Journal of Cell Science 102, 527-532 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

527

Origins of the parasitophorous vacuole membrane of the malaria parasite,

Plasmodium falciparum, in human red blood cells

A. R. DLUZEWSKI1, G. H. MITCHELL2, P. R. FRYER3, S. GRIFFITHS3, R. J. M. WILSON4

and W. B. GRATZER1

'Medical Research Council Muscle and Cell Motility Unit, King's College, 26-29 Drury Lane, London WC2B 5RL, UK2Department of Immunology, U.M.D.S., The Medical School, Guy's Hospital, London SEJ 9RT, UK^Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, UK4National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK

Summary

We have attempted to determine whether the parasito-phorous vacuole membrane, in which the malariaparasite (merozoite) encapsulates itself when it enters ared blood cell, is derived from the host cell plasmamembrane, as the appearance of the invasion process inthe electron microscope has been taken to suggest, orfrom lipid material stored in the merozoite. We haveincorporated into the red cell membrane a haptenicphospholipid, phosphatidylethanolamine, containing anNBD (N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)) group, sub-stituted in the acyl chain, and allowed it to translocateinto the inner bilayer leaflet. After invasion of theselabelled cells by the parasite, Plasmodium falciparum,immuno-gold electron microscopy was used to follow the

distribution of the labelled lipid; this was found to beoverwhelmingly in favour of the host cell membranerelative to the parasitophorous vacuole. Merozoites of P.knowlesi were allowed to attach irreversibly to red cellswithout invasion, using the method of pretreatment withcytochalasin. The region of contact between the mero-zoite and the host cell membrane was in all cases devoidof the labelled phosphatidylethanolamine. These resultslead us to infer that the parasitophorous vacuolemembrane is derived wholly or partly from lipid pre-existing in the merozoite.

Key words: malaria, merozoite, parasitophorous vacuole,lipid.

Introduction

The sequence of events that characterises the entry ofthe malaria parasite into the red blood cell has beendefined at the morphological level (Bannister et al.,1975; Aikawa et al., 1978; Miller et al., 1979; Aikawa etal., 1981). In essence the merozoite first makes randomcontact with the red cell surface; it then reorients, so asto bring its apical surface into apposition with the hostcell membrane. An invagination develops in the hostcell and deepens as the merozoite enters. An electron-dense junction, which forms during the attachmentphase, remains at the contact zone as the parasitemoves inwards; when the parasite is engulfed, the hostcell membrane closes behind it. The internalisedparasite is thus encapsulated in a so-called parasito-phorous vacuole.

The appearance of the invasion process in theelectron microscope strongly suggested that the parasit-ophorous vacuole membrane (PVM) is formed byeversion of the host cell membrane, and until recentlythis was generally assumed to be the case (see e.g. Holz,1977, for a review). The PVM, moreover, is devoid of

the major red cell membrane proteins (Atkinson et al.,1987) and this is true also of the membrane that boundsthe internal vacuole, which represents the first step information of the cavity in the host cell that eventuallyreceives the parasite (Dluzewski et al., 1989). Never-theless, counter-indications have accumulated thathave been interpreted as indicating a parasite-derivedorigin for the PVM; in particular, the rhoptries andmicronemes, which are organelles located near the apexof the merozoite, were shown to contain lamellardeposits, consisting almost certainly of lipid (Bannisteret al., 1986; Stewart et al., 1986). This material drainsthrough ducts into the area of contact with the host cellat the time of entry (Bannister and Mitchell, 1989) orattachment of the parasite (Aikawa et al., 1981). It alsoappears that fluorescent lipids, metabolically intro-duced into the parasite, appear in the PVM afterinvasion, indicating that parasite-derived lipid makes atleast some contribution to the PVM (Mikkelsen et al.,1988).

We have attempted to resolve the conflicting evi-dence about the origin of the PVM by immuno-electronmicroscopy, making use of a haptenic lipid, inserted

528 A. R. Dluzewski and others

into the red cell membrane before exposure to theparasite. Our results support the view that the PVM isderived partly or entirely from the parasite.

Materials and methods

Plasmodium falciparum parasites were cultured in vitro(Trager and Jensen, 1976) and synchronised by the sorbitolmethod (Lambros and Vandenberg, 1979). The duration ofinvasion experiments was 7 h. P. knowlesi parasites had beencryopreserved as ring-stage trophozoites in Callithrix jacchusred cells. These were thawed, rehydrated and culturedovernight to schizogony as described earlier (Bannister andMitchell, 1989). Invasion of human red cells was initiated byaddition of purified schizont preparations (Dluzewski et al.,1984) to the cells in RPMI 1640 culture medium containing10% human serum. Externally, irreversibly attached P.knowlesi result when cytochalasin B is added to themerozoites or rupturing schizonts before mixing with the redcells (Miller et al., 1979); the conditions of treatment were asdescribed previously (Dluzewski et al., 1989). Incubationswere for 90 min with 2 /.ig ml"1 cytochalasin B. Human targetcells were used.

The labelled lipid, NBD-PE, i.e. l-acyl-2-[6-[(7-nitrobenz-2-oxa-l,3-diazol-4-yl) amino] caproyl] phosphatidylethanola-mine, was obtained from Avanti Polar Lipids. It wasdispersed in the form of an ethanolic solution at 80 ^g ml~ in100 volumes of RPMI medium, to which packed, washed redcells were then added to give a haematocrit of 1% and allowedto incorporate into the membrane for 1 h at 37°C (Tanaka andSchroit, 1983). The cells were washed in RPMI. During theincubation with parasites this extraneous lipid is translocatedto the inner membrane leaflet (Devaux, 1988), and thisbecomes largely resistant to extraction by albumin-containingmedium (see below).

For preparation of anti-NBD antibodies, bovine immuno-globulin G (Sigma) was derivatised by reaction with NBD-C1(7-chloro-4-nitrobenzo-2-oxa-l,3-diazole); 1 mg reagent, dis-solved in ethanol, was added to 5 ml protein at a concen-tration of 2 mg ml"1 in 0.1 M sodium phosphate, pH 8.1, andthe reaction was allowed to proceed at room temperature for90 min. An excess of buffered Tris hydrochloride was addedand the protein was dialysed against 0.1 M sodium chloride,20 mM sodium phosphate, pH 7.4. A rabbit was inoculatedsubcutaneously and intramuscularly with the protein incomplete Freund's adjuvant, followed by two booster injec-tions with the antigen in Freund's incomplete adjuvant over aperiod of 4 months. Serum was taken 6 weeks after the lastboost.

Parasitised fluorescent cells in wet films under coverslipswere examined in a Zeiss microscope in epifluorescence in thepresence of p-phenylenediamine as anti-bleaching agent. Forelectron microscopy, cells were metabolically depleted beforefixation: this was found to reduce the extraction of theantigenic lipid in the course of ethanol-dehydration. Thus theparasitised cells were suspended in isotonic phosphate-buffered saline, containing 4 mM iodoacetate, pH 7.4; thesupernatant was then replaced by the same buffer, containing5 mM N-ethylmaleimide, pH 8.0. The cells were fixed in 0.5%glutaraldehyde in 0.1 M potassium phosphate, pH 7.4,followed by suspension in 50 mM ammonium chloride for 20min, and finally washed twice with 0.1 M potassiumphosphate, pH 7.4. The treated cells were dehydrated bysuccessive transfers to five solutions of increasing ethanolconcentration, up to 75% (v/v). The cells were equilibrated

with LR White monomer, transferred to gelatin capsules andthe resin was polymerised by warming at 50°C for 24 h.Sections were prepared and mounted on 200-mesh coppergrids.

The grids were floated on 50 mM potassium phosphatebuffer, pH 7.4, which contained 10 mg ml"1 bovine serumalbumin and 2.5 mg ml"1 Tween 20. They were thentransferred to the surface of a drop of antibody solution,diluted 1:20 - 1:40, or, in the case of controls, normal rabbitserum and antiserum, absorbed with isolated membranesfrom NBD-CI-labelled ghosts, and left in place for 1 h. Theywere washed by flotation on three changes of buffer and thenfloated on a solution of protein A, labelled with colloidal goldby conjugation with chlorauric acid (BDH), following themethod of Roth (1982). The grids were rinsed twice withbuffer, then with distilled water, air-dried and stained withuranyl acetate and lead citrate. The sections were coated withcarbon and examined in a Philips 300 electron microscope at80 kV accelerating voltage. The same procedure was used forthe detection of the transmembrane protein, band 3, asdescribed earlier (Dluzewski et al., 1989).

Results

If a labelled phospholipid is to be introduced into thered cell membrane bilayer, the derivatising group mustbe in the acyl chain and not the head-group (Struck andPagano, 1980). Moreover, an aminophospholipid modi-fied in this manner will be translocated to the innermembrane leaflet by the membrane-associated translo-case (Devaux, 1988). NBD-PE fulfilled these require-ments and gave rise to abundant fluorescence in thecell; a part of this remained in the membrane and wasonly slowly extracted on incubation with the culturemedium, which contains a high concentration of serumalbumin. Thus the cells remained brightly fluorescentover the 7 h period of incubation in the invasionexperiments, and even after 20 h there was only amoderate reduction in intensity. The labelled cells wereinvaded by P. falciparum with comparable efficiency tocontrol cells. Examination in the fluorescence micro-scope revealed no detectable excess fluorescence in theregion of the intraerythrocytic parasite, suggesting thatthe label had not entered the PVM; limitations ofcontrast and resolution, however, precluded definitiveor quantitative conclusions on the basis of this negativeobservation. We therefore had recourse to gold-labelling immuno-electron microscopy.

It is likely that a proportion of the lipid would beextracted in the course of dehydration and embedding,although the aminophospholipids may be stabilised bythe fixation step with glutaraldehyde. The results showin any event that enough lipid remains to givesatisfactory immuno-gold labelling in thin sections. Fig.1 shows labelling of the red cell plasma membrane,which is absent from sections treated with antiserumthat had been absorbed out with ghosts from NBDchloride-derivatised red cells. Equally the labelling hasno (or only a minimal) counterpart around theperiphery of the parasite or elsewhere in either cell. Thenumber of attached antibody molecules per unit length

Malarial parasitophorous vacuole 529

Fig. 1. Thin sections of red cells, labelled with haptenicallymodified phosphatidylethanolamine, infected with P.falciparum. The sections were incubated with anti-NBDantibody, followed by gold-labelled protein A; (a) and (b)show two different red cells, containing young ring-stageparasites (r). Note label on host cell membrane but not onparasitophorous vacuole membrane. Bars, 1 fan.

of host cell membrane and PVM contour is given inTable 1. These results strongly suggest that the PVM isnot in large degree derived from the host cellmembrane.

We then examined red cells bearing attached P.knowlesi merozoites: in this system the invasion cycle ofthe parasites, treated with cytochalasin B, is arrested atthe stage of irreversible attachment (Miller et al., 1979).With the human cells used here we did not observe theinternal vacuole in the host cell, opposite the zone ofattachment, that were formed in simian cells (Dlu-zewski et al., 1989). In Fig. 2 no haptenically labelledlipid is to be seen in the red cell membrane in the region

Table 1. Distribution of host cell phospholipid marker(NBD-PE) in the membranes of human red cells with

internalised P. falciparum and attached P. knowlesimerozoites

Total length"membrane (/an)

Gold particlesper /an

Internalised P. falciparumRed cell membraneb

Parasite membrane

Attached P. knowlesid

Red cell membraneRegion of attachment

479367

250

14

0.66±0.25c

0.08±0.09

0.56+0.260

'Total length of contour of red cell membrane andparasitophorous vacuole membrane examined (26 and 14 cellswere analysed in the P. falciparum and P. knowlesi experimentsrespectively).

bAs a control, antiserum absorbed out with NBD-Cl-labelledghosts was used. This gave 0.045±0.04 (s.d.) gold particles per /mi(total membrane length examined, 234 /an on sections of 11 cells).Non-immune serum was also applied and gave a gold particledensity of 0.16±0.07 per /jm of membrane (total length examined,168 /an on sections of 8 cells).

'Standard deviation.dGold particles were counted in successive 1 /im-length elements

from left-hand edge of point of attachment of parasite (asindicated in Fig. 3).

of contact between the attached parasite and the hostcell. To establish this as a general feature of theattached state, because of the sparsity of the label, itwas necessary to determine the distribution of goldparticles around the membrane contour, measuredfrom the left-hand boundary of the zone of contact (Fig.3). Here the first element on the left represents the partof the membrane in contact with the parasite, and is theonly one in which there is no antibody in any of the 14cells with attached parasites examined. To evaluate thesignificance of this observation we may calculate theprobability that this has happened by chance: if rparticles (summed over all cells examined) are distrib-uted between n membrane elements the probability(/\) of finding k of these r particles in the tth membraneelement (n > i > 1) is:

Pk = [r!/it!(r-Jfc) !]./r*. (1-/T1)1-*

For 132 particles, distributed randomly among 18membrane elements, the probability of finding noparticles in any one element (here by definition element1), Pp, is 5.3 x 1CT4. Thus the probability that the hostcell lipid marker is absent from the region of contact bychance can be disregarded. We infer that the parasite atthe stage of incipient invasion generates membranematerial, which appears to form a pool at the point ofattachment, that then normally expands inwards toform the vacuole into which the parasite passes.Because of the rapidity of the invasion process it may besurmised that the substance of the new membrane iswholly or partly derived from the pre-existing rhoptryand microneme contents (Bannister et al., 1986;Stewart et al., 1986; Bannister and Mitchell, 1989),rather than freshly synthesised lipid.

530 A. R. Dluzewski and others

a

Fig. 2. Thin sections of redcells, labelled with haptenicallymodified phosphatidylethanol-amine and bearing attached P.knowlesi merozoites (m); thesections were incubated withanti-NBD (a and b) antibodyand gold-labelled protein A, asbefore. Panels (a) and (b)show results for different cellsand (c) shows typical labellingof membrane with anti-band 3antibody for comparison. Noteabsence of label in the regionof contact between merozoitesand host cells. The arrow in(a) denotes the position of theleft-hand edge of the firstlength element (see Fig. 3).Bars, 1 /an.

Discussion

The debate about the provenance of the PVM (Joiner,1991) has focussed on the appearance presented by theinvasion process in the electron microscope (Aikawa etal., 1978, 1981), which undeniably suggests encapsula-tion of the invading merozoite by the host cellmembrane, and on the other hand on the followingitems of evidence, all indirect: (1) multiple invasion,which is not uncommon, would, on the above model,result in the elimination of a considerable proportion ofthe host cell membrane area (some 7% for eachmerozoite internalised in the case of P. knowlesi); (2)

the PVM is much more resistant than the red cell to lysisby saponin (Sherman and Hull, 1960), which suggests adifferent lipid composition; (3) the rhoptries andmicronemes contain abundant amounts (sufficient, asBannister and Mitchell (1989) have pointed out, togenerate a bilayer with the area of the PVM) of materialwith the appearance of multilamellar lipid bodies(Bannister et al., 1986; Stewart et al., 1986.); thismaterial drains from the organelles at the moment ofinvasion; (4) when fluorescent lipid precursors areintroduced into parasites and metabolically incorpor-ated into lipids, discernible fluorescence appears in thePVM following invasion (Mikkelsen et al., 1988); (5)

Malarial parasitophorous vacuole 531

^ 15 ,

8•s 10

6 12Distance (um)

18

Fig. 3. Cumulated distribution (14 cells, bearing attachedmerozoites) of antibody label in 1 (im length elements,measured from the left-hand edge of the parasite-host cellcontact zone (arrow in Fig. 2a), showing absence of labelin the contact zone (first length element).

the PVM in mature intracellular parasites (Atkinson etal., 1987) and in freshly internalised parasites, as well asthe internal vesicle membrane in red cells bearingirreversibly attached parasites (Dluzewski et al., 1989,and Fig. 2c), is devoid of major red cell membraneproteins. Thus either the endogenous proteins areswept away as the parasite invaginates the host cell(assuming that they are not eliminated in their entiretyby proteolysis even before invasion begins), or thePVM is, as our present results lead us to infer, derivedwholly or partly from parasite material alone.

Haldar and Uyetake (1992) have recently reportedthat a fluorescent carbocyanine dye, introduced into thered cell membrane, is carried in by the invadingparasite, as judged by the distribution of fluorescence.The fluorescent intensity appears much higher in theparasite than the host cell, and this is attributed to a"different membrane environment" or selective in-crease in dye concentration in the parasite membrane.In either event the implication would be that the lipidcomposition of the PVM is not that of the red cell. Inthe absence of quantitative data, there is not necessarilyany incompatibility between these data and ours.

The exchange of lipid by endocytic pathways or eventhrough the fluid phase between parasite and host cellcannot be excluded a priori, but the absence of theNBD-PE from the PVM appears to eliminate such aprocess, at least on the time scale of invasion. Later indevelopment (trophozoite stage) PE is evidently trans-ported from the host cell plasma membrane to theparasite (Haldar et al., 1989) and according to Pouvelleet al. (1991) bulk diffusion of extraneous solutes occursat this stage by way of a duct. Some migration of PCbetween the two membranes, presumably by a differentmechanism, has also been detected at an early stage ofdevelopment (Haldar et al., 1989).

This work was supported by the UNDP/World Bank/ WorldHealth Organization Special Programme for Research andTraining in Tropical Diseases. We thank Dr Gary Ward for anilluminating discussion.

Note added in proofColleau et al. (1991) have stated that the NBD-PE, bearingthe substituent in the C-6 position of the caproyl chain, doesnot undergo enzymic translocation to the inner membraneleaflet, as measured by back-extraction with serum albumin.

We found that after incubation a definite, though evidentlyminor proportion of the NBD-PE became resistant to back-extraction and was by implication in the inner leaflet.Whether this represents a level of translocation lower thandetected by Colleau et al. or possibly another isomer presentin the preparation is not at this stage clear.

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