Lactobacillus casei BL-23 produces membrane-derived vesicles containing DNA, RNA and proteins.

DC MARTÍNEZ-CASILLAS; MARIANA PIURI; ANA PAULA DOMÍNGUEZ-RUBIO; HORACIO CORTI; FEDERICO COLUCCIO-LESKOW; OSCAR E PÉREZ

La Plata

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications; 2016

Universidad Nacional de La Plata y CONICET

Outer membrane vesicles are a form of intracellular communication used by bacteria, eukaryotes, and archaea. Despite its nanometric size, between 10-400 nm, they are also known as microvesicles (MV) in Gram-positive bacteria. Little is known regarding their production and its cargo. They have been proposed to be involved in signaling between probiotic bacteria and their mammalian hosts, gaining a central role in the microbiome-gut-brain axis. The aim of this study was to isolate microvesicles from Lactobacillus casei BL-23, a probiotic bacterium and to carry out their characterization.L. casei BL-23 were grown in Man-Rogosa-Sharpe (MRS) medium at 37°C for 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 contamination with other bacterial cells during the concentration of the sample. Bacillus subtilis 168 was used as a positive control for its known micovesicles production by growing in brain heart infusion (BHI) medium at 37°C under continuous agitation for 18 h (100 ml). 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 UV absorbance using a NanoDrop and protein content was quantified by Lowry and analyzed by SDS polyacrylamide gel electrophoresis (10% resolving gel) followed by coomassie blue staining. Microvesicles were resuspended in PBS and their size and zeta potential were characterized by dynamic light scattering (DLS) (Zetasizer) and atomic force microscopy (AFM). 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 a small, clear pellet. Microvesicles contained cytoplasmic constituents such as DNA, RNA and proteins (n=2). Two major proteins were separated by SDS-electrophoresis (n=2). Microvesicle diameter was 47±3 nm (DLS, n=4) and 33±3 nm (AFM). The zeta potential resulted -8.7±1,9 mV in PBS at 25 °C (DLS, n=4).In the present work, we design an efficient protocol to isolate microvesicles from L. casei BL-23 and inform for the first time that L. casei BL-23 actively produced extracellular vesicles with a negative zeta potential, i.e. ensuring colloidal stability. Microvesicles are similar to those previously reported from others strains in terms of diameter and citoplasmatic content such as DNA, RNA and proteins.