Extracellular vesicles as indicators of environmental stress response in Lactiplantibacillus plantarum : a multi-platform study

Extracellular vesicles (EVs) are key mediators of bacterial communication and adaptation to environmental stress. Their size, cargo, and surface charge are influenced by several factors, including environmental conditions, bacterial physiology, and isolation methods, and are highly strain-specific. Herein, we investigated how exposure to bile, a physiological component of the gut environment, affects the production and properties of EVs released by the probiotic strain Lactiplantibacillus plantarum NCIMB 8826. Through ultracentrifugation followed by size-exclusion chromatography (SEC), we isolated highly purified L. plantarum EVs (LpEVs) and characterized them according to the Minimal Information for Studies of Extracellular Vesicles guidelines.

SEC purification significantly reduced the contents of contaminating proteins and peptidoglycans, improving the compositional purity of the isolated EVs. Compared with the parent bacteria, purified LpEVs exhibited distinct surface lipid profiles and zeta potential as well as remarkable stability across varying pH levels, elevated NaCl concentrations, and increasing detergent challenges. Under bile stress, the bacteria released larger LpEVs enriched in bile metabolism–related proteins, suggesting vesicle-mediated adaptation. Fourier-transform infrared spectroscopy further revealed bile-induced molecular alterations in LpEVs that differed from those in the parent bacteria.

These findings highlight bacterial EVs as dynamic environmental communicators that respond to stress and may modulate host–microbe interactions before detectable changes occur in the bacterial cells.