Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Instituto René Rachou, FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.
Cell Biol Int. 2021 May;45(5):1060-1071. doi: 10.1002/cbin.11551. Epub 2021 Feb 4.
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.
自由生活的棘阿米巴属原生动物是机会主义原生动物，会引起人类角膜、全身和脑部感染。大多数种内变异和致病性的机制尚不清楚。最近，棘阿米巴释放细胞外囊泡（EVs）的现象被报道。然而，不同菌株 EVs 的比较特征还没有报道。本研究旨在评估不同基因型的棘阿米巴产生的 EVs，比较它们的蛋白酶谱和免疫调节特性。通过超速离心从 4 株环境或临床分离株（基因型 T1、T2、T4 和 T11）中获得 EVs，通过纳米颗粒跟踪分析定量，并通过扫描和透射电子显微镜进行分析。通过酶谱法测定蛋白酶谱，通过腹腔巨噬细胞刺激测定 EVs 的功能特性（亚硝酸盐和细胞因子产生的测量）。尽管基因型不同，但所有菌株都释放了 EVs，并且在大小和/或浓度方面没有差异。EVs 表现出丝氨酸蛋白酶的主要活性（pH7.4 和 3.5），在 T4 和 T1 菌株中活性更高。来自环境、非致病性 T11 株的 EVs 表现出更具促炎特性，通过 TLR4/TLR2 诱导更高水平的亚硝酸盐、肿瘤坏死因子α和白细胞介素-6，比具有致病特性的菌株（T4、T1 和 T2）更高。用蛋白酶抑制剂预处理或加热处理的 EVs 可大大降低巨噬细胞中亚硝酸盐浓度的产生。这些数据表明，EVs 的免疫调节作用可能反映了它们的致病潜力，这取决于棘阿米巴菌株，并依赖于蛋白酶的完整性。
