Staphylococcus aureus derived extracellular vesicles modulate osteoblast-like cell immune responses independently of vesicle internalization

Staphylococcus aureus is a major opportunistic Gram-positive pathogen whose virulence is partly mediated by extracellular vesicles (EVs). Here, we examined the production, composition, and functionality of EVs released by the methicillin-resistant strain N315 under infection-mimicking conditions, focusing on their interaction with MG-63 human osteoblast-like cells. N315 EVs, purified by size exclusion chromatography, and characterized by nanoparticle tracking analysis and electron microscopy, contained a selectively enriched and diverse molecular cargo, including proteins, lipoproteins, DNA, RNA, lipoteichoic acid, and peptidoglycan. Confocal microscopy and protection assays confirmed the intravesicular localization of RNAs and proteins. EVs were recognized by TLR2 on MG-63 cells and selectively induced the expression of genes involved in Toll-like receptor, NF-κB, JAK/STAT, and inflammasome pathways. The induction of these pathways was confirmed at the protein level, as well as by increased IL-8 release and caspase-1 activation. EVs were internalized via a dynamin-dependent endocytosis mechanism and trafficked to late endosomes. Importantly, inhibition of uptake did not affect the expression of target genes, demonstrating an uncoupling between EVs internalization and transcriptional response. This work provides new insight into how S. aureus N315 EVs interact with osteoblast-like cells and modulate host responses, revealing both conserved mechanisms and strain- or cell type-specific differences.