Bulletin of the World Health Organization, 55 (2-3): 199-203 (1977)

A freeze-fracture study on the parasite-erythrocyteinterrelationship in Plasmodium knowlesi infectionsDIANE J. McLAREN,' L. H. BANNISTER,2 P. I. TRIGG,3 & G. A. BUTCHER 4

Freeze-fracture studies were made on the parasite and the erythrocyte in P. knowlesiinfections. There is a loss of transmembrane integral proteins from the plasma membraneof the schizont-infected erythrocyte and the intraerythrocytic parasite synthesizes newtransmembrane proteins as development proceeds. Formation of the parasitophorousvacuole includes changes in the number of integral proteins present in the vacuolar mem-brane, indicating that this membrane may be modified by and in part derived from theparasite.

Over the last few years, the development andelaboration of the freeze-fracture technique hasadded a new dimension to the study of biologicalmembranes. The interaction of apposed plasmamembranes is of particular importance in the host-parasite relationship, and in this context the malaria-infected erythrocyte provides an excellent modelfor studying intracellular parasitism in general. Inthe present investigation, we have used the freeze-fracture technique to examine the surface membranesof free merozoites, trophozoites, and schizonts ofP. knowlesi. In addition, we have studied alterationsin the erythrocyte plasma membrane and vacuolarmembrane induced by the invading, growing, andmultiplying parasite.

MATERIALS AND METHODS

Free merozoites were prepared according to themethod of Dennis et al. (1). Invasion studies weremade on merozoites incubated for 1-7 min withpacked erythrocytes as described by Bannister etal. (2).Normal erythrocytes and erythrocytes infected

with ring stages, trophozoites, and schizonts werecollected in heparinized heart blood from normaland infected rhesus monkeys.

1 Scientist, Division of Parasitology, National Institute forMedical Research, Mill Hill, London NW7 1AA, England.

2Senior Lecturer in Biology and Anatomy, Departmentof Biology, Guy's Hospital Medical School, London SEI9RT, England.

3Scientist, Division of Parasitology, National Institutefor Medical Research.

' MRC Senior Research Fellow, Department of Anatomy,Guy's Hospital Medical School.

Free and invading merozoites were fixed in 3%glutaraldehyde in 0.15 mol/litre phosphate-bufferedsaline (PBS), pH 7.4, for 2 h at 4°C, then washed inPBS and stored in 20% glycerol in PBS at 40C priorto freeze-fracturing. Infected and normal erythro-cytes were collected straight into 20% glycerol inPBS at 4°C, without prior fixation, and freeze-fractured 2 h later. Freeze-fracturing was performedin a Balzers BAF 300 apparatus using a doublereplica hinge device.

Replicas were examined in a Philips 300 electronmicroscope and photographic prints were preparedwith the white shadows pointing upwards. Theprints were all enlarged to a standard magnification.Estimates of intramembraneous particle numberswere made over areas of 1 /jm2 on the micrographs.Surface areas of normal erythrocytes and erythro-cytes infected with ring-stage parasites were cal-culated according to the method of Houchin et al.(3). Erythrocytes infected with trophozoites andschizonts were assumed to be spheres and theirsurface areas were calculated using the formula47rr2. This formula was also employed to calculatethe surface area of the parasite/vacuole complex.

RESULTS

The merozoiteThe free merozoite has been shown to have a

typical outer plasma membrane, beneath which lieintermediate and inner pellicular membranes (1, 4).The two fracture faces of each of these membraneswere revealed by freeze-fracturing and designated Por E faces (Fig. 1), according to the nomenclatureof Branton et al. (5). Since the two pellicular mem-

3597 199-

D. J. MCLAREN ET AL.

fracture plane

E XTE R IOR

outer plasma membrane

pellicular cytoplasm

intermediatepellicular membrag, e

innerpellicular membrane

lit 1II'HINIII 111IlIIl1l1l IIII!lliIIfi

CYTOPLASM

Fig. 1. Diagram showing the fracture faces revealed when the three surface membranes of a P. knowlesimerozoite are freeze-fractured.

branes are closely apposed and line a potentialcisterna, the two adjacent leaflets lining this cisternawere designated E faces. The intramembraneousparticle counts for each face are presented as ratiosin Table 1.

Table 1. Ratios of intramembraneous particle countsrecorded for the six fracture faces of the merozoitesurface membranes

Outer plasma Intermediate Inner pellicularmembrane pellicular membrane membrane

E face P face P face E face E face P face

5 55 46 1 1 13

The particles on the P face of the intermediatepellicular membrane were very small, whereasthose on all other faces were larger and of the same

order of size. The E faces of the intermediatepellicular membrane and the inner pellicular mem-

brane, which had the smallest number of particles,were both pitted.

InvasionThere was no apparent aggregation or loss of

particles on either the erythrocyte plasma membraneor the merozoite plasma membrane at the time ofcontact, or during the initial stages of invasion.However, by the time the merozoite had completed

invasion, there was a remarkable depletion ofparticles from both faces of the vacuolar membrane.

Normal and infected erythrocytesThe intramembraneous particles on both the P

and E faces of the normal erythrocyte plasma mem-brane showed a random distribution (Fig. 2a).Fig. 3 illustrates the fracture faces revealed byfreeze-fracturing an infected erythrocyte. Particlecounts made on the plasma membranes of erythro-cytes infected with ring stages and trophozoites didnot differ significantly from the counts obtainedfrom normal erythrocytes. In schizont-infectederythrocytes, there appeared to be a 20% reductionin particle density on the P face of the plasmamembrane, and the remaining particles becameclumped in a reticulate pattern (Fig. 2b). Using thetechniques described herein, we found no apparentchange in the surface area of the infected erythrocytethroughout parasite development.The parasitophorous vacuoleWhen the parasite reached the young ring stage,

significant numbers of intramembraneous particleswere again visible on the vacuolar membrane. The Pand E faces of the vacuolar membrane had 10%and 18 %, respectively, of the particles present in thetwo faces of the erythrocyte plasma membrane.These figures did not change appreciably as theparasite developed through to the schizont stage.The surface area of the vacuole increased, however,from about 13 ,Um2 at the young ring stage to about134 ,Um2 at the schizont stage.

200

FREEZE-FRACTURE STUDY OF PARASITE-ERYTHROCYTE RELATIONSHIPS

Fig. 2. (a) Replica of the P fracture face of the plasma membrane of an uninfected erythrocyte. (b) Replica of theP fracture face of the plasma membrane of a schizont-infected erythrocyte. (c) Replica of the P fracture face of theouter membrane of a P. knowlesi trophozoite.

EXTERIORfracture

plane.

6P faceJ#Z.E

-face P face 71tfi

'IaIEcec

erythrocyt eplasma membrane

hasmoglobin

vacu olar membrane

parasiteplasma membrane

Fig. 3. Diagram showing the fracture faces revealed when a P. knowlesi-infected erythrocyteis freeze-fractured.

201

D. J. MCLAREN ET AL.

The intraerythrocytic parasiteThe P and E faces of the original merozoite

plasma membrane could be identified in subsequenterythrocytic stages, although the P-face particlesbecame arranged in a reticulate pattern after penetra-tion (Fig. 2c). Particle counts increased on both facesof the parasite membrane as development proceeded.

DISCUSSION

The intramembraneous particles seen in freeze-fracture replicas represent intercalated proteins inthe lipid bilayer. In the erythrocyte, these proteinsare known to contain transmembrane integralglycoproteins (6). The 20% reduction in particledensity recorded for the P face of the plasma mem-brane of the schizont-infected erythrocyte can becorrelated with a report that schizont-infectederythrocytes have only half the number of con-canavalin A receptors present on the normalerythrocyte (Shakespeare et al., in preparation).Concanavalin A receptors are known to be associatedwith membrane intercalated particles (7). In sec-tioned specimens, the plasma membrane of theschizont-infected erythrocyte is extremely dense andthickened (D. J. McLaren, unpublished observa-tions, 1975). This morphological change, coupledwith the reduced level of integral protein in themembrane, may possibly account for the increasedosmotic fragility of the infected erythrocyte recordedby Herman (8). The fact that the P-face particles ofthe infected erythrocyte membrane become clumpedinto a reticulate pattern suggests that spectrin, aprotein network that controls the glycoproteincomplexes of the erythrocyte membrane and preventslateral movement of proteins, may have beenadversely affected by the parasite.With regard to the parasite membrane it is

interesting that, although the P-face particles becomearranged in a reticulate pattern, this does not seemto involve particle clumping. Mazzen et al. (9)have reported a similar configuration of P-faceparticles in the plasma membrane of intraerythrocytic

stages of P. chabaudi. The fact that the intra-erythrocytic parasite is able to synthesize newtransmembrane proteins is shown by the observa-tion that both the surface area of the parasite andthe particle counts. for the parasite membraneincrease during parasite development.The membrane lining the parasitophorous vacuole

is perhaps the most interesting, but also poses themost difficult problems of interpretation. When themerozoite first makes contact with the erythrocyteit induces the host cell to form an invagination (2,10). At this time, the invaginated erythrocytemembrane still has its full complement of intra-membraneous particles. However, when the mero-zoite has moved into and expanded the invaginationto form a parasitophorous vacuole, the vacuolarmembrane is almost devoid of particles. A reducedlevel of particles in the vacuolar membrane sur-rounding plasmodia have been reported previously(11, 12). Bannister et al. (13) have proposed thatmerozoite rhoptry material may, in aqueous sur-roundings, form lamellae, perhaps analogous toliposomes, that could be incorporated into theerythrocyte surface. This proposal might wellaccount for the particle-free appearance of thevacuolar membrane at invasion. As the parasitedevelops to the young ring stage, however, particlesdo appear on both faces of the vacuolar membrane,and although the particle count for each face remainsfairly constant throughout parasite development, thesurface area of the vacuolar membrane increases byabout 1500%. It is clear, therefore, that there is anincrease in the total number of intercalated proteinsin the vacuolar membrane. If we assume that theseproteins are not synthesized by the erythrocyte, wecan consider two alternative sources: (a) that theindividual components of the transmembrane pro-teins become expressed as separate entities, althoughparticle sizes do not indicate that this is the case;and (b) that the additional transmembrane proteinsare of parasite origin. If the latter is correct, then thevacuolar membrane may be modified by and inpart derived from the parasite itself.

UMt

ETUDE PAR CRYOFRACTURATION DES INTERACTIONS PARASITE/ERYTHROCYTEDANS LES INFECTIONS A PLASMODIUM KNOWLESI

On a procede 'a des etudes de cryofracturation sur les On constate que des proteines transmembranaires deparasites et les erythrocytes lors d'infections a P. knowlesi. structure sont soustraites a la membrane plasmique de

202

FREEZE-FRACTURE STUDY OF PARASITE-ERYTHROCYTE RELATIONSHIPS 203

1'erythrocyte infecte par le schizonte et que le parasiteintra-erythrocytaire synthetise de nouvelles proteinestransmembranaires au fur et a mesure de son ddveloppe-ment. La formation de la vacuole parasitophore s'accom-

pagne de modifications dans le nombre des proteines destructure de la membrane vacuolaire ce qui montre quecette membrane peut etre modifiee par le parasite et pro-venir en partie de celui-ci.

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1. DENNIS, E. D. ET AL. Parasitology, 71: 475-481(1975).

2. BANNISTER, L. H. ET AL. Parasitology, 71: 483-491(1975).

3. HoUCHIN, D. N. ET AL. Blood, 13: 1185-1191 (1958).4. AIKAWA, M. & STERLING, C. R. Intracellular

parasitic protozoa. New York, Academic Press, 1974.

5. BRANTON, D. ET AL. Science, 190: 54-56 (1975).6. NICHOLSON, G. L. Biochimica et biophysica acta,

475: 57-108 (1976).7. PINTO DA SILVA, P. & NICHOLSON, G. L. Biochimica

et biophysica acta, 363: 311-319 (1974).

8. HERMAN, R. Journal of parasitology, 55: 626-632(1969).

9. MAZZEN, L. ET AL. Journal ofprotozoology, 22: 55A(1975).

10. LADDA, R. L. ET AL. Journal of parasitology, 65:633-644 (1969).

11. LADDA, R. L. & STEERE, R. L. In: 27th AnnualProceedings of the Electron Microscopical Society ofAmerica, 1969, pp. 396-397.

12. MEZOELY, C. A. M. ET AL. Proceedings of theHelminthological Society of Washington, 39: 149-173(1972).

13. BANNISTER, L. H. ET AL. Journal of protozoology,(1977) (in press).