Background: The probiotic Lactobacillus reuteri is effective against infantile colic, alleviation of eczema and Helicobacter pylori colonization. L.reuteri develops biofilm in vitro, producing factors that give health benefit to the host although the mechanism by which commensal bacteria communicate with the host remains unclear. The purpose of this study was to detect and characterize membrane vesicles (MVs), bilayer structures containing several molecules generated by bacteria, produced by L.reuteri DSM 17938. Methods: The structure of MVs was evaluated by transmission and scanning electron microscope analysis. MVs were subsequently isolated by biofilm (bMVs) and planktonic (pMVs) phenotypes by ultracentrifugation and physicochemically characterized by dynamic light scattering (DLS) analysis. An enzymatic treatment was performed to determine MVs composition. eDNA was detected and quantified using the Quant-iTTMPicoGreendsDNA assay and NanoDropUV-VIS spectrophotometer. Proteins associated with MVs were extracted and quantified by BCA assay. Results: PicoGreen showed that eDNA was associated with MVs and that its concentration is higher in bMVs than in pMVs, although an inverse correlation was found in the protein concentration, suggesting a different role of MVs in the two phenotypes. The enzymatic treatment showed that lipids and proteins represent the main structural components of MVs. The DLS analysis demonstrated that L.reuteri generates MVs with sizes in the nanometer range and a broad size distribution. Conclusions: L.reuteri produces MVs whose main components are lipids and proteins; eDNA is also associated to MVs. The biological activity and composition of MVs may represent the starting point for future applications in the development of vesicles-based therapeutic systems.
Lactobacillus reuteri:production and characterization of membrane vesicles for future health applications
GRANDE, ROSSELLA;CELIA, Christian;DI MARZIO, Luisa;DI MARCANTONIO, Maria Carmela;LOCATELLI, Marcello;SAVINO, LUCA;MURARO, Raffaella;MINCIONE, Gabriella
2016-01-01
Abstract
Background: The probiotic Lactobacillus reuteri is effective against infantile colic, alleviation of eczema and Helicobacter pylori colonization. L.reuteri develops biofilm in vitro, producing factors that give health benefit to the host although the mechanism by which commensal bacteria communicate with the host remains unclear. The purpose of this study was to detect and characterize membrane vesicles (MVs), bilayer structures containing several molecules generated by bacteria, produced by L.reuteri DSM 17938. Methods: The structure of MVs was evaluated by transmission and scanning electron microscope analysis. MVs were subsequently isolated by biofilm (bMVs) and planktonic (pMVs) phenotypes by ultracentrifugation and physicochemically characterized by dynamic light scattering (DLS) analysis. An enzymatic treatment was performed to determine MVs composition. eDNA was detected and quantified using the Quant-iTTMPicoGreendsDNA assay and NanoDropUV-VIS spectrophotometer. Proteins associated with MVs were extracted and quantified by BCA assay. Results: PicoGreen showed that eDNA was associated with MVs and that its concentration is higher in bMVs than in pMVs, although an inverse correlation was found in the protein concentration, suggesting a different role of MVs in the two phenotypes. The enzymatic treatment showed that lipids and proteins represent the main structural components of MVs. The DLS analysis demonstrated that L.reuteri generates MVs with sizes in the nanometer range and a broad size distribution. Conclusions: L.reuteri produces MVs whose main components are lipids and proteins; eDNA is also associated to MVs. The biological activity and composition of MVs may represent the starting point for future applications in the development of vesicles-based therapeutic systems.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
