Extracellular vehicles (EVs) are nanospheres emitted by most prokaryotic and eukaryotic organisms into their surrounding environment. Extensive research has underscored the ability of bacterial EVs to function as molecular transporters and to modulate immune responses, thereby playing a critical role in host immune system regulation . This study aims to analyze the proteomic profile of EVs isolated from Edwardsiella piscicida (EpEVs) and investigate their immune responses.
The isolation of EpEVs was done by ultracentrifugation with transmission electron microscopy confirming their spherical morphology. The isolated EpEVs had an average size of 85.3 ± 1.8 nm and a zeta potential of -8.28 ± 0.41 mV. Proteomic analysis identified 1,487 proteins, with subcellular localization indicating that "cell" and "cell part" constituted 61.31% of the structural proteins. Key proteins associated with virulence, such as Type III secretion system components, flagellin, outer membrane proteins, and several chaperones involved in protein folding and stability, were also identified. Protein-protein interaction (PPI) predictions with E. tarda proteins revealed 673 interactions among the 1,487 identified EpEV proteins.
Exposure of fathead minnow (FHM) cells to Ep EVs up to 100 μg /mL resulted in cell proliferation without toxicity, while Raw 264.7 cells exhibited mild toxicity at 12.5 μg/mL. Fluorescent-labeled EpEVs demonstrated cellular internalization in FHM cells after 24 hours. In vitro gene expression analysis showed upregulation of interleukin (Il)6, IL1β, interferon (Ifn)β, and pro-inflammatory cytokines, along with simultaneous upregulation of the anti-inflammatory cytokine Il10. In vivo gene expression, except for heat shock protein (hsp )70, indicated upregulation of other genes, with a timely response suggesting EpEV-induced immune gene expression. Western blot analysis revealed elevated levels of tumor necrosis factor (Tnf)α at 6 hours post-treatment (hpt ) and increased Il10 expression at 24 hpt in spleens treated with 10 µg/fish EpEVs.
These findings confirm that EpEVs can be effectively isolated via ultracentrifugation and highlight the necessity of further investigation into their immunomodulatory mechanisms to develop novel therapeutic applications in fish medicine.