ABSTRACT
Free living amoeba of the genus Acanthamoeba are opportunist protozoan involved in corneal, systemic, and encephalic infections in humans. Most of the mechanisms underlying intraspecies variations and pathogenicity are still unknown. Recently, the release of extracellular vesicles (EVs) by Acanthamoeba was reported. However, comparative characterization of EVs from distinct strains is not available. The aim of this study was to evaluate EVs produced by Acanthamoeba from different genotypes, comparing their proteases profile and immunomodulatory properties. EVs from four environmental or clinical strains (genotypes T1, T2, T4, and T11) were obtained by ultracentrifugation, quantitated by nanoparticle tracking analysis and analyzed by scanning and transmission electron microscopy. Proteases profile was determined by zymography and functional properties of EVs (measure of nitrite and cytokine production) were determined after peritoneal macrophage stimulation. Despite their genotype, all strains released EVs and no differences in size and/or concentration were detected. EVs exhibited a predominant activity of serine proteases (pH 7.4 and 3.5), with higher intensity in T4 and T1 strains. EVs from the environmental, nonpathogenic T11 strain exhibited a more proinflammatory profile, inducing higher levels of Nitrite, tumor necrosis factor alpha and interleukin-6 via TLR4/TLR2 than those strains with pathogenic traits (T4, T1, and T2). Preincubation with EVs treated with protease inhibitors or heating drastically decreased nitrite concentration production in macrophages. Those data suggest that immunomodulatory effects of EVs may reflect their pathogenic potential depending on the Acanthamoeba strains and are dependent on protease integrity.
Subject(s)
Acanthamoeba/genetics , Acanthamoeba/metabolism , Extracellular Vesicles/immunology , Acanthamoeba/classification , Animals , Extracellular Vesicles/physiology , Female , Genotype , Immunologic Factors/immunology , Immunologic Factors/physiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Polymerase Chain Reaction , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolismABSTRACT
Free-living amoebae of the genus Acanthamoeba are causative agents of Acanthamoeba keratitis and amoebic encephalitis in humans, both of which are serious infections. The ability to produce proteases is one of the factors involved in the pathogenesis of Acanthamoeba infections. The aim of this study was to evaluate the secreted proteases of six Acanthamoeba strains from distinct genotypes (T1, T2, T4 and T11) maintained in prolonged axenic culture and following three successive passages in Madin-Darby Canine Kidney (MDCK) cells. Conditioned medium was obtained from cultures before and after interaction with the MDCK monolayers, resolved in SDS-PAGE containing gelatine, then subjected to quantitative azocasein assays. Zymography profiles varied between the strains, with the predominant proteases found to be serine-type proteases from 49 to 128 kDa. A T1 genotype strain isolated from dust showed quantitatively higher protease secretion compared to the other strains. No changes were detected in the zymography profiles of MDCK-interacted cultures compared to long-term axenic cultures. Two strains presented lower proteolytic activity post-MDCK interaction, while the remaining strains presented similar values before and after MDCK passages. In conclusion, this study confirms the predominance of serine-type protease secretion by Acanthamoeba, with distinct profiles presented by the different strains and genotypes studied. Also, interaction of trophozoites with MDCK cells did not alter the zymography pattern.
