Glycine-Induced Cargo Alterations of Bacterial Outer Membrane Vesicles by Multi-Omics Analysis

Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic with therapeutic potential in intestinal inflammatory disorders. Its secreted outer membrane vesicles (OMVs) are key mediators of host-microbe interactions and serve as promising platforms for vaccine development and drug delivery. Although glycine is known to enhance OMVs biogenesis in EcN, the resulting cargo alterations are less studied, which are of great importance for the downstream drug delivery and biomedical applications. In this study, we systematically explored the impacts of glycine on the physicochemical properties, yield, and molecular composition of OMVs via morphological characterization, non-targeted and stable isotope-tracing lipidomics. Results showed that glycine stimulation significantly increased the yield of OMVs, particle size, colloidal stability, and purity, while inhibiting bacterial growth. Lipidomic profiling revealed that glycine remodels the lipid composition of OMVs, particularly affecting glycerophospholipid and sphingolipid metabolism. Stable isotope tracing with D₃-serine demonstrated a metabolic shift in phospholipid synthesis from bacterial biomass toward OMVs production.