio. "P < 0.05, "" P < 0.01, ""P < 0.001.

parasitization. In order to study this, normal red cells were bound to the Affigel beads and lysed as described. An identical cell volume of infected cells at 70% parasitemia was bound to the same amount of Affigel beads and lysed as described. The lysate from the Affigel bead-bound infected cells was then added to the Affigel bead-bound normal cell membranes, and the vortex and sonication steps were carried out as described. Lipids were extracted from this preparation and analyzed. The lipid composition of these lysatetreated membranes (Fig. 2, control) were similar to that of normal cells (Fig. 2, normal), indicating that the lipid changes observed in the membrane obtained from infected red cells were a consequence of parasitization. In normal red cells, all of the cholesterol in the membrane was exchangeable (Fig, 3) within 5 h. Trophozoite- and schizont- infected cells (at 50% parasitemia) exchanged 29-33% less cholesterol, respectively, than normal cells over the same time period. No change in cholesterol content or cholesterol/phospholipid ratio was detected over the 5-h time course of the assay. As observed with the

109

110,

0 NORMAL & TROPHOZOITE /', SCHIZONT

100

~ o = ¢D

40-

2010 0

0

(~

/ . , i N **

//J* 1

2

3

4

5

TIME (hr)

Fig. 3. [sH]Cholesterol exchange was assayed as described in

Materials and Methods in normal, trophozoite-infectedand schizont-infectedhuman red cells (at 50% parasitemia). The results are expressedas % of the total cholesterolevchanged at each time point. Values are means ± $EM for 3--7experiments. Statisticaldifferenceswere determinedby Student's ttest. "P < 0.05, "'P < 0.01, ""P < 0.001.

lipid composition, the bulk of the decrease in exchange rate occurred during the first 24 h, with very little change observed during the subsequent 24 h of parasite growth. Discussion

The present work describes changes in the lipids of the erythrocyte membrane after parasitization of the red cell with the human malaria, P. falciparum. Critical to our approach has been the separation of the red cell membrane from other membranes of the infected cell prior to lipid analysis, and to determine, using membrane-specific markers and SDS-PAGE, the purity of such isolated membranes. The isolation of red cell membranes, almost entirely free of parasite membranes, is essential, because the mature malaria parasite contains up to 5 times as much total lipid as the host red cell membrane; consequently, small changes in the lipid composition of the host membrane might go undetected due to dilution by parasite lipids. Using the Affigel bead method, we have found it possible to obtain lipid preparations in which less than 10% of the total lipid can be attributed to parasite membranes, as determined by the ~9ecific activity of CPT (Table I). The purity of the isolated red cell membranes was confirmed by SDS-PAGE (Fig. 1), in which there

was a difference in only one band at about 150 kDa that was observed in the infected cell membrane preparation. This is consistent with previous reports from this laboratory using the Affigel bead method [11]. This low degree of parasite membrane contamination, as well as the high levels of parasitemia used, allow for a more definitive interpretation of the experimental data. The phospholipid composition and the cholesterol/phospholipid ratio of normal red cell membrane ghosts or those bound to Affigel beads (Fig. 2), are consistent with the values reported by others [23-26]. The decrease (approx. 47%) in sphingomyelin content and the decline (55%) in the cholesteroFphospholipid ratio observed in the host membrane of trophozoite- and schizont-infected cells (Fig. 2) could have a marked effect on the physical properties and physiological function of the infected red cell membrane. A decrease in sphingomyelin would be expected to decrease the order within the membrane (increase fluidi'~y) [27], and it has been shown that artificial membranes composed entirely of sphingomyelin are much less fluid than those which are pure PC [28,29]. Cholesterol depletion has also been reported to increase the fluidity of the inner leaflet of red cell membranes [30]. Changes in membrane fluidity in malaria-infected red cells have also been noted. For example, the membranes of red cells infected with P!asmodium berghei [31-33], P. lophurae [34] and P. falciparum [34, 35] have been shown to be increased in their fluidity. The results presented here are in contrast to those of Schwartz et al. [36] and Joshi et al. [37], who detected no differences in phospholipid composition between normal and malaria-infected erythrocyte membranes. However, Schwartz et al. [36] studied cells at low parasitemias (up to 18%) and analyzed the lipids from whole infected cells, not isolated red cell membranes. Under these conditions, we would not expect differences to be observed. Joshi et al. [37] did utilize the Affigel bead method to isolate membranes from highly parasitized cells. However, they quantitated a soluble enzyme, glutamate dehydrogenase, as their index of parasite contamination; this may have limited use in assessing the amount of parasite membrane present

110

in a given Affigel bead preparation. Therefore, the discaepancy between their results and those of the present study may be due to differences in the purity of the membranes used for analysis. In normal erythrocytes, all the membrane cholesterol is exchangeable with serum, and the rate of exchange begins to plateau at 2-3 h (Fig. 3). During the period of incubation, no change in cholesterol content was observed in normal cells. This is consistent with the reports of others using human erythrocytes [38,39] The reduced exchange rate for cholesterol in trophozoite- and schizont-infected cells may be explained simply by the change in sphingomyelin and cholesterol content of the red cell membrane of infected cells. Although cholesterol exchange was found to be independent of cholesterol concentration in membranes containing sphingomyelin [40,41], it is important to note that membranes which were depleted of sphingomyelin did not incorporate labeled cholesterol as well as those with higher sphingomyelin levels [42]. Furthermore, cholesterol has a higher 'affinity' for sphingomyelin [43], so a depletion of sphingomyelin from the membrane could result in the movement of cholesterol out of the membrane, leading to an impaired capacity of the membrane to solubilize cholesterol [42]. Another possible explanation for the reduced cholesterol exchange rate in the P. falciparum-infected cell is the cross-linking of aminophospholipids, a consequence of lipid peroxidation. This occurs in irreversibly sickled cells [22], e.g., a 25% decrease in cholesterol exchange was observed in irreversibly sickled cells and in normal red cells treated with malonyldialdehyde (MDA), a product of lipid peroxidation [22]. It is noteworthy that elevated MDA levels (and other products of lipid

peroxidation) have been detected in red cells infected with Plasmodium vinckei [44,45] as well as e. falciparum [46]. The results presented here are in contrast to those of Vial et al. [5] who detected no differences in cholesterol exchange in erythrocytes infected with P. falciparum. However, in those analyses, low parasitemias (7%) were used, and as the present work demonstrates, a parasitemia of 7% would be expected to display less than a 5% decrease in cholesterol exchange, a value not significantly different from those of the normal red cell. In summary, membranes of human red blood cells infected with the malaria parasite P. falciparum display a decrease in sphingomyelin content as well as a decreased cholesterol/phospholipid ratio. These changes would result in a more fluid and a more permeable red cell membrane, both of which have been observed in the malaria-infected cell. These lipid changes may also be responsible for the decrease in the rate of cholesterol exchange observed in these cells, either directly, by limiting the solubility of cholesterol in the lipid phase, or indirectly, by leaving the membrane more susceptible to peroxidation and lipid cross-linking. Further studies of the asymmetric distribution of cholesterol and phospholipids and of lipid translocation are needed in order to elucidate the mechanism of the lipid changes reported here in P. falciparum-infected red blood cell membranes.

Acknowledgements This work was supported by a grant from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

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der Veen, E.A. (1984) Effect of dietary cod liver oil on the lipid composition of human erythroeyte membranes. Scand. J. Clin. Invest. 44, 39-46. 25 Jain, S.K. (1986) Membrane lipid peroxidation in erythrocytes of the newborn. Clin. Chim. Acta 16i, 301-306. 26 Dougherty, R.M., Galli, C., Ferro-Luzzi, A. and Iacono, J.M. (1987) Lipid and phospholipid fatty acid composition of plasma, red blood cells, and platelets and how they are affected by dietary lipids: a study of normal subjects from Italy, Finland, and the USA. Am. J. Clin. Nutr. 45, 443-455. 27 Villalain, J., Ortiz, A. and Gomez-Fernandez, J.C. (1988) Molecular interaction between sphingomyelin and phosphatidylcholine in phospholipid vesicles. Biochim. Biophys. Acta 941, 55-62. 28 Cherry, R.J. (i976) Protein and lipid mobility in biological and model membranes. In: Biological Membranes (Chapmap, D. and Wallach, D.F.H., eds.), pp. 47-102, Academic Press, London. 29 Cooper, R.A., Durocher, R.J. and Leslie, M.H. (1977) Decreased fluidity of red cell membrane lipids in abetalipoproteinemia. J. Clin. Invest. 60, 115-121. 30 Chabanel, A., Flamm, M., Sung, K.L.P., Lee, M.M., Schachter, D, and Chien, S. (1983) Influence of cholesterol content on red cell membrane viscoelasticity and fluidity. Biophys. J. 44, 171-176. 31 Howard, R.J. and Sawyer, W.H. (1980) Changes in the membrane microviscosity of mouse red blood cells infected with Plasmodium berghei detected using n-(9-anthroyioxy) fatty acid fluorescent probes. Parasitology 80, 331-342. 32 Allred, D.R., St~rling, C.R. and Morse, lI, P.D. (1983) Increased fluidity of Plasmodium berghei-infected mouse red blood cell membranes detected by electron spin resonance spectroscopy. Mol. Biochem. Parasitol. 7, 27-39. ~ ..... Duportail, G., Laustriat, G. and Kuhry, J.G. (1986) Membrane fluidity changes in P. berghei-infected erythrocytes investigated with a specific plasma membrane fluorescent probe. Biochem. Int. 12, 21-31. 34 Sherman, I.W. and Greenan, J.R.T. (1984) Altered red cell membrane fluidity during schizogonic development of malarial parasites (Plasmodium falciparum and P. Iophurae). Trans. R. Soc. Trop. Med. Hyg. 78, 641-644. 35 Taraschi, T.F., Parashar, A., Hooks, M. and Rubin, H. (1986) Perturbation of red cell membrane structure during intracellular maturation of Plasmodium falciparum. Science 232, 102-104. 36 Schwartz, R.S., OIson, J.A., Raventos-Suarez, C., Yee, M., Heath, R.H., Lubin, B. and Nagel, R.L. (1987) Altered plasma membrane phospholipid organization in Plasmodium falciparum-infectcd human erythrocytes. Blood 69, 401-407. 37 Joshi, P., Dutta, G.P. and Gupta, C.M. (1987) An intracellular simian malarial parasite (Plasmodium knowlesi) induces stage-dependent alterations in membrane phospholipid organization of its host erythrocyte. Biochem. J. 246, 103-108. 38 Lange, Y. and D'Allessandro, J.S. (1978) The exchange-

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Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodium falciparum-infected red cells.

The membrane lipid composition and [3H]cholesterol exchange rate were studied in both normal human erythrocytes and those infected with the human mala...
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