The avian malaria parasite Plasmodium gallinaceum causes marked structural changes on the surface of its host erythrocyte

Abstract

Using a combination of atomic force, scanning and transmission electron microscopy, we found that avian erythrocytes infected with the avian malaria parasite Plasmodium gallinaceum develop approximately 60 nm wide and approximately 430 nm long furrow-like structures on the surface. Furrows begin to appear during the early trophozoite stage of the parasite's development. They remain constant in size and density during the course of parasite maturation and are uniformly distributed in random orientations over the erythrocyte surface. In addition, the density of furrows is directly proportional to the number of parasites contained within the erythrocyte. These findings suggest that parasite-induced intraerythrocytic processes are involved in modifying the surface of host erythrocytes. These processes may be analogous to those of the human malaria parasite P. falciparum, which induces knob-like protrusions that mediate the pathogenic adherence of parasitized erythrocytes to microvessels. Although P. gallinaceum-infected erythrocytes do not seem to adhere to microvessels in the host chicken, the furrows might be involved in the pathogenesis of P. gallinaceum infections by some other mechanism involving host-pathogen interactions.

Fig. 1

Typical SEM images contrasting the surface topography of noninfected (a) and P. gallinaceum-infected erythrocytes (b, c). Noninfected erythrocytes have a smooth surface. In contrast, the furrow-like surface structures are seen on infected erythrocytes. Bars in (a) and (b) represent 1 μm, in (c) 200 nm.

Fig. 2

Composite bright field and epifluorescence microscopy images of (a) P. falciparum-infected erythrocytes, (b) P. gallinaceum-infected erythrocytes, and (c) P. gallinaceum-infected erythrocytes treated with DNase I prior to staining with YOYO-1. Bar in (a) represents 10μm.

Fig. 3

Representative example of P. gallinaceum-infected erythrocytes stained with Giemsa (a–d) or YOYO-1 following DAase I treatment (e–h). A typical early stage of development is shown in (a) and (e). As the parasite develops from the trophozoite stage (b, f) to the schizont stage (c, g), merozoite maturation occurs. Unstained erythrocyte nuclei enable us to unambiguously identify the developmental stage, number and position of the parasites as shown in (e–g). Merozoites are visible in the schizont stage of development. Mature gametocytes are shown in (d) and (h). Bar in (d) represents 5μm.

Fig. 4

AFM images of P. gallinaceum-infected erythrocytes at various stages of development. The insert shows the same cell imaged by combined LM and YOYO-1 fluorescence following DNase I treatment. The cell surface lesion resulting from merozoite invasion (arrows in (a–c)) persist throughout parasite development. Recently invaded erythrocyte (a); ring stage of the parasite with no apparent furrowing of the erythrocyte surface (not shown); early trophozoite stage showing marked furrow development (b); late trophozoite stage showing both furrows and erythrocyte deformation resulting from parasite growth (c); schizont stage showing furrows similar to those on trophozoite-infected erythrocytes (d); mature gametocyte stage showing both furrows and increased erythrocyte deformation (e); doubly infected erythrocyte showing markedly increased numbers of furrows on the surface (f). The bars in (a) represent 3μm for composite LM-fluorescence images and 1μm for AFM images.

Fig. 5

Graphical representation of (a) furrow density on P. gallinaceum-infected erythrocytes at various developmental stages as well as on doubly-infected erythrocytes at the trophozoite stage, and (b) height, width and length of furrows on P. gallinaceum-infected erythrocytes at the early trophozoite, late trophozoite and gametocyte stages of development. Data in the graph are expressed as the mean ± S.E.M. Note that the feature of furrows on the surface remains constant even at the gamete stage.

Fig. 6

TEM images of P. gallinaceum-infected erythrocytes. Noninfected (a) and trophozoite stage (b–d) of parasite development showing marked modification of the infected erythrocyte surface membrane (arrows in (b–d)). A food vacuole and pigment are also visible. Note that vesicles can be visualized within the infected erythrocyte cytosol (d). Bars represent 500 nm.