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2009 OMIG, Abstract 10

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Partial characterization of collagenolytic enzymes secreted by Acanthamoeba virulent strains.
F.R.S. Carvalho1, L.C. Carrijo-Carvalho2, A.M. Chudzinski-Tavassi2, A.S. Foronda1, Freitas1
1Federal University of Sao Paulo, Department of Ophthalmology;
Butantan Institute, Laboratory of Biochemistry and Biophysics.

Purpose: Corneal infections, designed 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. Collagenases are principal proteases secreted by Acanthamoeba trophozoites, which could be involved in the inflammatory cascade and stromal degradation processes during corneal infection by the protozoa.  The collagenases of secreted and crude extracts of Acanthamoeba virulent strains were studied and compared 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 (n = 11), 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. Collagenolytic activities were analyzed after performing electrophoresis of crude extract protein and conditioned medium in 10% polyacrylamide gels, containing collagen as copolymerized substrate.
Results: By in vitro experiments, our results showed the ability of Acanthamoeba strains isolated from six patients to degrade type I collagen, a major component of corneal stroma. In addition, collagenolytic activities showed to be different among Acanthamoeba strains studied.
Conclusions: Heterogeneous extracellular collagenases are produced by different Acanthamoeba isolates and corroborated with severity of infection observed in the patients previously. The degradation process of corneal stroma could be related with secreted collagenases by trophozoites and this class of proteases could be used as a virulence marker in the differentiation of Acanthamoeba species and genotypes.
Financial support: FAPESP (Grant 08/53969-0), CAPES (PNPD Scientific Program), CNPq.
Disclosure code: N

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