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

Origins of the membrane of the parasite, 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, UK 2Department 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, UK 4National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK

Summary

We have attempted to determine whether the parasito- distribution of the labelled lipid; this was found to be phorous vacuole membrane, in which the malaria overwhelmingly in favour of the host parasite (merozoite) encapsulates itself when it enters a relative to the parasitophorous vacuole. Merozoites of P. red blood cell, is derived from the host cell plasma knowlesi were allowed to attach irreversibly to red cells membrane, as the appearance of the invasion process in without invasion, using the method of pretreatment with the electron microscope has been taken to suggest, or cytochalasin. The region of contact between the mero- from lipid material stored in the merozoite. We have zoite and the host cell membrane was in all cases devoid incorporated into the red cell membrane a haptenic of the labelled phosphatidylethanolamine. These results phospholipid, phosphatidylethanolamine, containing an lead us to infer that the parasitophorous vacuole NBD (N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)) group, sub- membrane is derived wholly or partly from lipid pre- stituted in the acyl chain, and allowed it to translocate existing in the merozoite. into the inner bilayer leaflet. After invasion of these labelled cells by the parasite, , Key words: malaria, merozoite, parasitophorous vacuole, immuno-gold electron microscopy was used to follow the lipid.

Introduction the major red cell membrane proteins (Atkinson et al., 1987) and this is true also of the membrane that bounds The sequence of events that characterises the entry of the internal vacuole, which represents the first step in the malaria parasite into the red blood cell has been formation of the cavity in the host cell that eventually defined at the morphological level (Bannister et al., receives the parasite (Dluzewski et al., 1989). Never- 1975; Aikawa et al., 1978; Miller et al., 1979; Aikawa et theless, counter-indications have accumulated that al., 1981). In essence the merozoite first makes random have been interpreted as indicating a parasite-derived contact with the red cell surface; it then reorients, so as origin for the PVM; in particular, the and to bring its apical surface into apposition with the host , which are organelles located near the apex cell membrane. An invagination develops in the host of the merozoite, were shown to contain lamellar cell and deepens as the merozoite enters. An electron- deposits, consisting almost certainly of lipid (Bannister dense junction, which forms during the attachment et al., 1986; Stewart et al., 1986). This material drains phase, remains at the contact zone as the parasite through ducts into the area of contact with the host cell moves inwards; when the parasite is engulfed, the host at the time of entry (Bannister and Mitchell, 1989) or cell membrane closes behind it. The internalised attachment of the parasite (Aikawa et al., 1981). It also parasite is thus encapsulated in a so-called parasito- appears that fluorescent lipids, metabolically intro- phorous vacuole. duced into the parasite, appear in the PVM after The appearance of the invasion process in the invasion, indicating that parasite-derived lipid makes at electron microscope strongly suggested that the parasit- least some contribution to the PVM (Mikkelsen et al., ophorous vacuole membrane (PVM) is formed by 1988). eversion of the host cell membrane, and until recently We have attempted to resolve the conflicting evi- this was generally assumed to be the case (see e.g. Holz, dence about the origin of the PVM by immuno-electron 1977, for a review). The PVM, moreover, is devoid of microscopy, making use of a haptenic lipid, inserted 528 A. R. Dluzewski and others into the red cell membrane before exposure to the with LR White monomer, transferred to gelatin capsules and parasite. Our results support the view that the PVM is the resin was polymerised by warming at 50°C for 24 h. derived partly or entirely from the parasite. Sections were prepared and mounted on 200-mesh copper grids. The grids were floated on 50 mM potassium phosphate buffer, pH 7.4, which contained 10 mg ml"1 bovine serum Materials and methods albumin and 2.5 mg ml"1 Tween 20. They were then transferred to the surface of a drop of antibody solution, Plasmodium falciparum parasites were cultured in vitro diluted 1:20 - 1:40, or, in the case of controls, normal rabbit (Trager and Jensen, 1976) and synchronised by the sorbitol serum and antiserum, absorbed with isolated membranes method (Lambros and Vandenberg, 1979). The duration of from NBD-CI-labelled ghosts, and left in place for 1 h. They invasion experiments was 7 h. P. knowlesi parasites had been were washed by flotation on three changes of buffer and then cryopreserved as ring-stage trophozoites in Callithrix jacchus floated on a solution of protein A, labelled with colloidal gold red cells. These were thawed, rehydrated and cultured by conjugation with chlorauric acid (BDH), following the overnight to schizogony as described earlier (Bannister and method of Roth (1982). The grids were rinsed twice with Mitchell, 1989). Invasion of human red cells was initiated by buffer, then with distilled water, air-dried and stained with addition of purified schizont preparations (Dluzewski et al., uranyl acetate and lead citrate. The sections were coated with 1984) to the cells in RPMI 1640 culture medium containing carbon and examined in a Philips 300 electron microscope at 10% human serum. Externally, irreversibly attached P. 80 kV accelerating voltage. The same procedure was used for knowlesi result when cytochalasin B is added to the the detection of the transmembrane protein, band 3, as merozoites or rupturing schizonts before mixing with the red described earlier (Dluzewski et al., 1989). cells (Miller et al., 1979); the conditions of treatment were as described previously (Dluzewski et al., 1989). Incubations were for 90 min with 2 /.ig ml"1 cytochalasin B. Human target cells were used. Results The labelled lipid, NBD-PE, i.e. l-acyl-2-[6-[(7-nitrobenz- 2-oxa-l,3-diazol-4-yl) amino] caproyl] phosphatidylethanola- If a labelled phospholipid is to be introduced into the mine, was obtained from Avanti Polar Lipids. It was dispersed in the form of an ethanolic solution at 80 ^g ml~ in red cell membrane bilayer, the derivatising group must 100 volumes of RPMI medium, to which packed, washed red be in the acyl chain and not the head-group (Struck and cells were then added to give a haematocrit of 1% and allowed Pagano, 1980). Moreover, an aminophospholipid modi- to incorporate into the membrane for 1 h at 37°C (Tanaka and fied in this manner will be translocated to the inner Schroit, 1983). The cells were washed in RPMI. During the membrane leaflet by the membrane-associated translo- incubation with parasites this extraneous lipid is translocated case (Devaux, 1988). NBD-PE fulfilled these require- to the inner membrane leaflet (Devaux, 1988), and this ments and gave rise to abundant fluorescence in the becomes largely resistant to extraction by albumin-containing cell; a part of this remained in the membrane and was medium (see below). only slowly extracted on incubation with the culture For preparation of anti-NBD antibodies, bovine immuno- medium, which contains a high concentration of serum globulin G (Sigma) was derivatised by reaction with NBD-C1 albumin. Thus the cells remained brightly fluorescent (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- over the 7 h period of incubation in the invasion tration of 2 mg ml"1 in 0.1 M sodium phosphate, pH 8.1, and experiments, and even after 20 h there was only a the reaction was allowed to proceed at room temperature for moderate reduction in intensity. The labelled cells were 90 min. An excess of buffered Tris hydrochloride was added invaded by P. falciparum with comparable efficiency to and the protein was dialysed against 0.1 M sodium chloride, control cells. Examination in the fluorescence micro- 20 mM sodium phosphate, pH 7.4. A rabbit was inoculated scope revealed no detectable excess fluorescence in the subcutaneously and intramuscularly with the protein in region of the intraerythrocytic parasite, suggesting that complete Freund's adjuvant, followed by two booster injec- the label had not entered the PVM; limitations of tions with the antigen in Freund's incomplete adjuvant over a period of 4 months. Serum was taken 6 weeks after the last contrast and resolution, however, precluded definitive boost. or quantitative conclusions on the basis of this negative Parasitised fluorescent cells in wet films under coverslips observation. We therefore had recourse to gold- were examined in a Zeiss microscope in epifluorescence in the labelling immuno-electron microscopy. presence of p-phenylenediamine as anti-bleaching agent. For It is likely that a proportion of the lipid would be electron microscopy, cells were metabolically depleted before extracted in the course of dehydration and embedding, fixation: this was found to reduce the extraction of the although the aminophospholipids may be stabilised by antigenic lipid in the course of ethanol-dehydration. Thus the the fixation step with glutaraldehyde. The results show parasitised cells were suspended in isotonic phosphate- in any event that enough lipid remains to give buffered saline, containing 4 mM iodoacetate, pH 7.4; the satisfactory immuno-gold labelling in thin sections. Fig. supernatant was then replaced by the same buffer, containing 5 mM N-ethylmaleimide, pH 8.0. The cells were fixed in 0.5% 1 shows labelling of the red cell plasma membrane, glutaraldehyde in 0.1 M potassium phosphate, pH 7.4, which is absent from sections treated with antiserum followed by suspension in 50 mM ammonium chloride for 20 that had been absorbed out with ghosts from NBD min, and finally washed twice with 0.1 M potassium chloride-derivatised red cells. Equally the labelling has phosphate, pH 7.4. The treated cells were dehydrated by no (or only a minimal) counterpart around the successive transfers to five solutions of increasing ethanol periphery of the parasite or elsewhere in either cell. The concentration, up to 75% (v/v). The cells were equilibrated number of attached antibody molecules per unit length Malarial parasitophorous vacuole 529

Table 1. Distribution of host cell phospholipid marker (NBD-PE) in the membranes of human red cells with internalised P. falciparum and attached P. knowlesi merozoites Total length" Gold particles membrane (/an) per /an Internalised P. falciparum Red cell membraneb 479 0.66±0.25c Parasite membrane 367 0.08±0.09 Attached P. knowlesid Red cell membrane 250 0.56+0.26 Region of attachment 14 0 'Total length of contour of red cell membrane and parasitophorous vacuole membrane examined (26 and 14 cells were analysed in the P. falciparum and P. knowlesi experiments respectively). bAs a control, antiserum absorbed out with NBD-Cl-labelled ghosts 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 particle density 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 (as indicated in Fig. 3).

of contact between the attached parasite and the host cell. To establish this as a general feature of the attached state, because of the sparsity of the label, it was necessary to determine the distribution of gold particles around the membrane contour, measured from the left-hand boundary of the zone of contact (Fig. 3). Here the first element on the left represents the part of the membrane in contact with the parasite, and is the only one in which there is no antibody in any of the 14 cells with attached parasites examined. To evaluate the significance of this observation we may calculate the probability that this has happened by chance: if r Fig. 1. Thin sections of red cells, labelled with haptenically particles (summed over all cells examined) are distrib- modified phosphatidylethanolamine, infected with P. uted between n membrane elements the probability falciparum. The sections were incubated with anti-NBD (/\) of finding k of these r particles in the tth membrane antibody, followed by gold-labelled protein A; (a) and (b) element (n > i > 1) is: show two different red cells, containing young ring-stage 1 1 parasites (r). Note label on host cell membrane but not on Pk = [r!/it!(r-Jfc) !]./r*. (1-/T ) -* parasitophorous vacuole membrane. Bars, 1 fan. For 132 particles, distributed randomly among 18 membrane elements, the probability of finding no particles in any one element (here by definition element of host cell membrane and PVM contour is given in 1), Pp, is 5.3 x 1CT4. Thus the probability that the host Table 1. These results strongly suggest that the PVM is cell lipid marker is absent from the region of contact by not in large degree derived from the host cell chance can be disregarded. We infer that the parasite at membrane. the stage of incipient invasion generates membrane We then examined red cells bearing attached P. material, which appears to form a pool at the point of knowlesi merozoites: in this system the invasion cycle of attachment, that then normally expands inwards to the parasites, treated with cytochalasin B, is arrested at form the vacuole into which the parasite passes. the stage of irreversible attachment (Miller et al., 1979). Because of the rapidity of the invasion process it may be With the human cells used here we did not observe the surmised that the substance of the new membrane is internal vacuole in the host cell, opposite the zone of wholly or partly derived from the pre-existing attachment, that were formed in simian cells (Dlu- and contents (Bannister et al., 1986; zewski et al., 1989). In Fig. 2 no haptenically labelled Stewart et al., 1986; Bannister and Mitchell, 1989), lipid is to be seen in the red cell membrane in the region rather than freshly synthesised lipid. 530 A. R. Dluzewski and others a

Fig. 2. Thin sections of red cells, labelled with haptenically modified phosphatidylethanol- amine and bearing attached P. knowlesi merozoites (m); the sections were incubated with anti-NBD (a and b) antibody and gold-labelled protein A, as before. Panels (a) and (b) show results for different cells and (c) shows typical labelling of membrane with anti-band 3 antibody for comparison. Note absence of label in the region of contact between merozoites and host cells. The arrow in (a) denotes the position of the left-hand edge of the first length element (see Fig. 3). Bars, 1 /an.

Discussion the PVM is much more resistant than the red cell to lysis by saponin (Sherman and Hull, 1960), which suggests a The debate about the provenance of the PVM (Joiner, different lipid composition; (3) the rhoptries and 1991) has focussed on the appearance presented by the micronemes contain abundant amounts (sufficient, as invasion process in the electron microscope (Aikawa et Bannister and Mitchell (1989) have pointed out, to al., 1978, 1981), which undeniably suggests encapsula- generate a bilayer with the area of the PVM) of material tion of the invading merozoite by the host cell with the appearance of multilamellar lipid bodies membrane, and on the other hand on the following (Bannister et al., 1986; Stewart et al., 1986.); this items of evidence, all indirect: (1) multiple invasion, material drains from the organelles at the moment of which is not uncommon, would, on the above model, invasion; (4) when fluorescent lipid precursors are result in the elimination of a considerable proportion of introduced into parasites and metabolically incorpor- the host cell membrane area (some 7% for each ated into lipids, discernible fluorescence appears in the merozoite internalised in the case of P. knowlesi); (2) PVM following invasion (Mikkelsen et al., 1988); (5) Malarial parasitophorous vacuole 531

^ 15, We found that after incubation a definite, though evidently 8 minor proportion of the NBD-PE became resistant to back- •s 10 extraction and was by implication in the inner leaflet. Whether this represents a level of translocation lower than detected by Colleau et al. or possibly another isomer present in the preparation is not at this stage clear. 6 12 18 Distance (um) Fig. 3. Cumulated distribution (14 cells, bearing attached References merozoites) of antibody label in 1 (im length elements, Aikawa, M., Miller, L. H., Johnson, J. and Rabbege, J. (1978). measured from the left-hand edge of the parasite-host cell Erythrocyte entry by malaria parasites. A moving junction between contact zone (arrow in Fig. 2a), showing absence of label erythrocyte and parasite. J. Cell. Biol. 77, 72-82. in the contact zone (first length element). Aikawa, M., Miller, L. H., Rabbege, J. and Epstein, N. (1981). Freeze-fracture study on the erythrocyte membrane during malarial parasite invasion. J. Cell Biol. 91, 55-62. the PVM in mature intracellular parasites (Atkinson et Atkinson, C. T., Ikawa, M., Perry, G., Fujino, T., Bennett, V., al., 1987) and in freshly internalised parasites, as well as Davidson, E. A. and Howard, R. J. (1987). Ultrastructural the internal vesicle membrane in red cells bearing localization of erythrocyte cytoskeletal and integral membrane irreversibly attached parasites (Dluzewski et al., 1989, proteins in Plasmodium falciparum-infected erythrocytes. Eur. J. Cell Biol. 45, 192-199. and Fig. 2c), is devoid of major red cell membrane Bannister, L. H., Butcher, G. A., Dennis, E. D. and Mitchell, G. H. proteins. Thus either the endogenous proteins are (1975). Structure and invasive behaviour of swept away as the parasite invaginates the host cell merozoites in vitro. Parasitology 71, 483-491. (assuming that they are not eliminated in their entirety Bannister, L. H. and Mitchell, G. H. (1989). The fine structure of secretion by Plasmodium knowlesi merozoites during red cell by proteolysis even before invasion begins), or the invasion. /. Protozool. 36, 362-367. PVM is, as our present results lead us to infer, derived Bannister, L. H., Mitchell, G. H., Butcher, G. A. and Dennis, E. D. wholly or partly from parasite material alone. (1986). Lamellar membranes associated with rhoptries in Haldar and Uyetake (1992) have recently reported erythrocyte merozoites of Plasmodium knowlesi: a clue to the mechanism of invasion. Parasitology 92, 291-303. that a fluorescent carbocyanine dye, introduced into the Colleau, M., Herve, P., Fellmann, P. and Devaux, P. F. (1991). red cell membrane, is carried in by the invading Transmembrane diffusion of fluorescent phospholipids, in human parasite, as judged by the distribution of fluorescence. erythrocytes. Chem. Phys. Lipids 57, 29-37. The fluorescent intensity appears much higher in the Devaux, P. F. (1988). Phospholipid flippases. FEBS Lett. 234, 8-12. parasite than the host cell, and this is attributed to a Dluzewski, A. R., Fryer, P. R., Griffiths, S., Wilson, R. J. M. and Gratzer, W. B. (1989). Red cell membrane protein distribution "different membrane environment" or selective in- during malarial invasion. /. Cell Sci. 92, 691-699. crease in dye concentration in the parasite membrane. Dluzewski, A. R., Ling, I. T., Rangachari, K., Bates, P. A. and In either event the implication would be that the lipid Wilson, R. J. M. (1984). A simple method for isolating viable composition of the PVM is not that of the red cell. In mature parasites of Plasmodium falciparum from cultures. Trans. Roy. Soc. Trop. Med. Hyg. 78, 622-624. the absence of quantitative data, there is not necessarily Haldar, K., De Amorim, A. and Cross, G. A. M. (1989). Transport of any incompatibility between these data and ours. fluorescent phospholipid analogues from the erythrocyte The exchange of lipid by endocytic pathways or even membrane to the parasite in Plasmodium flaciparum-infectcd cells. through the fluid phase between parasite and host cell /. Cell Biol. 108, 2183-2192. cannot be excluded a priori, but the absence of the Haldar, K. and Uyetake, L. (1992). The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes. NBD-PE from the PVM appears to eliminate such a Mol. Biochem. Parasitol. 50, 161-178. process, at least on the time scale of invasion. Later in Holz, G. G. (1977). Lipids and the malarial parasite. Bull W.H.O. 55, development (trophozoite stage) PE is evidently trans- 237-248. ported from the host cell plasma membrane to the Joiner, K. A. (1991). Rhoptry lipids and parasitophorous vacuole formation: a slippery issue. Parasitol. Today 7, 226-227. parasite (Haldar et al., 1989) and according to Pouvelle Lambros, C. and Vandenberg, J. P. (1979). Synchronization of et al. (1991) bulk diffusion of extraneous solutes occurs Plasmodium falciparum intraerythrocytic stages in culture. J. at this stage by way of a duct. Some migration of PC Parasitol. 65, 418-420. between the two membranes, presumably by a different Mikkelsen, R. B., Kamber, M., Wadwa, K. S., Lin, P-S. and mechanism, has also been detected at an early stage of Schmidt-Ullrich, R. (1988). The role of lipids in Plasmodium falciparum invasion of erythrocytes: a coordinated biochemical and development (Haldar et al., 1989). microscopic analysis. Proc. Nat. Acad. Sci. USA 85, 5956-5960. Miller, L. H., Aikawa, M., Johnson, J. G. and Shiroishi, T. (1979). This work was supported by the UNDP/World Bank/ World Interaction between cytochalasin B-treated malarial parasites and Health Organization Special Programme for Research and erythrocytes. Attachment and junction formation. J. Exp. Med. Training in Tropical Diseases. We thank Dr Gary Ward for an 149, 172-184. illuminating discussion. Pouvelle, B., Spiegel, R., Hsiao, L., Howard, R. J., Morris, R. L., Thomas, A. P. and Taraschi, T. F. (1991). Direct access to serum macromolecules by intraerythrocytic malaria parasites. Nature 353, Note added in proof 73-75. Roth, J. (1982). The protein A-gold (pAg) technique. A qualitative Colleau et al. (1991) have stated that the NBD-PE, bearing and quantitative approach for antigen location in thin sections. the substituent in the C-6 position of the caproyl chain, does Techniques in Immunocytochemistry, vol. 1 (ed. G.R. Bullock and not undergo enzymic translocation to the inner membrane D. Petrusz), pp. 108-133. Academic Press, London. leaflet, as measured by back-extraction with serum albumin. Sherman, I. and Hull, R. W. (1960). The pigment (hemozoin) and 532 A. R. Dluzewski and others

proteins of the avian malaria parasite Plasmodium lophurae. J. Tanaka, Y. and Schroit, A. J. (1983). Insertion of fluorescent Protozool. 7, 409-416. phosphatidylserine into the plasma membrane of red blood cells. J. Stewart, M. J., Schulman, S. and Vandenberg, J. P. (1986). Rhoptry Biol. Chem. 258, 11335-11343. secretion of membrane whorls by Plasmodium falciparum Trager, W. and Jensen, J. B. (1976). Human malaria parasites in merozoites. Am. J. Trop. Med. Hyg. 35, 37-44. continuous culture. Science 193, 673-675. Struck, D. K. and Pagano, R. E. (1980). Insertion of fluorescent phospholipids into the plasma membrane of a mammalian cell. J. Biol. Chem. 255, 5404-5410. (Received JO February 1992 - Accepted 23 March 1992)