Invasion of erythrocytes by merozoites of the monkey malaria, Plasmodium knowlesi, was investigated by electron microscopy. The apical end of the merozoite makes initial contact with the erythrocyte, creating a small depression in the erythrocyte membrane. The area of the erythrocyte membrane to which the merozoite is attached becomes thickened and forms a junction with the plasma membrane of the merozoite. As the merozoite enters the invagination in the erythrocyte surface, the junction, which is in the form of a circumferential zone of attachment between the erythrocyte and merozoite, moves along the confronted membranes to maintain its position at the orifice of the invagination. When entry is completed, the orifice closes behind the parasite in the fashion of an iris diaphragm, and the junction becomes a part of the parasitophorous vacuole. The movement of the junction during invasion is an important component of the mechanism by which the merozoite enters the erythrocyte.The extracellular merozoite is covered with a prominent surface coat. During invasion, this coat appears to be absent from the portion of the merozoite within the erythrocyte invagination, but the density of the surface coat outside the invagination (beyond the junction) is unaltered. KEY WORDS malarial parasitesPlasmodium knowlesi erythrocyte junction invasion mechanism electron microscopy cell interactionThe asexual malaria parasite infects erythrocytes and there develops to a mature schizont that is made up of many individual merozoites. Upon rupture of the schizont-infected erythrocyte, the merozoites are released and are capable of infecting other erythrocytes. In 1969 Ladda et al. (15) reported on the invasion of erythrocytes by merozoites of Plasmodium berghei and P. gallinaceum, and established that merozoites enter within an invagination of the erythrocyte membrane rather than by penetrating it. They found that merozoites approached erythrocytes with the apical end and formed a focal depression on the erythrocyte membrane. With deeper invaginati0n of the erythrocyte membrane, the resulting cavity conformed to the shape of the merozoite. The orifice of the invaginated erythrocyte membrane fused upon completion of the entry. They also noted that granular material (a surface coat) covered the entire surface of the extracellular merozoite and was removed upon completion of the merozoite 72 J. CELL BIOLOGY 9 The Rockefeller University Press 9

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Invasion of erythrocytes by malarial merozoites requires the formation of a junction between the merozoite and the erythrocyte . Migration of the junction parallel to the long axis of the merozoite occurs during the entry of the merozoite into an invagination of the erythrocyte . Freeze-fracture shows a narrow circumferential band of rhomboidally arrayed particles on the P face of the erythrocyte membrane at the neck of the erythrocyte invagination and matching rhomboidally arrayed pits on the E face . The band corresponds to the junction between the erythrocyte and merozoite membranes observed in thin sections and may represent the anchorage sites of the contractile proteins within the erythrocyte. Intramembrane particles (IMP) on the P face of the erythrocyte membrane disappear beyond this junction . When the erythrocytes and cytochalasin B-treated merozoites are incubated together, the merozoite attaches to the erythrocyte membrane and a junction is formed between the two, but the invasion process does not advance further and no movement of the junction occurs . Although there is no entry of the parasite, the erythrocyte membrane still invaginates. Freezefracture shows that the P face of the invaginated erythrocyte membrane is almost devoid of the IMP that are found elsewhere on the membrane, suggesting that the attachment process in and of itself is sufficient to create a relatively IMP-free bilayer.Invasion of the erythrocytes by malarial merozoites requires the formation of a junction between the merozoite and the erythrocyte (1). Migration of the junction parallel to the long axis of the merozoite occurs during the entry of the merozoite into an invagination of the erythrocyte . As observed by thinsection electron microscopy, this junction is a region of close apposition between the merozoite and the erythrocyte, where the inner leaflet of the erythrocyte membrane appears thickened (1) . The invaginated erythrocyte membrane beyond the junction has been shown by freeze-fracture to be devoid of intramembrane particles (IMP) (11). Cytochalasin-treated merozoites that can attach to but not enter into the erythrocyte also form a junction between the apical end of the merozoite and the erythrocyte (12). Even though the cytochalasin-treated merozoite never enters the erythrocyte, membrane invaginations occur within the erythrocyte in the region of the attached parasite. To further characterize the junctions and the erythrocyte membrane invaginations, we have analyzed them by freeze-fracture during normal invasion and after attachment of cytochalasin-treated merozoites to the erythrocytes . 5). The merozoites were released into a protein-free medium containing medium RPMl 1640/15 mM HEPES/34 .5 mM RC03 gassed with 5% C02 in air. 1 ml of the merozoite suspension contained -5 x 10' merozoites . Forinvasion studies, 200 pl of heat-inactivated fetal calf serum and 100 pl of rhesus monkey erythrocytes (5 x W/ml) in medium 199/10% fetal calf serum were added to 2 ml of the merozoite suspension . The cell susp...

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The objectives of this study were to describe the ultrastructure of granulocyte-Schistosoma mansoni egg interaction and to determine the role of reduced oxygen products as effectors of cell-mediated damage to the parasite target. Granulocytes attached to the parasites and closely applied their plasma membranes to the microspicules of the egg shell 30 min after mixing in the presence of immune serum. By 4 h, the egg shell was fractured and granulocyte pseudopodia extended toward the underlying miracidium. Granulocyte attachment to eggs resulted in release of O-(030-0.52 nmol/min per 2 X 106 cells) and accumulation of H202 (0.14-0.15 nmol/min) in the presence of antibody or complement. Granulocytes reduced egg tricarboxylic-acid cycle activity and hatching by 283±0.9 and 35.2±2.8%, respectively (cell-egg ratio of 1,000: 1). Exogenous superoxide dismutase (10 M&g/ml) inhibited granulocyte toxicity for egg metabolic activity (3.0±2.1% reduction in acetate metabolism vs. 283±0.9% decrease in controls without superoxide dismutase, P < 0.0005) and hatching (12.5±1.8% reduction, P < 0.0005), whereas catalase and heparin had no effect. Inhibitors of myeloperoxidase (1 mM azide, cyanide, and methimazole) augmented granulocyte-mediated toxicity of egg tricarboxylic-acid cycle activity (44-58% reduction in activity vs. 31 and 35% reduction in controls), suggesting that H202 released from cells was degraded before reaching the target miracidium. Oxidants generated by acetaldehyde (2 mM)-xanthine oxidase (10 mU/ml) also decreased egg metabolic activity and hatching by 62.0±9.0 and 38.7±73%, respectively. Egg damage by the cell-free system was partially prevented by superoxide dismutase (26.5±4.2% reduction in egg tricarboxylic-acid cycle activity) and completely blocked by catalase (0% reduction in activity). These data suggest that granulocyte-mediated toxicity for S. mansoni eggs is dependent on release of 02 or related molecules. These oxygen products, unlike H202, may readily reach the target miracidium where they may be converted to H202 or other microbicidal effector molecules.

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