The origin of parasitophorous vacuole membrane lipids in malaria ...

Journal of Cell Science 106, 237-248 (1993) Printed in Great Britain © The Company of Biologists Limited 1993

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The origin of parasitophorous vacuole membrane lipids in malaria-infected erythrocytes Gary E. Ward1,*, Louis H. Miller1 and James A. Dvorak2 1Laboratory

of Malaria Research and 2Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA *Author for correspondence

SUMMARY During invasion of an erythrocyte by a malaria merozoite, an indentation develops in the erythrocyte surface at the point of contact between the two cells. This inden tation deepens as invasion progresses, until the merozoite is completely surrounded by a membrane known as the parasitophorous vacuole membrane (PVM). We incorporated fluorescent lipophilic probes and phospholipid analogs into the erythrocyte membrane, and followed the fate of these probes during PVM formation with low-light-level video fluorescence microscopy. The concentration of probe in the forming PVM was indistinguishable from the concentration of probe in the

erythrocyte membrane, suggesting that the lipids of the PVM are continuous with and derived from the host cell membrane during invasion. In contrast, fluorescently labeled erythrocyte surface proteins were largely excluded from the forming PVM. These data are consistent with a model for PVM formation in which the merozoite induces a localized invagination in the erythrocyte lipid bilayer, concomitant with a localized restructuring of the host cell cytoskeleton.

INTRODUCTION

The adult erythrocyte appears to be incapable of receptor-mediated endocytosis (Haberman et al., 1967; Schekman and Singer, 1976; Zweig and Singer, 1979), and the mechanism of formation of the PVM is unknown. It has been reported that erythrocyte proteins (McLaren et al., 1977; Aikawa et al., 1981; Atkinson et al., 1987; Dluzewski et al., 1989) and lipids (Dluzewski et al., 1992) are excluded from the PVM, leading to the suggestion that the PVM is formed from substances stored in the rhoptries and secreted into the erythrocyte membrane during invasion (Bannister and Dluzewski, 1990; Dluzewski et al., 1992; Joiner, 1991). To study the origin of the PVM, we labeled the surface of the erythrocyte with a variety of fluorescent probes, and followed the fate of these probes during PVM formation by low-light-level video fluorescence microscopy. We found that while erythrocyte membrane proteins are indeed excluded from the PVM, the lipids of the forming PVM are indistinguishable from those of the host cell membrane.

Malaria parasites require both a vertebrate and an invertebrate host to complete their life cycle. In the vertebrate host, asexual-stage merozoites invade and multiply within erythrocytes. The parasite-induced modification and destruction of erythrocytes that occur during this stage of the life cycle give rise to the life-threatening aspects of the malaria infection. Consequently, it is important to understand and interdict the process whereby the merozoite invades and becomes established within the vertebrate host erythrocyte. Morphological studies at the light and electron microscope levels have revealed that invasion is a sequential, multistep process (Dvorak et al., 1975; Bannister and Dluzewski, 1990; Aikawa et al., 1978; Miller et al., 1979). Upon contact with an erythrocyte, the merozoite attaches and orients its anterior end towards the erythrocyte. A region of tight apposition, or ‘junction’ (Aikawa et al., 1978), develops between the membranes of the two cells. Membrane-bound organelles at the anterior end of the merozoite, the rhoptries, discharge their contents onto the erythrocyte surface, which begins to indent at the point of contact. The junction transforms from a localized patch to an orifice, through which the merozoite penetrates into a progressively deepening, membrane-bound vacuole. The membrane surrounding the fully internalized parasite is known as the parasitophorous vacuole membrane (PVM).

Key words: malaria, invasion, parasitophorous vacuole membrane, erythrocyte

MATERIALS AND METHODS Reagents PKH2, PKH26, and Diluents A and C were purchased from Zynaxis Cell Science, Inc. (Malvern, PA). Bisbenzimide Hoe33342, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES; Ultrol grade), cytochalasin B, adenosine diphosphate

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(ADP), and hypoxanthine were from Calbiochem (San Diego, CA). Octadecyl rhodamine B (R18), 1,1′-dihexadecyl-3-3′-3-3′tetramethylindocarbocyanine (DiIC16), fluorescein-5-thiosemicarbazide, Texas Red sulfonyl chloride, and 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) were from Molecular Probes, Inc. (Eugene, OR). 1-palmitoyl-2[6-[(7-nitro-2-1,3-benzoxadiazol4-yl)amino]caproyl]-sn-glycero-3-phosphoserine (NBD-PS) and 1-acyl-2[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-snglycero-3-phosphoethanolamine (NBD-PE) were from Avanti Polar Lipids (Alabaster, AL). Leupeptin and Nutridoma-NS were from Boehringer Mannheim Corp. (Indianapolis, IN), NuSerum I was from Collaborative Biomedical Products (Bedford, MA), Versilube silicon oil was from General Electric Co. (Waterford, NY), citrate phosphate dextrose anticoagulant (CPD) was from Baxter Healthcare Corp. (Deerfield, IL), Percoll was from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ), and prestained high molecular mass standards were from Bio-Rad Laboratories (Melville, NY). Phosphate-buffered saline (PBS) and horse serum were from BioFluids, Inc. (Rockville, MD). Gentamicin, fetal bovine serum, and RPMI-1640 (#430-1800EC) were from Gibco BRL Life Technologies, Inc. (Grand Island, NY). Acid-washed glass beads (60 min), they became weakly fluorescent (data not shown). Merozoites attached to human erythrocytes lacking the Duffy blood group antigen also became detectably fluorescent, but only after longer periods of attachment (>135 min). Free merozoites were not detectably labeled, even after 135 min. We followed the kinetics of fluorescent probe internalization during invasion by low-light level video microscopy, using both DIC and fluorescence optics. The DIC images of a typical interaction are shown in Fig. 2 (upper row). After initial attachment of the merozoite (Fig. 2A) and apical reorientation, the surface of the erythrocyte begins to indent at the point of contact. As the merozoite enters, this indentation grows progressively deeper to form the invasion pit (Fig. 2B), the neck of which constricts and ultimately closes once the parasite is completely inside the erythrocyte, to form the PVM (Fig. 2C, D; see also Aikawa

Fig. 1. Rhesus erythrocytes were incubated for 20 min (37°C) with P. knowlesi merozoites, in the absence (a-f) or presence (g-i) of 10 µg/ml cytochalasin B. Erythrocytes were labeled with PKH26 either prior to merozoite addition (a-c, g-i), or after 20 min incubation with merozoites and removal of free merozoites (d-f). In each case, a differential interference contrast (DIC) image (a, d, g), and the corresponding bisbenzimide Hoe33342 fluorescence image (revealing the parasite nucleus on a brightfield background; b, e, h), and PKH26 fluorescence image (c, f, i) are shown. The cells surrounding the central infected erythrocyte in (d) to (f) moved during sequential capture of the DIC, Hoe33342, and PKH26 images. Bar, 2.5 µm.

Fig. 2. DIC (top row) and fluorescence (bottom row) images of PVM formation during invasion of PKH26-labeled erythrocytes. (A) Merozoite is attached but not yet apically reoriented; note the lateral position of the rhoptries (arrow; Dvorak et al., 1975; Miller et al., 1979). (B-D) Sequential images captured during merozoite internalization, (B) 12 min 43 s, (C) 14 min 56 s, and (D) 17 min 51 s after mixing merozoites with labeled erythrocytes. Bar, 5 µm.

et al., 1978). During the process of invasion the erythrocyte often changes from a biconcave to a spherical shape (Fig. 2A-D; see also Dvorak et al., 1975). PKH26 fluores-

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Fig. 3. Fluorescence intensity profile histograms of the images shown in Fig. 2B-D. Fluorescence intensity was measured along the axis shown in the inset of each panel. In each image, the fluorescence intensity of the combined PVM and erythrocyte membrane surfaces is approximately twice that of the erythrocyte membrane alone (see text).

cence images corresponding to the DIC images are shown in the lower row of Fig. 2. The fluorescent probe is initially distributed uniformly throughout the erythrocyte membrane (Fig. 2A); the biconcave shape of the erythrocyte at this stage is characterized by the bright circle of fluorescence that appears at the steep transition in erythrocyte thickness. As the merozoite enters, the fluorescent probe demarcates the initial indentation of the erythrocyte membrane (not shown), the deepening invasion pit (Fig. 2B), and ultimately the fully formed PVM (Fig. 2D). These images demonstrate that the probe is indeed internalized at the time of invasion. Furthermore, since the posterior end of a partially internalized parasite is not fluorescent (e.g. see Fig. 2B), the probe is apparently internalized via the PVM, and not incorporated into the merozoite itself. The video microscopy system had been adjusted to produce a linear intensity output response for all of the images collected. Consequently, we were able to quantify fluorescence intensity profiles of the images to determine the relative distribution of PKH26 in the erythrocyte membrane and PVM. Intensity profiles of the images presented in Fig. 2 are shown in Fig. 3; fluorescence intensity was determined along the axis shown in the inset of each profile. The mean fluorescence intensity in regions encompassing the mem-

branes of both the erythrocyte and the PVM was approximately two-fold higher than the mean intensity in regions encompassing only the erythrocyte membrane. The two-fold difference in [erythrocyte + PVM] to erythrocyte fluorescence was highly reproducible: in 29 independent images of invading or recently internalized (95% of the total probe associated with the cells (Fig. 5A). When erythrocytes were depleted of ATP prior to labeling, to inhibit the pump responsible for translocating aminophospholipids from the outer to the inner leaflet (Daleke and Huestis, 1985; Devaux, 1991), less probe was resistant to back extraction (approximately 20% the level seen in control Fig. 5. (A) Rhesus erythrocytes labeled with 10 µg/ml NBD-PS were back extracted five times with fatty acid-free BSA as described in Materials and Methods. After each back extraction, the amount of probe associated with the cell pellet (●) and the amount of probe extracted into the supernatant (■) were determined spectrofluorimetrically. When the erythrocytes were ATP depleted prior to labeling with NBD-PS by a 30 min incubation at 37°C in 6 mM iodoacetamide + 10 mM inosine (Daleke and Huestis, 1985), the amount of non-backextractable probe remaining associated with the pellet (◆) was reduced. (B) Rhesus erythrocytes labeled with NBD-PS, washed, and back extracted 5 times with fatty acid-free BSA were incubated at 37°C (8.3% hematocrit) in MED1B containing 33% fetal bovine serum. Aliquots of the suspension were removed at various times and back extracted in 7.2 volumes PBS containing 1% fatty acid-free BSA. The amount of probe extracted into the supernatant (■) and the amount remaining associated with the cell pellet (●) were determined spectrofluorimetrically. (C) NBD-PS fluorescence (upper row), DIC (lower left) and bisbenzimide Hoe33342 fluorescence (lower right) images of PVM formation during invasion of an NBD-PS-labeled erythrocyte. Merozoites and erythrocytes were mixed together at t=0. A partially internalized merozoite is shown in the first panel (t=14:28); this merozoite was completely internalized by t=15:08. Bar, 2 µm.


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Fig. 6. Fluorescence intensity profile histograms of the images shown in Fig. 5C, upper row. NBD-PS fluorescence intensity was measured along the axis shown in the inset of each panel.

cells; Fig. 5A). After a 15 min incubation under the conditions necessary to visualize invasion, >90% of the probe remained resistant to back extraction (Fig. 5B). In video microscope-based invasion studies of NBD-PSlabeled erythrocytes, NBD-PS fluorescence was seen to demarcate both the forming and the fully-formed PVM (Fig. 5C, upper row). Merozoites that were only partially internalized were not fluorescent (data not shown), again indicating that the fluorescent probe was initially incorporated into the PVM, and not the parasite. No detectable transfer of NBD-PS from labeled erythrocytes to attached merozoites was seen after 20 min at 37°C (Fig. 5C, t=20:38). Quantitative analysis of the distribution of NBD-PS fluorescence during invasion showed that, like PKH26, the concentration of NBD-PS in the newly formed PVM was indistinguishable from the concentration in the erythrocyte membrane (Fig. 6A, B). Unlike PKH26, however, the amount of internalized NBD-PS steadily increased over the subsequent 10 minutes post-invasion (Fig. 6C, D); these differences in the post-invasion distribution of NBD-PS and PKH26 are shown graphically in Fig. 7. We were unable to determine by fluorescence microscopy whether

the NBD-PS, which accumulated post-invasion was confined to the PVM, or was also incorporated into the parasite itself. Patches of increased probe fluorescence on the erythrocyte surface at the point of merozoite attachment During the interaction between merozoites and PKHlabeled erythrocytes, we often observed patches of increased fluorescence on the erythrocyte surface at or near the point of merozoite attachment. This was most easily seen when cytochalasin B was used to inhibit internalization (Fig. 8 a/d, b/e; see also Fig. 1i), but it also occurred in the absence of cytochalasin B treatment (Fig. 8 c/f). Erythrocytes labeled with PKH2, PKH26, DiIC16 and NBDPS all exhibited this behavior, as did erythrocytes labeled with the non-exchanging lipophilic probe, R18. The patches of increased fluorescence were present on erythrocytes labeled with both self-quenched and unquenched concentrations of R18 (data not shown), and they were seen with merozoites attached to Duffy-positive human erythrocytes, but not with merozoites attached to Duffy-negative human erythrocytes.

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Fig. 7. The fluorescence intensities measured in Fig. 3 (PKH26) and Fig. 6 (NBD-PS) were expressed as the ratio of intensity in the combined [PVM + erythrocyte] membranes to that of the erythrocyte membrane alone, and plotted versus time postinvasion. For both NBD-PS (●) and PKH26 (■), the concentration of probe in the PVM is similar to the concentration in the erythrocyte membrane (i.e. fluorescence intensity ratio = 2) immediately after invasion. However, the concentration of PKH26 associated with the parasite and/or PVM remains constant over the first 5 min post-invasion, while that of NBD-PS steadily increases. Similar results were obtained with different sets of images from different invasion events (n=8 for PKH26, n=5 for NBD-PS).

Erythrocyte membrane proteins are excluded from the forming PVM In contrast to our results with lipophilic membrane probes, it has been shown by freeze-fracture and immunoelectron microscopy that erythrocyte membrane proteins are excluded from the PVM (McLaren et al., 1977; Aikawa et al., 1981; Atkinson et al., 1987; Dluzewski et al., 1989). To confirm and extend these observations, we fluorescently labeled erythrocyte surface proteins and followed the fate of the labeled proteins during invasion, while simultaneously monitoring the fate of erythrocyte lipids using PKH2 or PKH26. Fluorescein-5-thiosemicarbazide was used to label sialic acid residues on the glycophorins (Fig. 9A, lane a′; see also Golan et al., 1986), and the amino-reactive reagents DTAF (Sheetz et al., 1980) and Texas Red sulfonyl chloride were used to label Band 3 (and several other minor proteins; Fig. 9A, lane b′). All three reagents gave qualitatively similar results: labeled erythrocyte surface proteins were largely excluded from the newly formed PVM (e.g. see Fig. 9B, d). Images of partially internalized merozoites (e.g. see Fig. 9B, e-h) reveal that the exclusion of proteins occurs during vacuole formation: the invasion pit, though stained with PKH2 (Fig. 9B, g), is largely free of labeled protein (Fig. 9B, h). DISCUSSION Internalization of PKH26, PKH2 and DiIC16 during invasion The PVM has not been isolated from malaria-infected erythrocytes and, as a consequence, little is known about its macromolecular composition. Immunoelectron microscopy and freeze-fracture studies have shown that erythrocyte membrane proteins are essentially absent from the PVM

Fig. 8. DIC (a-c) and corresponding fluorescence (d-f) images of merozoites attached to PKH26-labeled erythrocytes, in the presence (a/d, b/e) and absence (c/f) of 10 µg/ml cytochalasin B. Images were captured 20 min (a/d, b/e) and 25 min (c/f) after mixing merozoites and labeled erythrocytes. In each case, there is a localized patch of increased PKH26 fluorescence near the point of contact between the two cells. Bar, 5 µm.

(McLaren et al., 1977; Aikawa et al., 1981; Atkinson et al., 1987; Dluzewski et al., 1989). It has been reported that the PVM is also largely depleted of host membrane lipids (Dluzewski et al., 1992). We studied the composition of the PVM by labeling the erythrocyte membrane with a variety of fluorophores and determining the distribution of these probes during invasion by fluorescence microscopy. In initial experiments with several fluorescent lipophilic probes, we found that the probes were associated with the internalized parasite shortly after invasion (Fig. 1c). A similar result was described by Haldar and Uyetake (1992). It was not clear from these initial experiments how probe originally inserted into the erythrocyte membrane was internalized, i.e. through incorporation into the forming PVM or via transfer into the invading parasite itself. PKH2, PKH26, and DiIC16 reportedly undergo little or no intermembrane exchange once they have been incorporated into a membrane bilayer (Horan and Slezak, 1989; Slezak and Horan, 1989; Honig and Hume, 1989). However, we detected transfer of these probes from labeled rhesus or Duffy-positive human erythrocytes to attached merozoites after a 60 minute incubation at 37°C. A reduced rate of transfer was observed using labeled Duffy-negative human erythrocytes, and no detectable transfer was observed between labeled erythrocytes and free merozoites. A gap of approximately 10 nm separates the membrane of a Duffypositive erythrocyte from that of an attached merozoite, whereas the separation is approximately 150 nm for a Duffy-negative erythrocyte (Miller et al., 1979). These data suggest that while PKH2, PKH26, and DiIC16 are relatively non-exchanging, if two membranes are sufficiently tightly apposed some transfer can occur across the aqueous space between them. Furthermore, in many of our post-invasion images the internalized probe was not confined to a rim surrounding the newly invaded parasite, but exhibited a heterogeneous substructure. Haldar and Uyetake (1992) reported a similar observation, and demonstrated directly that at some time during or after invasion the probe was transferred to the parasite itself.


A

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B

Fig. 9. (A) Erythrocytes labeled with (a, a′) fluorescein thiosemicarbazide or (b, b′) Texas Red sulfonyl chloride were lysed in hypotonic buffer to remove cytoplasmic proteins, and the resultant erythrocyte ghosts were analyzed by SDS-PAGE. Total ghost proteins were visualized with Coomassie Blue (a, b), and fluorescently-derivatized proteins were visualized directly by UV transillumination (a′, b′). Positions of molecular mass markers (in kDa) are indicated. (B) Invasion into erythrocytes double-labeled with Texas Red sulfonyl chloride and PKH2. Panels (a-d) show a fully internalized parasite (20 min after mixing merozoites with the labeled erythrocytes), and (e-h) show a pair of partially internalized merozoites (9 min after mixing). DIC images are shown in panels (a, e); (b, f) show the corresponding bisbenzimide Hoe33342 fluorescence on a DIC or brightfield background, (c, g) show PKH2 fluorescence, and (d, h) show Texas Red fluorescence. Bar, 5 µm.

To resolve the issue of how the probes became internalized, we developed methods for following their distribution in real time during the process of invasion. The results showed that the probes are in fact internalized via the PVM during invasion (Fig. 2). In addition, we found that the concentration of probe in the erythrocyte membrane and in the forming PVM were essentially indistinguishable (Fig. 3). If the probes used accurately reflect the behaviour of endogenous erythrocyte lipids, these results demonstrate that the lipid composition of the PVM is similar to that of the erythrocyte membrane and suggest that the lipids of the PVM are derived directly from the host cell membrane during invasion. Are NBD-phospholipids excluded from the PVM? It was recently reported that following invasion of NBDPE-labeled human erythrocytes by P. falciparum merozoites, the PVM surrounding the young (