title: Cytotoxic activity and degradation patterns of structural proteins by corneal isolates of Acanthamoeba spp.
abstract: BACKGROUND
  Proteolytic enzymes secreted by trophozoites (amoebic secretome) are suggested as the main virulence factor involved in the severity of Acanthamoeba keratitis. The degradation profile of the main glycoprotein components of anterior and posterior portions of the cornea and the cytopathic effect of secretomes on endothelial cells by contact-independent mechanism were evaluated.
  
  
  METHODS
  Trophozoites were isolated primarily from corneal tissue samples (n = 11) and extracellular proteins were collected from axenic cell culture supernatants. The molecular weights of proteolytic enzymes were estimated by zymography. Enzymatic cleavage of laminin and fibronectin substrates by amoebic secretome was investigated and cluster analysis was applied to the proteolysis profiles. Primary cultures of endothelial cells were used in both qualitative and quantitative assays of cytophatogenicity.
  
  
  RESULTS
  Differential patterns of proteolysis were observed among the Acanthamoeba secretomes that were analysed. The uniformity of laminin degradation contrasted with the diversity of the proteolysis profiles observed in the fibronectin substrate. Acanthamoeba secretome extracted from four clinical isolates was shown to be toxic when in contact with the endothelial cell monolayer (p < 0.01). Induction of apoptosis and membrane permeability, at different percentual values, were suggested as the main mechanisms that could induce endothelial cell death when in contact with amoebic secretome.
  
  
  CONCLUSIONS
  Our results provide evidence that virulence factors secreted by Acanthamoeba trophozoites can be related to an increased pathogenicity pattern by an independent contact-trophozoite mechanism, through induction of endothelial cell death by apoptosis at a higher percentage than providing the lack of cell viability by the membrane-associated pore-forming toxin activity.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Paulista School of Medicine, Federal University of Sao Paulo, São Paulo, Brazil.
authors.forename: Viviane Peracini
authors.initials: VP
authors.lastname: Sant'ana
authors.forename: Linda Christian
authors.initials: LC
authors.lastname: Carrijo-Carvalho
authors.forename: Annette Silva
authors.initials: AS
authors.lastname: Foronda
authors.forename: Ana Marisa
authors.initials: AM
authors.lastname: Chudzinski-Tavassi
authors.forename: Denise
authors.initials: D
authors.lastname: de Freitas
authors.forename: Fábio Ramos Souza
authors.initials: FR
authors.lastname: de Carvalho
mesh_terms: D015823:Acanthamoeba Keratitis / Q000469:parasitology*; D048248:Acanthamoeba castellanii / Q000201:enzymology* / Q000302:isolation & purification / Q000472:pathogenicity; D017209:Apoptosis / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000469:parasitology; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007797:Laminin / Q000378:metabolism*; D059748:Proteolysis; D015800:Protozoan Proteins / Q000633:toxicity*; D057057:Serine Proteases / Q000633:toxicity*; D014404:Tubulin / Q000378:metabolism*; D037521:Virulence Factors
chemical_list: D005353:Fibronectins; D007797:Laminin; D015800:Protozoan Proteins; D014404:Tubulin; D037521:Virulence Factors; D057057:Serine Proteases