Mapping the proteomic landscape and anti-inflammatory role of Streptococcus parauberis extracellular vesicles

Bacterial extracellular vesicles (BEVs) are nanoscale membrane-bound structures involved in intercellular communication and transport of bioactive molecules. In this study, we described the proteomic insight and anti-inflammatory activity of Streptococcus parauberis BEVs (SpEVs). Proteomics analysis of SpEVs identified 6209 distinct peptides and 1039 proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated enrichment in pathways related to the biosynthesis of aminoacyl tRNA, amino acids, and secondary metabolites. Based on the predicted protein-protein interactions, we discovered key immunological proteins such as IL12A, IL12B, IL8, CD28, and NF-κB between SpEVs and human proteins. Functionally, SpEVs exhibit strong anti-inflammatory activity in LPS-stimulated Raw 264.7 cells by reducing the production of key inflammatory mediators. These include nitric oxide (NO), reactive oxygen species (ROS), inflammatory cytokines such as TNFα and IL6, as well as inflammation-related proteins like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). qRT-PCR and immunoblotting results clearly indicate that SpEVs modulate the NF-κB and MAPK pathways to induce anti-inflammatory activity. Furthermore, in vivo experiments with zebrafish larvae demonstrated that SpEVs treatment reduced the NO and ROS production with minimal cell mortality. Finally, we validated the anti-inflammatory activity of SpEVs in vivo by systematically assessing the inhibition of NO production, reduction in ROS generation, prevention of cell death, and modulation of NF-κB and MAPK signaling pathways. In conclusion, SpEVs contain rich in unique proteins that play crucial roles in mediating anti-inflammatory effects.