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

Introduction: 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. Methods: Here, we systematically compared BMVs from the ESKAPE pathogens Results: 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. Discussion: 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.