Surface proteins of schizont-infected erythrocytes and merozoites of Plasmodium falciparum

Molecular Biochemical Parasitology, 5 (1982) 5 5 - 64 Elsevier Biomedical Press

55

SURFACE PROTEINS OF SCHIZONT-INFECTED ERYTHROCYTES AND MEROZOITES OF PLASMODIUM FALCIPAR UM

MARGARET PERKINS

Rockefeller University, 1230 York Ave., New York, NY 10021, U.S.A. (Received 21 September 1981; accepted 29 October 1981)

Schizont-infected erythrocytes and merozoites were isolated from in vitro cultures of the human parasite, Plasmodium falciparum labeled with various radioactive substrates. The isolated merozoites were viable since they were able to reinvade fresh erythrocytes. On the basis of sensitivity to specific enzymes, eleven proteins synthesised by the parasite, were localised on the surface of the schizontinfected erythrocyte. Eight of these were glycoproteins, six of which appeared to represent three doublets. Five merozoite surface proteins were identified on the basis of their sensitivity to trypsin and chymotrypsin, treatments which also rendered the merozoite incapable of erythrocyte invasion. Merozoites appeared not to contain any glycoproteins; all of the glycoproteins synthesised by the parasite were apparently transported to the surface of the schizont-infected erythrocyte. Key words: Plasmodiumfalciparum, Surface proteins, Schizont-infected erythrocytes, Schizont-infected merozoites

INTRODUCTION The free form o f the erythrocytic stage o f the malarial parasite, the merozoite, invades its host cell the erythrocyte and asexually develops through several stages into a multinucleate schizont. The mature schizont ruptures the red cell and releases merozoites which are then able to infect erythrocytes. The infected erythrocyte undergoes considerable morphological and metabolic changes (see Ref. 1 for review). Changes in the surface o f erythrocytes infected with the human malarial parasite Plasmodium falciparum [2] and numerous other species [ 3 - 8 ] have been documented. These studies have relied upon surface labeling techniques which have the disadvantage that modified or newly exposed erythrocyte surface proteins may be labeled in addition to parasite derived proteins. There has been no biochemical characterization o f the surface o f the merozoite o f P. falciparum, due to the difficulties in obtaining merozoites free o f schizont material. However merozoites of the simian malaria P. knowlesi have been shown to be agglutinated by sera from infected animals and sensitive to a number o f reagents including trypsin [9,10]. The aim o f the present work was to identify the surface proteins o f schizont infected erythrocytes and isolated merozoites from metabolically labeled in vitro cultures o f P. falciparum. 0166-6851/82/0000-0000/$02.75

© 1982 Elsevier BiomedicalPress

56 MATERIALS AND METHODS

Cultivation and in vitro labeling of Plasmodium falciparum.P, falciparum (FCR-3/ Gambia) was cultured in vitro according to the method of Trager and Jensen [ 11 ] in 100 mm Petri dishes. The medium used was RPMI 1640 (Gibco, Long Island, N.Y.)-HEPES supplemented with 10% human serum (referred to as RPS medium). To collect merozoites in appreciable amounts it was first necessary to synchronize the cultures so that the majority of schizonts spontaneously released merozoites within a very short period. Synchronization was done by first treating cultures with sorbitol [ 12] and then by repeated separation of rings and trophozoites by flotation in gelatin [ 13 ]. The trophozoite infected cells were selected every 4 0 - 4 2 h in this manner (usually 5 times) until the parasite development was synchronous within 3 - 4 h. Cultures were allowed to reach 15% parasitemia before merozoites were collected. 8 h prior to the estimated time of merozoite release, the medium was removed and replaced with 10 ml of RPS medium containing one of the following substrates; (a) [3s S]methionine, 10 /aCi/ml of medium (New England Nuclear > 4 0 0 Ci/mmol); [a H]leucine 10 gCi/ml 60 Ci/mmol); [a H]glucosamine 40/aCi/ ml (New England Nuclear, 19.0 Ci/mmol); [aH]mannose, 25 #Ci/ml (NEN, 15 Ci/mmol). Collection of merozoites. Rupture of infected red cells and merozoite release were monitored from Giemsa stained blood smears. When the merozoites began to be released from the erythrocytes, the medium from the labeled cultures was removed and replaced with 4 ml of RPS medium. The cultures were then returned to the candle jar for 1 h. Typically 10 ml of packed erythrocytes in 80 ml of RPS medium was used for merozoite collection. The merozoites released during this period were collected from the medium in the following manner. Cultures were centrifuged at 2000 rpm for 5 min at room temperature and the supernatant, containing the merozoites was incubated for 3 min at 37°C with 10 ml (packed volume) of anti-erythrocyte antibody conjugated to Sepharose 4B. The antierythrocyte antibody was prepared in rabbits and cross-linked to cyanogen bromide activated Sepharose 4B. Erythrocyte membranes and intact schizont-infected erythrocytes bound to the Sepharose and were removed by centrifugation at 1000 rpm for 3 min and discarded. The supernatant was centrifuged at 3000 rpm for 5 min and the merozoite pellet resuspended in phosphate-buffered saline (PBS, sodium phosphate dibasic, potassium phosphate monobasic final concentration of 0.1 M with respect to phosphate, 1% (w/w) sodium chloride, pH 7.2) at a concentration of 10 a merozoites/ml. Typically, from 10 ml of packed infected erythrocytes used it was routinely possible to obtain 5 × 10 a merozoites during a 1 h collection. Schizont infected erythrocytes and uninfected erythrocytes, removed by the initial centrifugation were returned to the candle jar for a second collection. Schizonts from the same synchronous cultures were pulse labeled with equivalent amounts of radioactivity for 8 h and harvested 4 h prior to the estimated time ofmerozoite release. Enzymatic treatment of merozoites and schizonts. Merozoites (2.5-5 × 107/ml) were

57 treated with trypsin (Miles 3X crystallized 4611 U/mg) at concentrations of 1,5 and 10 /ag/ml for 5 min after which time soybean trypsin inhibitor (Sigma Chemical Co., 10/~g/ ml) was added: chymotrypsin (Miles 2X crystallized 2480 U/mg) at (10/ag/ml): or with neuraminidase (Vibrio cholerae Gibco, 500 U/ml) at 5 U/ml for 5 rain. These treatments were carried out at 37°C. The enzyme treated merozoites were resuspended in 10 ml of RPS medium and centrifuged at 3000 rpm for 5 min. Radioactivity labeled samples were prepared for electrophoresis. Samples of merozoites to be used in the reinvasion assay were immediately added to fresh erythrocytes as described below. To compare samples of labeled schizont-infected erythrocytes pretreated with enzymes to samples of labeled merozoites, the following method was employed. Synchronous cultures of labeled schizonts (36--40 h after invasion) were washed three times in PBS by centrifugation at 3000 rpm, and resuspended at a concentration of 6 X 10a cells/ml. The following enzymes were added to separate samples: trypsin (0.1 mg/ml) for 15 min followed by soybean trypsin inhibitor (0.2 mg/ml); neuraminidase from Vibrio cholerae (10 U/ml) for 15 min; pronase (Sigma 0.1 mg/ml) for 10 min. All enzyme incubations were done at 37°C. After incubation the schizonts were washed three times in RPS medium, once in RPM I and lysed in 20 volumes Tris-HC1 buffer (10 raM, pH 8.0) at 4°C. Parasites entrapped in erythrocyte membrane were collected by centrifugation at 3000 rpm for 10 min at 4°C and prepared for electrophoresis as described below.

Reinvasion assay. The capacity of isolated merozoites to invade ezythroeytes was assayed by adding untreated merozoites and merozoites treated with various enzymes to fresh erythrocytes in 10 mm microtitre wells. The erythrocytes were incubated for 4 h at 37°C, after which time they were washed three times in RPS medium by centrifugation at 3000 rpm, to remove extraceUular merozoites. Blood smears were made and ring stage parasites were counted per 10 000 cells. Only merozoites which had formed dearly visible rings were considered to have reinvaded.

SDS-polyacrylamide electrophoresis. Equivalent amounts (250 ~g of protein) of schizont and merozoite samples were heated to 100°C for 5 min with 10% SDS and mixed with sample buffer (10% glycerol, 0.1 M Tris-HC1, pH 6.8, 2% SDS) for a further 2 min at 100°C. The samples were electrophoresed on a 5-15% gradient SDS-polyacrylamide gel using the buffers of Laemmli [14]. Protein was estimated by the method of Lowry [15]; tritium was detected by fluorography [16]. RESULTS AND DISCUSSION

The surface proteins of the schizont-infected erythrocyte. Fig. 1 (lane a) shows the incorporation of [as S] methionine into the malarial parasite at the schizont stage. Uninfected erythrocytes did not incorporate any radioactive label. None of the labeled proteins appeared to be sensitive to trypsin or neuraminidase (Fig. lb and c); three proteins of low molecular weight, (indicated by arrows on left in Fig. la), were cleaved by pronase (Fig.

58

a

b

c

d

e

f

'2O0

130 94

67

Fig. 1. Autoradiography of SDS-PAGE of [3SS]methionine-labeled schizont-infected erythrocytes and merozoites of P. falciparum. All cultures were labeled for 8 h. (a) Untreated schizonts labeled for 8 h from 32-40 h after reinvasion; (b) schizonts treated with trypsin (0.1 mg/ml for 15 min); (c) schizonts treated with neuraminidase (10 U/ml); (d) schizonts treated with pronase (0.1 mg/ml); (e) isolated merozoites; (f) merozoites treated with trypsin (10 ~tg/ml). The molecular weight markers are spectrin (200 000), #-galactosidase (130 000), phosphorylase b (94 000), serum albumin (67 000) and ovalbumin (43 000), (Coomassie blue-stained lane of the gel not shown).

ld). The selective removal of these proteins suggests that they were on the surface o f the schizont-infected erythrocyte. There were eight major species o f glycoproteins labeled with [3H]glucosamine (Fig. 2a). Six o f these appeared to represent doublets. For this reason the glycoproteins were designated GPI-V, the doublets o f GPIII-V referred to as a' for the larger and b ' for the smaller mol. wt. species o f the two as indicated in Fig. 2.

59

Fig. 2. SDS-PAGE of [3H]glucosamine- and [3H]mannose-labeled sehizont-infected erythrocytes. (a-e) [3Hlglucosamine label (40/JCi/ml for 8 h); (f) [3 Hlmannose label 25 ~Ci/ml for 8 h. (a) [3H]glucosamine-labeled schizonts; (b) treated with trypsin (0.1 mg/ml); (c) sehizonts treated with neurarninidase (10 U/ml); schizonts (d) isolated merozoites; (e) schizonts treated with pronase (0.1 nag/ ml); (f) [3H]mannose-labeled schizonts. The reel. wt. markers are the same as those used in Fig. 1. A similar distribution of glycoproteins in in vitro cultured schizonts ofP. falciparum has been reported [2]. [3H]Mannose was incorporated into GPI and GPIII but only weakly into GPII, GPIV and GPV (Fig. 2f). The heavily [3H]mannose.labeled band < 43 000 in

60 Fig. 2f is at the dye front of that gel which was electrophoresised separately from the [3H]glucosamine-labeled samples. GPIIIa, GPIVb and GPVa were cleaved by trypsin (Fig. 2b) and all glycoproteins were affected to some extent by pronase (Fig. 3e) suggesting that all were on the surface of the infected erythrocyte. This is the first demonstration of metabolically labeled, i.e. parasite synthesised proteins and glycoproteins on the surface of schizont-infected erythrocytes of P. falciparum. The small amount of [all] glucosamine label seen in the merozoites (Fig. 2d) was considered to represent contamination with schizont-infected erythrocyte membranes. As the digestion with pronase removed only three of a total of more than forty [aSS]methionine-labeled proteins resolved in Fig. 1 it is unlikely that any cell lysis occurred as a result of this treatment. Neuramindase treatment (Fig. 2c) did not appear to affect the [3H]glucosamine labeled proteins indicating that these contained little sialic acid; this clearly differentiates them from the major sialoglycoproteins on the erythrocyte surface. All the [3H i mannose-labeled glycoproteins were also affected by pronase (not shown). Further investigations will be required to determine if the double bands of GPIII-V are indeed doublets, related metabolically. Their differential sensitivity to trypsin strongly suggests that they occur in different forms on the erythrocytes surface.

Surface proteins of extracellular merozoites. Fig. le shows the electrophoretic pattern of [3SS]methionine-labeled merozoites isolated from in vitro cultures. Of the fifty or so proteins resolved in this gel, 10/~g/ml of trypsin selectively removed four (shown by arrows in Fig. If). Three of the trypsin labile proteins had mol. wts. < 200 000 and a fourth had a mol. wt. of 94 000. Correlated with this was the effect of mild enzymatic treatment of P. falciparum merozoites on reinvasion, summarised in Table I. Of the merozoites added to fresh erythrocytes 8-10% invaded, Treatment with trypsin significantly affected reinvasion, in contrast, neuraminidase treatment had no effect. This is consistent with the apparent lack of glycoproteins in isolated merozoites (Fig. 2d). TABLE I Effect of enzymatic treatment of merozoites on erythrocyte invasion. Experiment treated

Merozoites invaded per 10 000 erythrocytes Untreated

1 2

410 260

Trypsin treated*

Neuraminidase**

1

5

10

400 250

164 140

0 0

5 420 240

In experiment 1, 5 X 107 untreated merozoites were added to IOs erythrocytes; 8% of those added formed rings. In experiment 2, 2.5 X 107 merozoites were added to 108 erytrocytes and 10% of these formed rings.

* pg/ml. ** U/ml.

61

Fig. 3a shows merozoites labeled with [3 H]leucine and merozoites treated with chymo. trypsin (Fig. 3b). Chymotrypsin also effectively removes the high molecular weight protiens C~ 200 000) and in addition cleaved a protein with a reel. wt. of 130 000. The absence of glycoproteins in the isolated merozoite cannot be attributed to the loss o f protein as equivalent amounts of merozoite protein were applied to the gels shown in Fig. le and Fig. 2d. Selective loss of the glycoproteins from the surface of the merozoite also seems unlikely as 8-10% of the isolated merozoites were invasive and therefore must have had their outer surface intact. However the strongest evidence that the merozoites do not contain glycoproteins is that it has been shown that all the proteins labeled with [3 H]glucosamine and [3 H]mannose were on the surface of the schizont-infected erythro-

Fig. 3. Autoradiography of SDS-PAGE of [3H]leucine-labeled merozoites. Labeling and culture conditions are described in the methods. (a) Isolated merozoites; (b) merozoites treated with chymotrypsin (10 ~g/ml). Mol. wt. markers are the same as in Fig. 1.

62 cyte. As the intracellular merozoite and infected erythrocyte plasma membrane do not form junctions it seems unlikely that they share common membrane proteins. It was reported previously [2] that a 'merozoite-enriched' fraction contained several of the glycoproteins which in the present work were shown to be on the surface of the schizont.infected erythrocyte. A possible explanation of the apparent presence of glycoproteins in the merozoite fraction could be that there was considerable contamination of the merozoite preparation with schizont material. An important point made in the present study is that the isolated merozoites were free of contaminating infected erythrocytes: the electrophoretic patterns of schizonts and merozoites [Figs. 1 (a and e) and 2 (a and d)], were quite different. This is especially true of samples labeled with [3H]glucosamine, indicating that there was minimal contamination of merozoites with schizonts. Furthermore, Coomassie blue stained gels of the samples used in Fig. le (not shown) revealed no spectrin or other erythrocyte membrane proteins. Several studies using lectins or lectins conjugated to ferritin also suggested an absence of significant amounts of exposed saccharides on the surface ofP. lophurae [8] and P. bergheimerozoites [17]. It is of interest to compare the present work with that of Perrin and coworkers who have used both monoclonal antibodies [ 18,19 ] and sera from individuals infected with malaria [20] to identify the protective antigens of P. falciparum. These workers found that antibodies secreted by several different hybridoma cell lines, which inhibit parasite growth in vitro, immunoprecipitate specific proteins with mol. wts. > 200 000 [18], 96 000, 41 000 and 36 000 [19]. A protein with a mol. wt. of 200 000 is one of five proteins immunoprecipitated by immune serum [20] and may well be homologous to the high molecular weight proteins (> 200 000) identified in the present study as being on the merozoite surface. Perrin and coworkers also have evidence that the protein with a mol. wt. of 96 000 was on the merozoite surface [19] and this may be the same as the prominent merozoite surface protein (mol. wt. 94 000) shown here to be sensitive to trypsin. In summary, on the basis of enzyme sensitivity, five proteins on the surface ofmerozoites isolated from in vitro cultures ofP. falciparum have been identified. Trypsin treatment of isolated merozoites inhibited their ability to invade fresh erythrocytes raising the possibility that all or some of these surface proteins are involved in erythrocyte attachment and or invasion of the parasite. Recent studies suggest that during invasion merozoites attach to glycophorin A on the erythrocyte surface [21] and it will be of interest to see if this sialoglycoprotein binds specifically to any of the merozoite surface proteins identified in the present study. Eleven major proteins, eight of these being glycoproteins, were localised on the surface of the schizont-infected erythrocyte. ACKNOWLEDGEMENTS I would like to thank Dr. W. Trager for his continued support of the project. I would especially like to thank Drs. I. Sherman and M. Muller for a critical reading of this manuscript. This investigation received financial support from the United Nations Development Programme/World Bank/World Health Organisation Special Programme for Training in Tropical Diseases.

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