Characterization of the proteomic content and immunomodulatory properties of the extracellular vesicles derived from the probiotic Propionibacterium freudenreichii CIRM-BIA129

Extracellular vesicles (EVs) are ubiquitous mediators of the intercellular communication and consist in an export system involved in essential processes of life, such as nutrition, adaptation, defense and host-microbe interaction. EVs are spherical non-replicating nanoparticles composed of a lipid bilayer and an internal aqueous lumen that shelters a diverse set of molecules, including proteins, nucleic acids, lipids, glycans and other metabolites. The research on bacteria-derived EVs focused initially in Gram-negative and pathogenic bacteria, but the scenario changed in the last years, including EVs derived from other groups, such as Gram-positive and non-pathogenic bacteria. A relevant Gram-positive and probiotic bacterium is Propionibacterium freudenreichii, which has been long and safely used in the production of Swiss-type cheese, organic acids and vitamin B12. P. freudenreichii has emerged in the probiotics landscape due to notable properties, such as anti-pathogenic and anti-cancer activities, modulation of the microbiota composition and immunomodulation. Some of these properties were attributed to surface-exposed or secreted factors. Therefore, we aimed to investigate whether P. freudenreichii produced EVs that could mediate its beneficial properties, particularly immunomodulation. Accordingly, the bacterium was cultured in milk ultrafiltrate (UF) or yeast extract-lactate (YEL) medium until the beginning of the stationary phase. Protocols for EV purification based on size-exclusion chromatography (SEC) or density gradient ultracentrifugation (UC) were applied. The resulting samples presented nano-sized particles with typical morphology, confirming EV production by P. freudenreichii. EV protein content was investigated with shotgun proteomics approaches, unveiling a diverse set of proteins that included immunomodulatory and cytoplasmic proteins. The protein cargo and interactomics analysis indicated potential immunomodulatory roles for P. freudenreichii-derived EVs. That was confirmed with the EV-mediated reduction of IL-8 release and NF-κB activity in human intestinal epithelial cells treated with bacterial LPS, indicating an anti-inflammatory role. Moreover, EVs properties were dependent on the culture medium used to grow bacteria, since they varied for EVs derived from UF or YEL, including EV size, abundance, protein content and anti-inflammatory activity. Furthermore, the method used for EV purification also impacted EVs properties, particularly protein content. Although there were exclusive proteins for some conditions, hundreds of proteins were identified in all the studied conditions, indicating the existence of a conserved core proteome. Altogether, these findings contribute to the understanding of the mechanisms underlying the probiotic effect in P. freudenreichii and reveal novel possibilities for the developing of optimizable and safe nanotechnological delivery systems, potentially holding a great impact in human health.