Plasma-derived exosomes of Edwardsiella piscicida challenged olive flounder (Paralichthys olivaceus): Characterization and miRNA profiling for potential biomarkers screening

Exosomes are released from multiple cell types as part of their normal physiology as well as during acquired abnormalities. In this study, we investigated the effect of pathogenic Edwardsiella piscicida infection on olive flounder (Paralichthys olivaceus) exosomes at morphometric, physicochemical, and molecular levels. Unique cup-shaped exosomes were isolated from the plasma of non-infected (PBS-Exo) and E. piscicida experimentally challenged (Ep-Exo) olive flounder using ultracentrifugation. The average particle size, concentration, and zeta potential were 150.9 +/- 6.9 nm, 5.67 x 10(10) particles/mL, and -25.6 +/- 1.36 mV for PBS-Exo while 138.7 +/- 1.9 nm, 1.22 x 10(11) particles/mL, and -35 +/- 1.82 mV for Ep-Exo, respectively. Expression of tetraspanin markers (CD81, CD9, and CD63) confirmed the presence of olive flounder exosomes. Differentially expressed (DE) known (9) and novel (29) miRNAs (log(2) fold change >= 1; p < 0.05) were identified in the Ep-Exo that could be potential as diagnostic biomarkers for the infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the predicted target genes of the DE miRNAs were highly enriched in metabolic and immune roles. Both PBS-Exo and Ep-Exo were non-toxic in vitro (up to 100 mu g/mL) and in vivo (up to 400 mu g/mL). Compared to the vehicle, PBS-Exo at 50 mu g/mL induced Nf-kB (>1.50-fold) while at 100 mu g/mL, Il8, Il10, Nf-kB, P53, and Inf were induced (>1.50-fold) in fathead minnow cells (FHMs). This suggests that the PBS-Exo contains molecules that moderately stimulate gene expression. In the future, validating the exact olive flounder immune response target genes that interact with DE miRNAs in Ep-Exo will be crucial for investigating the host-pathogen interactions, immune defense mechanisms, and therapeutic targets for olive flounder against E. piscicida infection.