Quantitative proteomic analysis and functional characterization of Acanthamoeba castellanii exosome-like vesicles

Background: Pathogenic protozoans use extracellular vesicles (EVs) for intercellular communication and host manipulation. Acanthamoeba castellanii is a free-living protozoan that may cause severe keratitis and fatal granulomatous encephalitis. Although several secreted molecules have been shown to play crucial roles in the pathogenesis of Acanthamoeba, the functions and components of parasite-derived EVs are far from understood. Methods: Purified EVs from A. castellanii were confirmed by electron microscopy and nanoparticle tracking analysis. The functional roles of parasite-derived EVs in the cytotoxicity to and immune response of host cells were examined. The protein composition in EVs from A. castellanii was identified and quantified by LC-MS/MS analysis. Results: EVs from A. castellanii fused with rat glioma C6 cells. The parasite-derived EVs induced an immune response from human THP-1 cells and a cytotoxic effect in C6 cells. Quantitative proteomic analysis identified a total of 130 proteins in EVs. Among the identified proteins, hydrolases (50.2%) and oxidoreductases (31.7%) were the largest protein families in EVs. Furthermore, aminopeptidase activities were confirmed in EVs from A. castellanii. Conclusions: The proteomic profiling and functional characterization of EVs from A. castellanii provide an in-depth understanding of the molecules packaged into EVs and their potential mechanisms mediating the pathogenesis of this parasite.