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D. Freitas, F. R. S. Carvalho, L. C. Carrijo-Carvalho, A. Chudzinski-Tavassi, A. S. Foronda; Serine Peptidases as Major Virulence Factors of Acanthamoeba Trophozoites Isolated From Corneal Surface. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3911.
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Corneal infections, designated as keratitis, due to free-living amoebae have potentially devastating consequences. In general, Acanthamoeba spp is the main etiological agent of amoebic keratitis. Extracellular proteases are the major virulence factors, which are commonly involved in invasion of corneal tissue by trophozoites. The proteases of secreted and crude extracts of Acanthamoeba pathogenic strains were studied and partially characterized by protease inhibition assays.
The research was conducted in accordance with the tenets of the Declaration of Helsinki. Appropriate institutional review board approval was obtained, and informed consent obtained from all patients. Clinical isolates of Acanthamoeba spp (n = 8) were obtained from corneal scraping of different patients, while non-pathogenic A. castellanii strain was obtained from American Type Culture Collection (ATCC 30011) was used as a control of the experiments. Acanthamoeba trophozoites forms were used for all assays. After three days, Acanthamoeba cell monolayer was observed in culture flasks and the supernatant fluid was collected, filtered and dialyzed for 48 hours against saline. Protease activities were analyzed after performing electrophoresis of crude protein extracts in polyacrylamide gels, containing gelatin as copolymerized substrate. Protease inhibition assays were done using both serine and metalloproteinase inhibitors.
Acanthamoeba isolates produce distinct enzymatic profiles with different molecular-weight mass, with emphasis in the occurrence of low-molecular weight enzymes. The activity of extracellular peptidases secreted by all Acanthamoeba pathogenic isolates was blocked by serine protease inhibitor while metalloproteinase inhibitor did not affect proteolytic activity.
Acanthamoeba trophozoites were isolated from patients with acute amoebal keratitis. All clinical isolates investigated were able to secrete proteolytic enzymes of serine peptidase class. Our findings corroborated with other studies, thus suggesting this class of enzymes as a major virulence marker in the differentiation of Acanthamoeba species and genotypes.
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