Encystment of Acanthamoeba is Associated with the Reduction in the Expression of the Mannose–Binding Protein

Abstract

Abstract: : Purpose: The mannose–binding protein (MBP) expressed in the cell membrane of Acanthamoeba trophozoites is thought to play a key role in the pathogenesis of Acanthamoeba keratitis by mediating the adhesion of parasites to host cells. Encystment leads Acanthamoeba to a dormant, non–pathogenic state. The goal of the present study was to compare the expression pattern of the MBP in Acanthamoeba trophozoites and cysts. Methods: Acanthamoeba parasites (A. castellanii MEEI, 0184) were cultured in PYG media (>95% trophozoites). Encystment (>95% cysts) was induced by incubating trophozoites in encystment media (95–mM NaCl, 5–mM KCl, 8–mM MgSO₄, 0.4–mM CaCl₂, 1–mM NaHCO₃, 20–mM Tris–HCl, pH 9.0). Acanthamoeba binding to Man–BSA and corneal epithelial cells, as well as determination of cytopathic effect (CPE) were assessed as described in our published study (J. Biol. Chem., Vol. 273, 15838–15845). The amoeba MBP was isolated by affinity chromatography and was used to prepare polyclonal antibodies in chicken (anti–MBP IgY). Trophozoites and cysts were immunostained in cell suspension using anti–MBP IgY (1x10⁶ cells in 1 ml of PBS, antibody dilution 1:100) and FITC–labeled goat anti–chicken IgY (antibody dilution 1:250). After immunostaining, the cells were smeared onto glass slides, fixed with 2.5% paraformaldehyde and were visualized under the light and confocal microscope. For electron microscopy, the cells were treated as described above except that gold–conjugated donkey anti–chicken IgY (antibody dilution 1:100) was used as secondary antibody, then cells pellets were fixed and cut in thin sections. Results: Acanthamoeba trophozoites bound to Man–BSA and to corneal epithelial cells, and produced potent CPE in a α–mannose inhibitable manner. Anti–MBP IgY inhibited the adhesion of parasites both to Man–BSA and to host cells and also the amoeba–induced CPE. In immunocytochemistry experiments, cell membranes of trophozoites stained intensely with anti–MBP. In contrast, the antibody did not react with the amoeba cysts. Electron microscopy confirmed that MBP is expressed in the cell membrane of Acanthamoeba trophozoites. Conclusions: Acanthamoeba–MBP is expressed in the cell membrane of Acanthamoeba trophozoites but is not detected in Acanthamoeba cysts. These studies support our hypothesis that the expression of MBP is a main factor conferring the pathogenicity to Acanthamoeba trophozoites.

Keywords: Acanthamoeba • keratitis • microscopy: light/fluorescence/immunohistochemistry