Electron Microscopy of Knobs in Plasmodium falciparum-Infected Erythrocytes

Abstract

Erythrocytes infected by certain species of malarial parasites show two types of alterations of erythrocyte membranes such as electron-dense protrusions called knobs and caveola-vesicle complexes (Aikawa et al., 1975, Am. J. Path. 79: 285; Aikawa, 1977, Bull. WHO 55:2). The knobs are found on the membrane of erythrocytes infected with falciparum-type malaria parasites. These knobs form focal junctions with the endothelial cell membranes or with the knobs of other erythrocytes, resulting in the sequestration of these infected erythrocytes along the vascular endothelium (Trager et al., 1966, Bull. WHO 35: 883; Luse and Miller, 1971, Am. J. Trop. Med. Hyg. 20: 655; Aikawa et al., 1972, Z. Zellforsch. 124: 72; Udeinya et al., 1981, Science 213: 555). The erythrocyte membrane covering the knobs is immunologically different from the rest of the erythrocyte membrane (Kilejian et al., 1977, Exp. Parasitol. 42: 157; Langreth et al., 1979, J. Exp. Med. 150:1241; Chulay et al., 1981, Am. J. Trop. Med. Hyg. 30: 12). The erythrocytes infected with small trophozoites show few knobs, but they increase in number as the parasite grows within the erythrocyte. Because the morphology of these knobs is still not fully understood, we have attempted to characterize them by transmission and scanning electron microscopy together with freeze-fracture and etch techniques. Plasmodiumfalciparum (Camp strain) in Aotus erythrocytes and P. falciparum (Thailand and Liberian strains) in human erythrocytes were obtained from culture (Udeinya et al., 1981, Science 213: 555). The infected erythrocytes were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 4% sucrose, washed in 0.1 M cacodylate buffer and postfixed for 1 hr in 1% osmium tetroxide. After fixation, the samples were centrifuged and then processed for thin section transmission and scanning electron microscopy. Freeze-fracturing and etching were performed on P. falciparum in human RBC's. Fracturing was carried out at -120 C under a vacuum of 2 X 10-7 Torr. Etching was performed at -100 C, under a vacuum of 2 X 10-7 Torr for 2 min. The surfaces obtained were replicated with platinum and carbon. Replicas were cleaned as described previously (Aikawa et al., 1981, J. Cell Biol. 91: 55). Transmission electron microscopy was performed with a JEOL 100CX electron microscope. Scanning electron microscopy was performed with a JEOL 100CX electron microscope with a scanning unit at 20 kV. Thin-section electron microscopy showed many knobs on the erythrocytes infected with P. falciparum (Fig. 1). Each knob is a cone-shaped, electron-dense structure measuring 30 to 40 nm in height and 9 to 100 nm in width and is covered by the erythrocyte membrane. The base is not sharply demarcated, but gradually merges into the erythrocyte cytoplasm. Scanning electron microscopy showed numerous cone-shaped knobs evenly distributed over the entire erythrocyte surface (Fig. 2). The distribution pattern of the knobs was more easily detectable by scanning electron microscopy than by thin-section transmission electron microscopy. Therefore, scanning electron microscopy appears to be a useful and easy method for the study of knob distribution. Freeze-fracture and etching demonstrated that the knobs were protruded structures and were evenly distributed over the erythrocyte membrane. The P face of the infected, human RBC membrane showed evenly distributed intramembrane particles (IMP) and no aggregation or depletion of IMP could be observed over the erythrocyte membrane covering the knobs (Fig.