Staphylococcus aureus derived extracellular vesicles trigger multiple inflammatory pathways in host cells and deliver their RNA cargo following their internalization and lysis within late endosomes

Staphylococcus aureus is a major opportunistic Gram-positive pathogen whose virulence can be partly mediated by extracellular vesicles (EVs). These nanosized particles carry a diverse cargo and can play key roles in host-pathogen interactions. Here, we investigated the production, composition, and host cell interactions of EVs secreted by the methicillin-resistant S. aureus strain N315, one of the most widely studied clinical isolates. To mimic infection, EVs produced from RPMI supplemented with 10% LB were purified by size exclusion chromatography, and characterized by nanoparticle tracking analysis and electron microscopy. N315 EVs contained a diverse molecular cargo, selectively enriched, and including proteins, lipoproteins, DNA, RNA, lipoteichoic acid, and peptidoglycan. Protection assays and confocal microscopy confirmed the intravesicular localization of RNAs and proteins. N315 EVs were recognized by multiple host pattern recognition receptors (PPRs) in human MG-63 cells, located at the cell surface (TLR1, TLR2, TLR4, TLR6), within endosomes (TLR3, TLR7, TLR4) and in the cytoplasm (NOD2). EV exposure promoted the selective upregulation of these PRRs, activated the NF-{kappa}B and JAK/STAT signaling pathways, and induced the expression of pro-inflammatory cytokines such as IL1{beta}, IL6, and CXCL8. N315 EVs were internalized via a dynamin-dependent endocytosis mechanism and trafficked to late endosomes, where RNA cargo release occurred. Importantly, inhibition of EVs uptake altered cytokine gene expression, confirming that internalization is required for immunomodulation. This work provides a better understanding of the mechanisms of interaction of S. aureus EVs with host cells and sheds light on the fate of EVs and their cargo once internalized by cells. IMPORTANCEAntibiotic resistance, coupled with the limited discovery of new antimicrobial agents, poses a growing threat to public health. Staphylococcus aureus is one of the six highly virulent and antibiotic-resistant bacterial species responsible for both severe hospital- and community-acquired infections. This pathogen demonstrates a remarkable ability to adapt to diverse hosts and environments and to produce a broad arsenal of virulence factors. Gaining deeper insight into its pathogenic mechanisms and host interactions may reveal novel therapeutic targets and alternative treatment strategies. Over the past decade, a key advancement has been the identification of extracellular vesicle production by S. aureus and its critical role as a new secretion system for virulence factors. This study shed light on the immunogenic effect of EVs and their role as a vehicle for the delivery of their contents into host cells. These adjuvant-like properties open new avenues for research and potential intervention.