Supplementary file 1_Proteomic profiling reveals growth condition-driven functional differences in bacterial membrane vesicles from planktonic and biofilm cultures.docx

Notably, exopolysaccharide biosynthesis proteins of the Psl, Pel, and Ica systems were more abundant in planktonic than in biofilm BMVs. P. aeruginosa BMVs isolated from biofilms carried heme and iron acquisition systems, proteins of the oxidative stress defense, and the secreted proteases LasB and AprA, while planktonic S. aureus BMVs were enriched in secreted virulence factors, highlighting species-level differences.

Functionally, all preparations reduced metabolic activity in epithelial cells with only limited lactate dehydrogenase release, identifying metabolic suppression rather than membrane rupture as the dominant effect. In the case of P. aeruginosa, biofilm-derived BMVs suppressed metabolic activity significantly more than planktonic ones. In human macrophages, planktonic BMVs elicited the strongest IL-1β and TNF-α responses, whereas biofilm-derived vesicles induced earlier secretion of IL-6 and, for P. aeruginosa, a distinctly reduced release of the anti-inflammatory cytokine IL-10.

These effects persisted under polymyxin B, which reduced but did not abolish the overall cytokine responses.</p>Discussion<p>This indicates that vesicle cargo does not simply mirror the biofilm matrix, pointing to a contribution of vesicle-associated components beyond lipopolysaccharide. Together, these data establish a comparative framework linking bacterial growth mode to BMV composition and host cell response, advancing the understanding of vesicle-mediated processes in chronic infection.</p>