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

PALAVECINO, M; PÉREZ, OE; MARTÍNEZ, JH; PIURI, M; DOMÍNGUEZ RUBIO AP

Barcelona

Congreso; Annual Meeting of the International Society for Extracellular Vesicles; 2018

International Society for Extracellular Vesicles

Bacillus subtilis 168 is a regular resident of the mammalian gastrointestinal (GI) microbiome and have 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 signaling 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 specifics 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 cell barrier was investigated in an in vitro model of human Caco-2 cells. B. subtilis 168 was grown in BHI medium at 37°C under agitation for 18h. Cells were removed from the culture by centrifugation. Supernatant was then concentrated using a 100kDa filter membrane. The concentrated supernatant was spun at 110000g for 2h to pellet EVs. Isolated EVs were stained with CFSE. Human colon carcinoma Caco-2 cells were differentiated for 14 days (100% confluence). EVs uptake was analyzed as the number of EVs labeled inside the cell by Confocal Laser Scanning Microscopy (CLSM). 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.Intact EVs uptake in Caco-2 cells was linear for up to 30 min: y=1.02x-1.25 and R²=0.97 (p0.05).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 signaling 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.