Differential Proteomic Analysis of Extracellular Vesicles Produced by Granulicatella adiacens in Biofilm vs. Planktonic Lifestyle

<b>Background:</b> Gram-positive bacteria, once considered incapable of producing extracellular vesicles (EVs) due to their thick peptidoglycan layer, are now known to secrete EVs that transport virulence factors and modulate host immunity. These EVs contribute to bacterial pathogenicity by facilitating biofilm formation, immune evasion, and inflammation. <i>Granulicatella adiacens</i>, an oral commensal associated with infective endocarditis, represents a clinically relevant model to study EV-mediated virulence. <b>Objectives:</b> This study's aim was to investigate whether the proteomic composition and immunomodulatory activity of <i>G. adiacens</i> EVs differ between biofilm and planktonic lifestyles, thereby contributing to distinct pathogenic behaviours. <b>Methods:</b> EVs isolated from <i>G. adiacens</i> CCUG 27809 cultures were characterized using nano LC-ESI-MS/MS, followed by comprehensive bioinformatic and cytokine assays. <b>Results:</b> Quantitative proteomic profiling identified 1017 proteins, revealing distinct signatures between biofilm- and planktonic-derived EVs. Principal component analysis showed clear segregation between the two states, with biofilm EVs enriched in proteins linked to stress adaptation, adhesion, and structural integrity, while planktonic EVs exhibited growth- and metabolism-related proteins. A total of 114 virulence-associated proteins were identified, including several novel candidates. Functionally, EVs from both conditions significantly induced pro-inflammatory cytokines IL-8 and IL-1β in a dose-dependent manner (<i>p</i> < 0.05), whereas IL-17 remained unchanged. <b>Conclusions:</b><i>G. adiacens</i> EVs exhibit lifestyle-dependent proteomic and immunomodulatory differences, underscoring their role in host-pathogen interactions and endocardial infection. These findings provide a foundation for future mechanistic and in vivo studies exploring EV-mediated virulence and potential therapeutic modulation.