PURPOSE: To determine whether the expression of Acanthamoeba mannose-binding protein (MBP) is associated with the pathogenicity of the parasite in vitro. METHODS: Both active trophozoites and dormant cysts of a pathogenic strain of A. castellanii were analyzed for their ability to bind to corneal epithelium, express MBP, and produce a cytopathic effect (CPE) on host cells. In addition, host cell binding, CPE-inducing ability, and MBP expression pattern of trophozoites of four different isolates of Acanthamoeba with various degrees of in vitro pathogenicity were analyzed. Binding assays were performed with radiolabeled parasites; CPE assays were performed with rabbit corneal epithelial cells as host cells; and the expression of MBP was detected by affinity chromatography of parasite extracts on mannose affinity columns and by immunohistochemical and Western blot analyses. RESULTS: Trophozoites of A. castellanii bound avidly to corneal epithelial cells in a mannose-inhibitable manner, whereas cysts exhibited little binding. The lack of binding of the cysts to host cells was associated with the downregulation of MBP, along with the concomitant loss of CPE. Analysis of trophozoites of five different species of Acanthamoeba exhibiting various degrees of pathogenic potential revealed that the ability of parasites to bind to host cells and produce CPE is directly correlated with the expression of the MBP. Acanthamoeba strains that bound avidly to host cells and produced potent CPE, robustly expressed MBP. In contrast, parasite strains that produced only weak CPE, expressed markedly reduced levels of MBP. CONCLUSIONS: The data demonstrating that the pathogenic potential of Acanthamoeba directly correlates with the expression level of the MBP in conjunction with our published studies showing that Acanthamoeba MBP is a major virulence protein suggest that the amoeba lectin has the potential to serve as a marker of pathogenicity.
PURPOSE: To determine whether the expression of Acanthamoebamannose-binding protein (MBP) is associated with the pathogenicity of the parasite in vitro. METHODS: Both active trophozoites and dormant cysts of a pathogenic strain of A. castellanii were analyzed for their ability to bind to corneal epithelium, express MBP, and produce a cytopathic effect (CPE) on host cells. In addition, host cell binding, CPE-inducing ability, and MBP expression pattern of trophozoites of four different isolates of Acanthamoeba with various degrees of in vitro pathogenicity were analyzed. Binding assays were performed with radiolabeled parasites; CPE assays were performed with rabbit corneal epithelial cells as host cells; and the expression of MBP was detected by affinity chromatography of parasite extracts on mannose affinity columns and by immunohistochemical and Western blot analyses. RESULTS: Trophozoites of A. castellanii bound avidly to corneal epithelial cells in a mannose-inhibitable manner, whereas cysts exhibited little binding. The lack of binding of the cysts to host cells was associated with the downregulation of MBP, along with the concomitant loss of CPE. Analysis of trophozoites of five different species of Acanthamoeba exhibiting various degrees of pathogenic potential revealed that the ability of parasites to bind to host cells and produce CPE is directly correlated with the expression of the MBP. Acanthamoeba strains that bound avidly to host cells and produced potent CPE, robustly expressed MBP. In contrast, parasite strains that produced only weak CPE, expressed markedly reduced levels of MBP. CONCLUSIONS: The data demonstrating that the pathogenic potential of Acanthamoeba directly correlates with the expression level of the MBP in conjunction with our published studies showing that Acanthamoeba MBP is a major virulence protein suggest that the amoeba lectin has the potential to serve as a marker of pathogenicity.