EXTRACELLULAR VESICLES PRODUCED BY PROBIOTIC BACTERIA: CHARACTERIZATION AND VISUALIZATION BY HIGH PERFORMANCE MICROSCOPY TECHNIQUES

ANA PAULA DOMINGUEZ RUBIO; CECILIA D´ANTONI; CECILIA SAMANIEGO LÓPEZ; CARLA SPAGNUOLO; MARIANA PIURI; OSCAR E PÉREZ

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias; 2022

Sociedad Argentina de Investigación Clínica (SAIC), la Sociedad Argentina de Inmunología (SAI) y la Sociedad Argentina de Fisiología (SAFIS).

Extracellular vesicles (EVs) are composed of a lipid bilayer with cytosolic components such as DNA, RNA and proteins. Bacterial nanosized EVs have been proposed to be involved in signaling between probiotic bacteria and their mammalian hosts. The aim of this study was to analyze the EVs produced by Lacticaseibacillus casei BL23 (47±3nm) and Bacillus subtilis 168 (142±14nm) by Atomic Force Mycroscopy (AFM), Transmission Electronic Microscopy (TEM), Confocal Laser Scanning Microscopy (CLSM) and Super-resolution Microscopy (SRM).L. casei BL23 was grown in MRS medium at 37°C for 48h and B. subtilis 168 was grown in BHI medium at 37°C under 200rpm agitation for 18h. Cultures were spun at 4,000g for 25 min at 4°C. The supernatant was filtered, concentrated using a 100kDa filter and centrifuged at 110,000g for 2h at 4°C. The pellet was resuspended in PBS for AFM and TEM, or labeled with CFSE or boroxol for CLSM and SRM.Topography of EVs obtained by AFM showed EVs with spherical shape morphology for both strains. To elucidate details regarding shape and ultrastructure, TEM images were analyzed. L. casei BL23 EVs showed bilayered membranes and an electron-dense luminal content; consistent with the notion that vesicles contain bioactive cargo such as proteins or nucleic acids However, B. subtilis 168 EVs did not always show a central electron-dense core. Both techniques confirmed the size distribution of these EVs (n=3, p>0.05). CLSM images of EVs stained with CFSE showed an identical signal to EVs shedding from the bacteria, but EV size resulted smaller than the theoretical resolution of the microscope. By SRM we observed spherical EVs, with the certainty that the fluorescence comes only from a single EV.The results contribute to the characterization of bacterial EVS. The expression and encapsulation of biomolecules into EVs could represent a scientific novelty with applications in food, nutraceuticals and clinical therapies.