Extracellular vesicles derived from Enterococcus faecalis: inflammatory activation does not require internalization

Abstract Background Enterococcus faecalis is a common gut commensal Gram-positive bacterium that can act as an opportunistic pathogen and is frequently associated with severe infections community-acquired and nosocomial. Bacteria-derived extracellular vesicles (EVs) emerge as key mediators of host-bacteria communication with immunomodulatory roles and mechanistic participation in pathophysiological processes. However, the impact of E. faecalis -derived EVs ( Ef -EVs) on host cells and their potential role in shaping host responses during infection remain unclear. Methods Ef -EVs from the E. faecalis DSM 20478 type strain and four independent clinical bloodstream isolates were isolated via ultracentrifugation and size exclusion chromatography. EVs were characterized by nanoparticle tracking analysis and cryogenic transmission electron microscopy. Immunomodulatory effects of Ef -EVs were studied in vitro on NF-κB/AP-1 reporter cells, primary human monocyte-derived macrophages, and human umbilical vein endothelial cells, and by transcriptomic analysis of macrophages isolated from in vivo EV-treated zebrafish larvae. EV-induced signaling mechanisms were studied using uptake inhibitors as well as bottom-up assembled bacterial EVs functionalized with synthetic bacterial ligands. EV-induced metabolic reprogramming in macrophages was investigated by RNA-Seq and live-cell metabolic analyses using the Seahorse XFe-96 Flux Analyzer. Results We found that Ef -EVs can induce pro-inflammatory responses in host macrophages via Toll-like receptor 2 (TLR2) signaling, as demonstrated using TLR2 transgenic cell lines and a TLR2-blocking antibody. Using uptake inhibitors as well as bottom-up assembled bacterial EVs functionalized with synthetic bacterial ligands as a minimalistic approach to study mechanisms of EV signaling, we demonstrated that Ef -EVs target the plasma membrane TLR2 to induce inflammation in a process uncoupled from their internalization. Furthermore, we found that Ef -EVs induce metabolic reprogramming towards a pro-inflammatory, glycolytic phenotype. Conclusion Our findings reveal a mechanism by which Gram-positive bacterial EVs modulate immune signaling and metabolic pathways, advancing our understanding of host-pathogen communication.