Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines, mitigates the inflammatory response in vitro and in vivo. These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e. modulating NF-κB transcription factor activity and IL-8 release) which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and hence the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical and functional properties of EVs produced from cells cultivated on laboratory Yeast Extract Lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant, smaller in diameter and displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacteria interactions.Proteomics characterization of EVs protein content was carried out on a Qexactive coupled to NanoLiquid Chromatography.
Three independent biological replicates of P. freudenreichii CIRM-BIA 129 purified EV were digested for NanoLC-ESI-MS/MS analysis. Approximately 50 µg of EVs were pelleted at 150,000 g for 2 h at 4°C and suspended in a solution of 6 M Guanidine-HCl (Sigma-Aldrich), 50 mM Tris-HCl (pH 8.0) (VWR C) and 2 mM DTT (Sigma-Aldrich). The EVs were heated at 95oC for 20 min and cooled in 50 mM NH4HCO3 (pH 7.8) (Sigma-Aldrich).
