Transcytosis of extracellular vesicles produced by Bacillus subtilis 168 in human intestinal Caco-2 cell monolayers

JIMENA H MARTÍNEZ; OSCAR E PÉREZ; MARCOS PALAVECINO; ANA PAULA DOMÍNGUEZ-RUBIO; MARIANA PIURI

Barcelona

Congreso; ISEV 2018; 2018

International Society of Extracellular Vesicles

Background: Bacillus subtilis 168 is a regular resident of the mammaliangastrointestinal (GI) microbiome and has been used in food fermentations,being awarded the status of ?Generally Recognized As Safe? (GRAS).Extracellular vesicles (EVs) have been proposed to be involved in signallingbetween probiotic bacteria and their mammalian hosts. B. subtilis 168 producesEVs whichwere on the nanometric size range (50?300nm). EVs carriedcytoplasmic components, such as specific proteins, which suggest a role forthe EVs in the bacteria?GI cells interface. We hypothesize that transcytosis ofEVs across intestinal epithelial cells is a crucial step in the host-probioticcommunication. To test this, the ability of EVs produced by the probioticstrain B. subtilis 168 to cross intestinal epithelial cell barrier was investigated inan in vitro model of human Caco-2 cells.Methods: B. subtilis 168 was grown in BHI medium at 37°C underagitation for 18 h. Cells were removed from the culture by centrifugation.Supernatant was then concentrated using a 100-kDa filter membrane.The concentrated supernatant was spun at 110000 g for 2 h topellet EVs. Isolated EVs were stained with carboxyfluorescein succinimidylester. Human colon carcinoma Caco-2 cells were differentiated for14 days (100% confluence). EVs? uptake was analysed as the number ofEVs labelled inside the cell by confocal laser scanning microscopy.Transcytosis was studied as the fluorescence measured in the collectedmedium from the transwell lower chamber and EVs were also observed.The cytotoxicity of the EVs was evaluated using MTT assay.Results: Intact EVs uptake in Caco-2 cells was linear for up to 30 min:y = 1.02 × −1.25 and R2 = 0.97 (p < 0.05). In transcytosis studies, fluorescencewas recorded after 120 min elapsed and increased 50% at 240 min (n= 3).Wealso found intact EVs in the collected medium from the lower chamber of thetranswell. EVs did not significantly reduce cell viability (p > 0.05).Summary/Conclusion: EVs produced by the probiotic strain B. subtilis168 crossed intestinal epithelial cell barrier of human Caco-2 cells. Thisevidence suggests that EVs could play a key role in signalling between GIbacteria and mammalian hosts. The expression and further encapsulationof proteins into EVs of GRAS bacteria could represent a scientificnovelty, with applications in food and clinical therapies.