MICROVESICLES PRODUCED BY LACTOBACILLUS CASEI BL23 CONTAINED PROTEINS ASSOCIATED WITH ITS PROBIOTIC ACTIVITY

ANA PAULA DOMINGUEZ RUBIO; JIMENA H.MARTINEZ; MARCOS PALAVECINO; MARIANA PIURI; OSCAR E. PEREZ

CABA

Congreso; LXII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2017

Extracellular vesicles in Gram positive bacteria they are alsoknown as microvesicles (MVs) and they have been proposed tobe involved in signaling between probiotic bacteria and their mammalian hosts. The aim of this study is to investigate the isolation andcharacterization of MVs released from the probiotic strain L. caseiBL23 which is a regular resident of the mammalian gastrointestinal(GI) microbiome.L. casei BL23 was grown in MRS medium at 37°C for 48h. Cellswere removed from the culture by centrifugation. Supernatant wasthen concentrated using a 100 kDa flter membrane. The concentrated supernatant was spun at 110000 g for 2h to pellet MVs. MVshave been characterized by Dynamic Light Scattering (DLS), AtomicForce Microscopy (AFM) and Transmission Electronic Microscopy(TEM). The shedding of MVs was investigated using CFSE stainingand further visualization by Confocal Laser Scanning Microscopy(CLSM). Total DNA, RNA and protein were isolated from MVs usingQuickZol. Proteins were analyzed by HPLC coupled to mass spectrometry.CLSM imaging of L. casei BL23 showed a protrusion on cells consistent with the shedding of MVs to the extracellular medium. MVssize obtained from DLS data were consistent with measurementsfrom AFM and TEM images (30-50 nm). The negative surface charge (-9±2 mV) could make the MVs stable by virtue of electrostaticrepulsion. AFM images suggests that MVs might have a shape ranging from spherical to cup shape morphology. In TEM images weobserved MVs with bilayered membranes and electron dense luminal contents; consistent with the presence of DNA, RNA and proteins. Proteomic analysis identifed 103 proteins in the MVs includingp40 and p75 that have been associated with its probiotic activity.MVs could play a key role in signaling between GI bacteria andtheir mammalian hosts. The expression and encapsulation of proteins into MVs could represent a scientifc novelty, with applications infood, nutraceuticals and clinical therapies