Comparative analysis of different extracellular vesicles secreted by Echinococcus granulosus protoscoleces

Extracellular vesicles (EVs) are heterogeneous populations of different membrane-wrapped vesicles in size and encapsulated cargo and have recently emerged as a crucial carrier with the functions in intercellular communication, being involved in host-parasite interactions. However, Echinococcus granulosus EVs are not fully described. To separate EVs with a different size, the culture supernatant of E. granulosus protoscoleces (PSCs) was sequentially centrifuged at 2,000g, 10,000g and 110,000g, and the resulting precipitates were accordingly named as 2K, 10K and 110K EVs, respectively. The size and morphology of three different EVs were identified using ZETASIZER NANO and transmission electron microscopy (TEM), respectively. Then mass spectrometry was applied to define protein cargo of EVs and EV internalization was assessed using fluorescent microscopy and flow cytometry. The results showed that 2K EVs mainly ranged from 450 to 950 nm in diameter, 10K EVs ranged from 220 to 390 nm and 110K EVs from 60 to 150 nm. A total of 901 EV proteins were identified, 328 of which were commonly found in the three types of EVs. GO analysis revealed that these proteins were mainly involved in binding (44%) and catalytic activity (44%). Three types of EVs were different in biomarkers (Enolase and 14-3-3) and in reactivity with anti-echinococcosis positive serum. Moreover, 110K EVs were more easily internalized by hepatic cells than 10K EVs as well as 2K EVs (p < 0.0001). These results reveal the physical and biological discrepancy among 2K, 10K and 110K EVs, suggesting a distinct role in host-parasite interactions.