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

• Published in: Journal of extracellular vesicles 2018-01, Vol.7, p.178-178
• Main Authors: Rubio, Ana Paula Domínguez, Martínez, Jimena Hebe, Palavecino, Marcos, Piuri, Mariana, Pérez, Oscar Edgardo
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; Bacteria; Cell culture; Centrifugation; Colon; Confocal microscopy; Cytotoxicity; Epithelial cells; Extracellular vesicles; Fermented food; Intestine; Lymphocytes B; Microbiomes; Probiotics
• ISSN: 2001-3078

Background: Bacillus subtilis 168 is a regular resident of the mammalian gastrointestinal (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 signalling between probiotic bacteria and their mammalian hosts. B. subtilis 168 produces EVs which were on the nanometric size range (50-300 nm). EVs carried cytoplasmic components, such as specific proteins, which suggest a role for the EVs in the bacteria-GI cells interface. We hypothesize that transcytosis of EVs across intestinal epithelial cells is a crucial step in the host-probiotic communication. To test this, the ability of EVs produced by the probiotic strain B. subtilis 168 to cross intestinal epithelial cellbarrierwas investigated in an in vitro model of human Caco-2 cells. Methods: B. subtilis 168 was grown in BHI medium at 37°C under agitation 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 to pellet EVs. Isolated EVs were stained with carboxyfluorescein succinimidyl ester. Human colon carcinoma Caco-2 cells were differentiated for 14 days (100% confluence). EVs' uptake was analysed as the number of EVs labelled inside the cell by confocal laser scanning microscopy. Transcytosis was studied as the fluorescence measured in the collected medium 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 x -1.25 and R2 = 0.97 (p < 0.05). In transcytosis studies, fluorescence was recorded after 120 min elapsed and increased 50% at 240 min (n = 3). We also found intact EVs in the collected medium from the lower chamber of the transwell. EVs did not significantly reduce cell viability (p > 0.05). Summary/Conclusion: EVs produced by the probiotic strain B. subtilis 168 crossed intestinal epithelial cell barrier of human Caco-2 cells. This evidence suggests that EVs could play a key role in signalling between GI bacteria and mammalian hosts. The expression and further encapsulation of proteins into EVs of GRAS bacteria could represent a scientific novelty, with applications in food and clinical therapies.