Proteomic profiling reveals growth condition-driven functional differences in bacterial membrane vesicles from planktonic and biofilm cultures

Abstract

Bacterial membrane vesicles (BMVs) are key mediators of host-pathogen interaction and intercellular communication. However, their characterization has been conducted almost exclusively under planktonic growth conditions, even though biofilm formation is the predominant mode of bacterial persistence in chronic and device-associated infections. How the shift from planktonic to biofilm growth reshapes BMV composition and function, and how this is affecting the host, remains poorly understood. Here, we systematically compared BMVs from the ESKAPE pathogens Staphylococcus aureus and Pseudomonas aeruginosa , representing Gram-positive and Gram-negative bacteria, across both growth conditions. Particle sizes and number-weighted size distributions were obtained by dynamic light scattering and nanoparticle tracking analysis, respectively. Cryogenic electron micrographs showed intact, membrane-enclosed vesicles for all four isolations. Biofilm-derived BMVs exhibited a more negative surface charge compared to their planktonic counterparts. Quantitative proteomics identified growth mode as a strong determinant of cargo: planktonic BMVs were enriched in ribosomal proteins, whereas biofilm-derived BMVs carried central metabolic enzymes. 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. 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.