Plasmodium falciparum: Assay in vitro for inhibitors of merozoite penetration of erythrocytes

EXPERIMENTAL

PARASITOLOGY

51,

400-407

(1981)

Plasmodium falciparum: Assay in Vitro for Inhibitors Merozoite Penetration of Erythrocytes MARTIN *Division

M. WEISS,* JOEL D.~PPENHEIM,~

of Parasitology,

TDepartment Avenue,

of Microbiology, New York, New

(Accepted

AND JEROME P. VANDERBERG*

New York University York 10016, U.S.A.

for publication

of

Medical

Center,

550 First

29 May 1980)

WEISS, M. M., OPPENHEIM, J. D., AND VANDERBERG, J. P. 1981. Plasmodium falAssay in vitro for inhibitors of merozoite penetration of erythrocytes. Experimental ciparrtm: 51, 400-407. Highly synchronous cultures of the erythrocytic stages of PlasParasitology modium falciparum were used both to assay penetration of merozoites into human red blood

cells, and to subsequently study the inhibitory effects of various substances on penetration. While several sugars exhibited no inhibitory effect, fucose, glucosamine-HCI, and N-acetyl glucosamine, when added to synchronous cultures at the schizont stage, inhibited invasion. On further testing fucose and glucosamine-HCI were found to be toxic to the intracellular growth and development of the parasite; only N-acetyl glucosamine had an inhibitory effect solely related to the inhibition of merozoite penetration. Glycophorin A, the major glycoprotein of the red blood cell surface, had no inhibitory effect at low concentrations, but had a slight effect at higher (500 &ml) levels. INDEX DESCRIPTORS: Malaria: PIasmodium fafalciparum; Malaria; Protozoa, parasitic; Synchronous cultures, in vitro; Merozoite penetration; Sugar inhibition: N-Acetyl glucosamine; N-Acetyl galactosamine; N-Acetyl mannosamine; Fucose; Lactose; Galactose; Mannose; Glucosamine-HCl: Glycoprotein; Glycophorin A; Erythrocytes, human. INTRODUCTION

Malaria parasites generally exhibit strong host specificity, a given malarial species usually being able to infect only a limited number of hosts of closely related species (Garnham 1966). This specificity appears to be determined in part by the host red blood cell (McGhee 1953, 1953a, Butcher et al. 1973; Miller et al. 1973). Studies on the interactions between red blood cells and the merozoites of Plasmodium vivax and P. knowlesi suggest that the parasite interacts with specific receptors on the surface of the erythrocyte (Miller et al. 1975). However, we have not yet arrived at a comparable understanding of parasite-erythrocyte membrane interactions for P. falciparum merozoites. A considerable amount of evidence suggests that cell-to-cell interactions are mediated by the carbohydrate moieties of cell surface glycoproteins and glycolipids, and that the specificity of these carbohy400 0014-4894/81/030400-08$02.00/O Copyright All rights

@ 1981 by Academic Press, Inc. of reproduction in any form reserved.

drates largely determines the specificity of the cellular interactions (Winzler 1970). In light of this, and in view of the recent developments in cultivation of P. fulciparum (Trager and Jensen 1976), we decided to investigate the effects of various sugars and the major human erythrocyte membrane glycoprotein, glycophorin A, on the penetration of P. falciparum merozoites into human erythrocytes. The availability of a technique for synchronization of P. falciparum grown in vitro (Lambros and Vanderberg 1979) now permits the sequential analysis of different developmental steps during the life cycle of the parasite. It is now possible to analyze the deleterious effects of a substance added to the culture medium and to distinguish between its effect on intraerythrocytic development and its inhibitory effect on merozoite penetration. The purpose of this paper is (1) to describe our assay procedure, (2) to report on the effects of several sugars and a purified erythrocyte surface

Plasmodiumfalciparum:

MEROZOITE PENETRATION IN VITRO

glycoprotein on cultures of P. falciparum, and (3) to show how our procedure may be used in the future to investigate erythrocyte surface macromolecules that might play a role as receptors for P. falciparum merozoites.

401

on the penetration of merozoites into red blood cells, various dilutions of the test materials were added to cultures of schizonts approximately 34-36 hr after the second, or fine-tuned synchronization. Blood films were prepared from each culture just prior to adding the test materials, and 24 hr MATERIALS AND METHODS afterwards were stained with 5% Giemsa’s The FCR-3 strain (Jensen and Trager stain (Fisher Scientific, Springfield, N.J.) at 1978) of Plasmodium falciparum was cul- pH 7.2 for lo- 15 min. Parasitemia was detured in Petri dishes maintained within can- termined by counting 10,000 red blood cells dle jars (Jensen and Trager 1977). We used per slide, and was expressed as the number 35 x lo-mm dishes containing 1.5 ml of a of parasites per 100 erythrocytes. The per10% hematocrit of red blood cells in RPM1 centages of the different stages of parasite 1640 with Hepes (Gibco, Grand Island, development were determined by counting N.Y.) and with 10% human serum. The red 100 parasitized red blood cells and desigblood cells (type A+ collected in citratenating them as ring, trophozoite, or schizphosphate-dextrose solution) were ob- ont stages. The effects of these additives on tained from the New York Blood Center. the cultures were determined in two ways, When the cultures reached 4-6% para- first, by the number of new rings which sitemia, they were synchronized by sor- arose from the schizont cultures, and secbitol treatment as described by Lambros ond, by the concomitant increase in and Vanderberg (1979). In brief, 34 hr after parasitemia during this transition from the initial sorbital synchronization, at a schizont to ring form. Those agents which significantly inhibtime when the cultures were in a stage of schizont to ring transition, the parasites ited schizont-ring transition were tested were again treated with sorbitol to sharpen further for their toxic effect on intracellular development of the parasite from the ring to and “fine tune” the synchrony. Substances which were tested for their the schizont stage. For this, various dilupossible inhibitory effects on penetration of tions of the test materials were added to merozoites into red blood cells were: N- cultures of ring forms shortly after synacetyl glucosamine (containing CO. 1% chronization with sorbitol. Blood films glucosamine), glucosamine - HCl (with were prepared, stained, and counted, as
2.3 4.2 0.04 0.03

5 10 100 50 100 100 100 100 100 100

Fucose

N-Acetyl Galactose galactosamine Galactosamine - HCI Lactose Mannose N-Acetyl mannosamine

9.8 11.7 10.5 10.5 14.6 9.9

13.1 13.1 13.1 13.1

13.1 13.1 13.1

13.1 13.1 13.1 13.1 13.1

Multiplication rate of nontreated control cultures at 24 hr

None None None None None None

82 68 100 100

100 100 94

100 100 94 15 2

Percentage inhibition of multiplication”

o Means of two to four experiments, with two replicate cultures per experiment. * Sugars were added to synchronized cultures at schizont stage of development and with parasitemias of 0.2-0.7%. ’ Multiplication rate = % parasitemia 24 hr after addition of sugar/% parasitemia at zero time. ” Percentage inhibition = (% parasitemia of controls at 24 hr) - (% parasitemia of experimentals at 24 hr)/% parasitemia of controls at 24 hr

9.1 10.9 9.6 11.1 15.3 10.2

0.04 0.05 0.8

100 50 25

Glucosamine-HCI

0.04 0.02 0.8 11.1 10.3

100 50 25 10 5

Multiplication rate of sugar-treated cultures at 24 hrc

N-Acetyl glucosamine

Sugarh

Concentration bw

TABLE I Inhibitory Effects of Various Sugars on Penetration of Plasmodium falciparum Merozoites in Vitro”

X

100.

z z

s

%

“?

”E 0 z E E

E

m

Plasmodium

falciparum:

MEROZOITE PENETRATION IN VITRO

sugars tested (Table I) had no significant effects on culture multiplication rates at levels up to 100 m&f. The deleterious effects of the three sugars shown in Table I required a differentiation between an inhibitory effect on red blood cell penetration by merozoites, and a toxic effect on intracellular development of the parasite. Therefore, these sugars were added to synchronized cultures of ring stages to test the effects of these sugars on the intracellular development of the rings to trophozoites and schizonts over a 24-hr period. N-Acetyl glucosamine, at levels up to 100 mM, was shown to have no significant effects on intracellular maturation of the parasites (Table II). Glucosamine-HCl and fucose, however, were clearly toxic to the intracellular parasites. At levels of 50 n&f and above, they completely inhibited parasite development from rings to schizonts (Table II). Glycophorin A was also tested in our system (Table III). This material had no significant effect on culture multiplication rates when added at levels of 200 pg/rnl and below. However, an inhibitory effect was observed at higher concentrations (400-500 pg/ml). Glycophorin A, when added at concentrations up to 500 pg/ml, was not observed to have any deleterious effects on the intracellular growth and development of the parasite.

403

motrypsin, blocks invasion by P. knowlesi merozoites. This implies that the receptors on the erythrocyte are proteins or glycoproteins. Moreover, different species of parasites seem to interact with different surface receptors. For instance, Duffy-negative red blood cells are not invaded by P. knowlesi or P. vivax merozoites, but are susceptible merozites to invasion by P. falciparum (Miller et al. 1977). In addition, pretreatment of erythrocytes with trypsin or chymotrypsin causes dissimilar effects on the subsequent susceptibility of these cells to invasion by P. knowlesi as compared with P. falciparum (Miller et al. 1977). Cell surface glycoproteins are known to play a role in many specific and characteristic interactions between cells (reviews in Bradshaw ef al. 1975; Society for Experimental Biology 1978). It has been well established that the specificity of these cellular interactions is determined by the carbohydrate moieties of the glycoproteins. This specificity has been taken advantage of by using exogenous carbohydrates to competitively inhibit the binding between cell surface glycoproteins and the macromolecules that interact with them. For example, the inhibition of cell agglutination by simple sugars is a technique that has been widely used to probe the specificity of the carbohydrate binding sites of lectins (Lis and Sharon 1973). Exogenous sugars DISCUSSION have also been used in this manner to inThe susceptibility of erythrocytes to in- hibit cellular interactions in a wide variety vasion by Plasmodium spp. merozoites of biological systems, including virus-cell varies depending upon the species of eryth- interactions (Gelb and Lemer 1965; Hughes 1973), nitrogen-fixing bacteria-plant cell rocytes and the species of parasite (McGhee 1953, 1953a; Butcher ef al. 1973; associations (Bohool and Schmidt 1974), Miller et al. 1973). This strongly suggests a phagocytosis of red blood cells by Acanrecognition system that is dependent upon thamoeba (Brown et al. 1975), mating inthe presence of specific receptors for the teractions in yeast and Chlamydomonas parasite on the surface membrane of the (Crandall and Brock, 1968; Wiese and erythrocyte. Additional support for this has Shoemaker 1970)) recognition among celcome from Miller ef al. (1973), who showed lular slime molds (Rosen et al. 1975), inthat treatment of human erythrocytes with teractions between bacterial pili and other the proteolytic enzymes, pronase and chy- cells (Old 1972; Korhonen 1979), and

100 50 25 10 5 loo 50

Glucosamine - HCl

Rings 96 98 99 91 99 99 93 96 94 97 92 100 100

0.4 0.5 0.5 0.5 0.5 0.6 0.6 0.5 0.6 0.6 0.6 0.4 0.3

4 1 2 1 2 0 0

2 1 0 2 1 0

2 1 1 1 0 1 3 3 4 2 6 0 0

Schizonts

Trophozoites

Percentage distribution of stages

Percentage parasitemia

‘* Means of two to three experiments, with two replicate cultures per experiment. b Sugars were added to synchronized cultures at ring stage of development. c Only degenerate parasites observed. d Rings appeared degenerated.

Fucose

100 50 25 10 5

None (control) N-Acetyl glucosamine

Sugar”

Concentration mm

Zero time

I3 I 61 0 0 62 5 5 0 0

02 2 00 0 0 13” 5” 2 0 0

0.6 0.6 0.5 0.5 0.6 0’ 0’ 0.2 0.3 0.4 0’ 0’

5

Trophozoites

0

Rings

0 0 25 90 93 0 0

99 94

95 93 91

95

Schizonts

Percentage distribution of stages

0.4

Percentage parasitemia

24 hr after addition of sugar

TABLE II Effects of Various Sugars on the in Vitro Intracellular Development of Plasmodium falciparum from the Ring to the Schizont Stage”

2 z E K 8

L5

>

”E

:

i

3 w5 0

Plasmodiumfalciparum:

MEROZOITE PENETRATION

TABLE III Effects of Glycophorin A on Penetration of Plasmodium falciparum Concentration of glycophorin Ab b.4mU 500 400 200 100 50 25 0 (Control)

INVITRO

405

Merozoites in Vitro”

Multiplication rate of culture at 24 hr’

Percentage inhibition of multiplicationd

3.6 4.3 4.1 5.6 5.1 5.3 5.0

28 14 6 None None None None

u Means of two experiments, with two to four replicate cultures per experiment. b Glycophorin A, prepared according to the method of Marchesi and Andrews, was added to synchronized cultures at schizont state of development and with parasitemias of 0.2-0.7%. c Multiplication rates of cultures assessed at 24 hr after addition of glycophorin. Multiplication rate = % parasitemia at 24 hr/% parasitemia at zero time. d Percentage inhibition = (% oarasitemia of controls at 24 hr) - (% parasitemia of experimentals at 24 hrs)/ % parasitemia of controls at.24 hr x 100.

aggregation of sponge cells (Weinbaum and Burger 1973). With these biological precedents in mind, we attempted to utilize this approach in our investigation of the recognition and invasion of human erythrocytes by P. falciparum merozoites. Our results showed that three sugars, N-acetyl glucosamine, glucosamine- HCl, and fucose interfered with the parasite’s growth and development between merozoite release from schizonts and new ring formation (Table I). In order to determine whether the interference with penetration was due to a specific blockage of invasion, or a generalized toxic effect, further experiments were performed. Fucase and glucosamine -HCl were found to exhibit general toxic effects on growth and development of the parasite (Table II). N-Acetyl glucosamine, however, while inhibiting invasion, induced no similar toxic effects on the intracellular growth of P. falciparum. The generalized toxic effects of glucosamine-HCl and fucose do not exclude the possibility that these sugars may also specifically inhibit merozoite invasion. The fact that N-acetyl glucosamine strikingly inhibits merozoite invasion, while sugars of very similar structure, namely,

N-acetyl galactosamine and N-acetyl mannosamine do not inhibit invasion, strongly suggests the specificity of this inhibitory process. We extended these studies by similarly testing glycophorin A. This sialoglycoprotein was chosen because it is easily isolated and purified in large quantities from red blood cell ghosts (Marchesi and Andrews 1971), and it is the major erythrocyte glycoprotein on the outer surface of the red blood cell (Marchesi et al. 1976). Our results with glycophorin A confirm Miller’s (1973) earlier finding that this glycoprotein does not inhibit merozoite invasion at about 100 @ml. However, at higher concentrations we found up to 28% inhibition of parasite multiplication (Table III). We do not consider this to be definitive evidence that glycophorin A is the receptor on the erythrocyte for merozoite invasion. If this were so, we would have expected the inhibition to occur at much lower levels than we observed. These results suggest either a nonspecific effect by glycophorin A or the presence of a contaminant that is actually responsible for the inhibition. The assay system we have described is a highly sensitive approach for investigating

WEISS,

406

OPPENHEIM,

AND

VANDERBERG

merozoite or ervthrocvte membrane re- BRADSHAW, R. A., FRAZIER, W. A., MERRELL,R. C., AND GOTTLIEB, D. I. 1976. “Surface Membrane ceptors involved in erythrocyte penetraReceptors. Interface Between Cells and Their Envition. This sensitivity is due to the high deronment.” Plenum Press, New York. gree of synchrony obtained by the double BROWN, R. C., BASS, H., AND COOMBS, J. P. 1975. sorbitol treatment (Lambros and VanderCarbohydrate binding proteins involved in phagocytosis by Acanthamoeba. Nature (London) berg 1979). In most of these experiments we 254, 434-435. initiated our studies with cultures containBUTCHER,G. A., MITCHELL, G. H., AND COHEN, S. ing greater than 95% schizonts or rings. At 1973. Mechanism of host specificity in malarial inthe end of the 24-hr experimental period, fection. Nature (London) 244, 40-41. the transition of the control parasites to the CRANDELL, M., AND BROCK, T. D. 1968. Molecular aspects of specific cell contact. Science 161, subsequent developmental stage was close 473-47s. to 100% (for example see Table II). Any GARNHAM, P. C. C. 1966. “Malaria Parasites and interruption of the growth and development Other Haemosporidia.” Blackwell, Oxford. of the ring stages, or inhibition of invasion GELB, L. D., AND LERNER, A. M. 1965. Rheovirus by newly released merozoites from schizhaemagglutination: Inhibition by N-acetyl glucosamine. Science 147, 404-405. onts is readily apparent and easily quantitated. The use of synchronous cultures in HUGHES,R. C. 1973. Glycoproteins as components of cellular membranes. Progress in Biophysics and this assay enables us to distinguish between Molecular Biology 26, 191-268. the inhibitory effects of exogenous agents JENSEN, 3. B., AND TRACER, W. 1977. Plasmodium on merozoite penetration and the generfalciparum in culture: Use of outdated erythrocytes and a description of the candle jar method. Journal alized toxic effects of these agents on ofParasitology 63, 883-886. intracellular parasite development. JENSEN, J. B., AND TRACER, W. 1978. Plasmodium Our findings suggest that N-acetyl in culture: Establishment of additional falciparum glucosamine may be an important compostrains. American Journal of Tropical Medicine and nent of the glycoprotein receptor involved Hygiene 27, 743 -746. in recognition and penetration of human KORHONEN, T. K. 1979. Yeast cell agglutination by purified enterobacterial pili. FEMS Microbiology erythrocytes by Plasmo’dium falciparum Letters 6, 421-425. merozoites. Of course, our data do not LIS, H., AND SHARON, N. 1973. The biochemistry of exclude the possibility that this sugarplant lectins (Phytohemagglutinins). Annuul Review containing receptor is actually a component of Biochemistry 42, 541-574. of the merozoite, and that it interacts with LAMBROS, C., AND VANDERBERG, J. P. 1979. Synchronization of Plasmodium falciparum erythrocytother macromolecules on the surface of the 65, ic stages in culture. Journal of Parasitology red blood cell. Further studies with our ex418-420. perimental system may enable us to identify MCGHEE, R. B. 1953. The infection by Plasmodium this receptor. lophurae of duck erythrocytes in the chick embryo. ACKNOWLEDGMENTS

This work was supported in part by a contract from the U.S. Agency for International Development, by Public Health Service Grant HL 21328, a Young Investigator Research Award from the National Heart, Lung and Blood Institute to Dr. Joel D. Oppenheim, and by U.S. Army Medical Research and Development command under Contract DADA 17 73 C 3027 and is contribution No. 1571 to the Army Research Program on Antiparasitic Drugs. REFERENCES

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MEROZOITE

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