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Proteolytic enzymes toxicity of Acanthamoeba spp.: an in vitro study

Grant number: 15/06852-4
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): June 01, 2015
Effective date (End): May 31, 2016
Field of knowledge:Health Sciences - Collective Health - Public Health
Principal Investigator:Fábio Ramos de Souza Carvalho
Grantee:Guilherme Moraes de Moura do Val
Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
Associated research grant:11/51626-1 - Acanthamoeba spp. toxins as virulence factors in infections of the ocular surface, AP.JP


Infections of the corneal surface by free-living amoebae of Acanthamoeba genus, amoebic keratitis, are characterized by acute pain and disability of the patient's visual acuity. Adhesion of trophozoites to epithelial cells of the cornea following injury and tissue invasion processes are important in the establishment of the infection. The damages to the corneal tissue cells and by amoebae are generally associated with the processes of phagocytosis and the secretion of enzymes. In a recent study, our research group has shown great diversity of proteolytic profiles associated with different clinical isolates of the protozoan, suggesting a specific and differential pattern of virulence for each clinical isolate. However, the pathogenic effect of these proteases in cell models of the cornea is unknown. Based on the hypothesis that proteases produced by Acanthamoeba spp not only can play a role in the pathogenesis of the disease and cytopathogenicity of corneal epithelial cells, but can also be applied as molecular markers for differentiation of pathogenic and non-pathogenic isolates, we propose the development of this study hoping to achieve the following objectives: (I) to study the effect of chemotaxis, from the direct interaction parasite-host of trophozoites from different clinical isolates of Acanthamoeba spp in corneal cell viability; (II) to evaluate the cytopathogenicity of different Acanthamoeba protease profiles in corneal cells; (III) to study the effect of Acanthamoeba spp proteases from parasite-host interaction indirectly, in corneal cells, and (IV) to evaluate quantitatively the effect of dose / response concentration of Acanthamoeba protease on the viability of different cell lineages of the human cornea. Results are expected to assist in the understanding of different aspects related to the genesis of amoebic keratitis, such as different virulence expressed by clinical isolates of Acanthamoeba spp in cell lineages of the human cornea; the direct and indirect effects of proteolytic enzymes secreted by the protozoan in the structure and specialized cells of the cornea function and finally knowing the spectrum of action of the enzymes secreted by the parasite in vitro, the results from this study may open promising perspectives on the development and application of chemical compounds and / or specifically targeted for biological inhibitory activity of extracellular proteases from Acanthamoeba, minimizing possible toxic effects on the cells of the cornea.

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