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2008 OMIG, Abstract 18

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Zymographic profiles of Acanthamoeba strains isolated from human corneal tissue
Denise de Freitas1, Fábio Ramos de Souza Carvalho1, Linda Christian Carrijo Carvalho2, Ana Marisa Chudzinsky-Tavassi2, Annette Silva Foronda1
1. Federal University of Sao Paulo, Department of Ophthalmology; 2. Butantan Institute, Laboratory of Biochemistry and Biophysics

Purpose:  Proteases represent a class of enzymes which are related with pathogenicity and cytolysis of Acanthamoeba species and genotypes. The proteases of secreted and crude extracts of Acanthamoeba pathogenic strains were studied and compared them with proteases from non-pathogenic A. castellanii strain.
Methods: The research was conducted in accordance with the tenets of the Declaration of Helsinki. Approval of the study was obtained from the local institutional review boards. Clinical isolates of Acanthamoeba were obtained from corneal scraping of different patients, while non-pathogenic A. castellanii strain was obtained from American Type Culture Collection (ATCC 30011). All amoebae were grown without shaking, at room temperature, in 5 ml of Neff medium for 72 hours. After three days, the supernatant fluid was collected, filtered and dialyzed for 48 hours against Sodium Chloride buffer. Protein concentration was quantified by the method of Bradford. Protease activities were analyzed after performing electrophoresis of crude extract protein and conditioned medium in 10% polyacrylamide gels, containing gelatin as copolymerized substrate.
Results: Our results showed that Acanthamoeba trophozoites produce distinct extracellular proteases. Protease activities showed to be different among Acanthamoeba strains sourced from keratitis patients.
Conclusions:Heterogeneous extracellular proteases are produced by different Acanthamoeba isolates and corroborated with other studies which showed that this class of enzymes could be used as a virulence marker in the differentiation of Acanthamoeba species and genotypes

Disclosure Code: N


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