Background
Extracellular vesicles (EVs) released by helminths play an important role in parasite-host communication. However, little is known about the characteristics and contents of the EVs of Fasciola gigantica, a parasitic flatworm that causes tropical fascioliasis. A better understanding of EVs released by F. gigantica will help elucidate the mechanism of F. gigantica-host interaction and facilitate the search for new vaccine candidates for the control and treatment of fascioliasis.
Conclusion
The results of this study suggest that proteins within the exosome-like vesicles of F. gigantica have immunomodulatory, immune evasion and virulence functions. This knowledge may lead to new strategies for immunotherapy, vaccination and the diagnosis of fascioliasis.
Methods
Two different populations of EVs (15k EVs and 100k EVs) were purified from adult F. gigantica culture media by ultracentrifugation. The morphology and size of the purified EVs were determined by transmission electron microscopy (TEM) and by the Zetasizer Nano ZSP high performance particle characterization system. With the aim of identifying diagnostic markers or potential vaccine candidates, proteins within the isolated 100k EVs were analyzed using mass spectrometry-based proteomics (LC-MS/MS). Mice were then vaccinated with excretory/secretory products (ESPs; depleted of EVs), 15k EVs, 100k EVs and recombinant F. gigantica heat shock protein 70 (rFg-HSP70) combined with alum adjuvant followed by challenge infection with F. gigantica metacercariae. Fluke recovery and antibody levels were used as measures of vaccine protection.
Results
TEM analysis and nanoparticle tracking analysis indicated the successful isolation of two subpopulations of EVs (15k EVs and 100k EVs) from adult F. gigantica culture supernatants using differential centrifugation. A total of 755 proteins were identified in the 100k EVs. Exosome biogenesis or vesicle trafficking proteins, ESCRT (endosomal sorting complex required for transport) pathway proteins and exosome markers, heat shock proteins and 14-3-3 proteins were identified in the 100k EVs. These results indicate that the isolated 100k EVs were exosome-like vesicles. The functions of the identified proteins may be associated with immune regulation, immune evasion and virulence. Mice immunized with F. gigantica ESPs, 15k EVs, 100k EVs and rFg-HSP70 exhibited a reduction in fluke burden of 67.90%, 60.38%, 37.73% and 56.6%, respectively, compared with the adjuvant control group. The vaccination of mice with F. gigantica 100k EVs, 15k EVs, ESP and rFg-HSP70 induced significant production of specific immunoglobulins in sera, namely IgG, IgG1 and IgG2a.