Extracellular vesicles produced by Bacillus subtilis release RNA and cross in human intestinal Caco-2 cells

MARTÍNEZ, JH; PÉREZ, OE; DOMÍNGUEZ RUBIO, AP; D´ANTON, CL; MARCILLA, A; SOLER, C; PIURI, M

Workshop; International Society for Extracellular Vesicles 2020 Workshop EVs in Immunology; 2020

International Society for Extracellular Vesicles

IntroductionThe gastrointestinal tract (GIT) mucosal surface is the primary interface between gut bacteria and internal host tissues. A vital communication between kingdoms in the mammalian GIT is supposed to occur through an exchange of EVs that may interact in a triangular way between gut bacteria and the host. The genus Bacillus includes well-known pathogenic bacteria like B. anthracis, the etiological agent of anthrax, and B. cereus which is associated with human food poisoning, as well as non-pathogenic bacteria like B. subtilis. In the case of B. anthracis and B. cereus, the entry into the GIT is an essential part of their virulent life cycle. In the other hand, B. subtilis is a regular resident of the mammalian GIT and is considered as GRAS (Generally Recognized As Safe). The aim of this work was to explore whether B. subtilis EVs (70?230 nm) can be transported across a polarized cell monolayer and transfer RNAs.MethodsB. subtilis EVs were isolated by ultracentrifugation, treated with RNAse A, and double-labelled with SYTO RNAselect green for RNA and BODIPY TR Ceramide red for membrane bilayer. Labelled EVs were added to Caco-2 monolayers, which were counterstained with DAPI. Fluorescence was evaluated by Confocal Laser Scanning Microscopy (CLSM) up to 120 min. In addition, Caco-2 cells differentiated for 14 days were analyzed in a transwell system to study the transcytosis process. Fluorescence was measured in the collected medium from the transwell lower chamber at different time points, and EVs observed by CLSM. Results B. subtilis EVs were internalized by host cells and their RNA content was released into the nucleus of Caco- 2 cells, while EVs-membrane bilayer remained in the cytosol. In transcytosis experiments, intact EVs were collected from the lower chamber of the transwell, and fluorescence first detected after 120 min of incubation, and it increased up to 30% at 240 min. ConclusionsIn this work, we show that RNA contained in B. subtilis EVs could be internalized into the nucleus of eukaryotic cells, and describe the transport of EVs across differentiated Caco-2 cells to enterocyte-like cells. Our findings suggest that this could be an initial step which could allow the EVs entrance the bloodstream. Future studies will focus on the role of RNA in signaling between GIT bacteria and mammalian hosts. The expression and further encapsulation of RNAs into EVs of GRAS bacteria represents a scientific novelty with future applications in clinical therapies.