Microvesicles from Probiotic Lactobacillus casei BL2. Detection and characterization

FEDERICO COLUCCIO-LESKOW; KARINA D MARTÍNEZ; PAULA DOMÍNGUEZ-RUBIO; OSCAR E PÉREZ; MARIANA PIURI

Buenos Aires

Simposio; Frontiers in Physical Sciences; 2016

Centro de Latinoamericano de Formación Interdisciplinaria y MinCyT.

Membrane vesicle are a demonstrated form of communication used by bacteria, eukaryotes, and archaea (Al-Nedawi et al. 2015). Little knowledge exist about the production and role of membrane vesicles in Gram-positive bacteria. The study of microvesicles (MV) is likely to yield crucial information as to which components are involved in signaling between prokaryotes such as probiotic bacteria and mammalian host and help further unravel the pathways involved in the microbiome-gut-brain axis (14, 53, 54). Microvesicles fall between 100-1000 nm diameters in size. The aim of this study was to explore the detection of microvesicles form BL23 strain, a probiotic bacterium type and carry out their characterization.Lactobacillus casei BL23 were grown in Man-Rogosa-Sharpe (MRS) medium 37°C and were harvested at 48 h (800 ml). Cultures were spun at 4,000 × g for 25 min at 4°C to remove cells. The resulting supernatant was then filtered through a series of decreasing pore-size membrane (0.8, 0.65 and 0.45 µm) and the filtrate was concentrated using Amicon ultrafiltration system with 100 kDa filter. The concentrate was then further filtered through a 0.45 µm syringe filter to remove larger cellular debris or aggregated material. The filtered supernatant was then centrifuged at 110,000 × g for 2 h at 4°C and washed twice with phosphate buffered saline (PBS). The vesicle pellet was resuspended in PBS or Quick-Zol reagent. MRS was also used as negative control to discard that the culture was contaminated with other bacterial cells during the concentration of the sample. Bacillus subtilis 168 was used as a positive control of micovesicles production. B. subtilis 168 were grown in brain heart infusion (BHI) medium 37°C under continuous agitation and were harvested at 18 h (100 ml). Microvesicles resuspended in PBS were characterized by dynamic light scattering (DLS) in the size and zeta potential (Zetasizer). Total RNA, DNA and protein were isolated from L. casei BL23 microvesicles using Quick-Zol (Kalium Technologies) reagent following the manufacturer?s instructions. RNA and DNA were quantified by NanoDrop and proteins was quantified by the method of Lowry and analyzed by SDS polyacrylamide gel electrophoresis (10% resolving gel). The gel was subsequently stained with coomassie blue. L. casei BL-23 produced extracellular vesicles at 48 h (n=12). Macroscopic pellet appearance denoted differences among L. casei BL-23 and B. subtilis 168. Centrifugation of B. subtilis 168 culture supernatants produced a large, reddish-brown vesicle pellet whereas centrifugation of L. casei BL-23 culture supernatant produced less amount of a clear pellets. Microvesicle diameter was 34?94 nm and the zeta potential resulted -8,7±1,9 mV in PBS at 25 °C (n=4). Microvesicles contained cytoplasmic constituents such as DNA, RNA and proteins (n=2). Proteins were separated by SDS- electrophoresis according to their molecular weight and there were identified two major vesicle protein bands (n=2).In the present work, we inform for the first time that L. casei BL-23 actively produced extracellular vesicles with a negative zeta potential, i.e. ensuring colloidal stability. Microvesicle was similar to those previously reported from others strains in terms of diameter and citoplasmatic content such as DNA, RNA and proteins.