Abstract
Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered to be a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. Among probiotic Gram-positive bacteria, some Propionibacterium freudenreichii strains exhibit anti-inflammatory activity, notably via surface proteins such as the surface-layer protein B (SlpB). We have hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host. In order to demonstrate their production in this species, EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, and their physicochemical characterization, using transmission electron microscopy and nanoparticle tracking analysis (NTA), revealed shapes and sizes typical of EVs. Proteomic characterization showed that EVs contain a broad range of proteins, including immunomodulatory proteins such as SlpB. In silico protein-protein interaction predictions indicated that EV proteins could interact with host proteins, including the immunomodulatory transcription factor NF-κB. This potential interaction has a functional significance because EVs modulate inflammatory responses, as shown by IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells. Indeed, EVs displayed an anti-inflammatory effect by modulating the NF-κB pathway; this was dependent on their concentration and on the proinflammatory inducer (LPS-specific). Moreover, while this anti-inflammatory effect partly depended on SlpB, it was not abolished by EV surface proteolysis, suggesting possible intracellular sites of action for EVs. This is the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. This study has enhanced our understanding of the mechanisms associated with the probiotic activity of P. freudenreichii and identified opportunities to employ bacterial-derived EVs for the development of bioactive products with therapeutic effects.
Highlights
Intercellular communication is an essential biological process that involves several soluble biomolecules that may be secreted, surface-exposed or packed inside extracellular vesicles (EVs) (Gho and Lee, 2017; Toyofuku, 2019)
The release of EVs by Lactobacillus species is well documented; Lactobacillus reuteri DSM 17938-derived EVs are associated with extracellular DNA-dependent biofilm formation (Grande et al, 2017) and EVs secreted by Lactobacillus casei BL23 have been reported and shown to contain diverse biomolecules which include nucleic acids and proteins previously associated with its probiotic effects, such as p40 and p75 (Rubio et al, 2017)
A total of 319 proteins was identified consistently in EVs derived from UF medium cultures (Supplementary Table S1), which corresponds to 11% of the whole theoretical proteome of P. freudenreichii CIRM-BIA 129
Summary
Intercellular communication is an essential biological process that involves several soluble biomolecules that may be secreted, surface-exposed or packed inside extracellular vesicles (EVs) (Gho and Lee, 2017; Toyofuku, 2019). EVs are lipid bilayer nanoparticles which range in size from 20 to 300 nm and are released by cells from all living kingdoms (Brown et al, 2015; Kim et al, 2015a; Liu et al, 2018a). They play a pivotal role in cell-to-cell communication through their ability to transport bioactive molecules (proteins, nucleic acids, lipids, metabolites) from donor to recipient cells. EVs derived from other probiotic species, such as Bifidobacterium longum KACC 91563, impact host cell responses by inducing mast cell apoptosis, which has implications for the treatment of food allergies (Kim et al, 2016). Probiotic strains of Escherichia coli release outer membrane vesicles (OMVs) that are involved in reinforcement of the gastrointestinal epithelial barrier (Alvarez et al, 2016), the regulation of inflammatory responses and intestinal homeostasis, via the NOD1-signaling pathway (Cañas et al, 2018)
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