Gene expression of corneal cells submitted to Acanthamoeba infection

Abstract

Infections caused by virulent strains of Acanthamoeba spp. are commonly related to the properties of invasion and destruction of the host tissue. Maintaining the structural stability of the corneal epithelium is associated with cell adhesion processes-cell and cell-substrate, each cell type has specific junctional structures in accordance with the position and function of the epithelial layer. The processes of cell adhesion and maintenance of the corneal epithelium are associated to the basement membrane, comprising different extracellular matrix components such as collagen type IV and VII, laminin, fibronectin and proteoglycans (perlecam). In amoebic keratitis Acanthamoeba trophozoites adhere to the surface of the epithelium, previously damaged, and invade the stroma, degrading it mainly by the secretion of proteolytic enzymes. For this reason, the process of stromal invasion by the parasite involves, besides the tissue cell death, degradation of components of the extracellular matrix (ECM) components which are responsible for maintaining the integrity of the various layers of the cornea, including the basement membrane. Despite the different pathogenesis of Acanthamoeba keratitis in the microbial etiology, mechanisms protozoan cell interaction with the specialized cells of the cornea, for the functional role of proteases in the synthesis of extracellular matrix components and tissue healing process by host cells remain little known. In this context, this research project proposes to (i) study the functional role of extracellular proteolytic enzymes of Acanthamoeba spp. in the synthesis of extracellular matrix components by specialized cells of the cornea; (ii) to study the functional role of extracellular proteolytic enzymes of Acanthamoeba spp. in the synthesis of components necessary adherence to the process of cell-cell adhesion in the epithelium of the cornea; (iii) studying the functional role of extracellular proteolytic enzymes from Acanthamoeba in the synthesis of proteins associated with wound healing and tissue regeneration by specialized corneal cells, and (iv) process to evaluate the effect of enzyme inhibitors on gene expression in cells associated with cornea processes of synthesis of extracellular matrix, cell adhesion and tissue healing. The results of this study provide the basis for comprehensive investigation of the mediators of virulence expressed by the protozoa in three separate events, but interconnected in corneal cells of the host. At first, the effect of the degradation of components secretome accession present in the basal membrane and extracellular matrix synthesis of corneal epithelial cells will be investigated. Moreover, the degradation profile of the components involved in tissue regeneration and healing for different secretomes Acanthamoeba spp. be evaluated. Thus, the direct effect (interaction trophozoite - corneal cell) or indirect (enzyme interaction - corneal cell) amebic of extracellular proteases in expression of genes encoding the synthesis of extracellular matrix components, cell adhesion, inflammatory cytokines and chemokines factor growth and signal transduction in specific cell culture of human cornea, will be studied by PCR in real time. Detailed research into the molecular mechanisms by which the secretome of Acanthamoeba spp. acts in the degradation of structural components of the corneal epithelium can bring a breakthrough in the understanding of the process of virulence of the protozoan, profiles healing and tissue regeneration across the different secretomes examined and the severity of the disease observed in patients, and generate a model of in vitro enzymatic inactivation of the parasite secretome in corneal cells, in order to open scaling perspectives for further in vivo assays. (AU)