Abstract
In recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The aim of this study was to characterize the EVs released by the dairy isolate Lactiplantibacillus plantarum BGAN8 and to gain an insight into the putative mechanism of EVs uptake by intestinal epithelial cells. The cryo-TEM observation undoubtedly demonstrated the release of EVs (20 to 140 nm) from the surface of BGAN8, with exopolysaccharides seems to be part of EVs surface. The proteomic analysis revealed that the EVs are enriched in enzymes involved in central metabolic pathways, such as glycolysis, and in membrane components with the most abundant proteins belonging to amino acid/peptide ABC transporters. Putative internalization pathways were evaluated in time-course internalization experiments with non-polarized HT29 cells in the presence of inhibitors of endocytic pathways: chlorpromazine and dynasore (inhibitors of clathrin-mediated endocytosis—CME) and filipin III and nystatin (disrupting lipid rafts). For the first time, our results revealed that the internalization was specifically inhibited by dynasore and chlorpromazine but not by filipin III and nystatin implying that one of the entries of L. plantarum vesicles was through CME pathway.
Highlights
Archaea, Bacteria and Eukarya produce and release extracellular vesicles (EVs) that are membrane-contained structures playing a relevant role in the inter-communications established among these tree domains of life; there are still many unsolved questions on this field[1]
In the case of bacteria, most studies have been performed with Gram-negatives and two types of bacterial EVs have been described: outer membrane vesicles (OMVs), derived from outer membrane that enclose periplasmic components, and outer-inner membrane vesicles (O-IMVs) formed by the protrusion of both, outer and cytoplasmic, membranes that could be enriched in cytoplasmic c omponents[3,4]
Bacterial EVs or membrane vesicles (MV); some authors propose the name of cytoplasmic (CMVs)[7], whereas others use the term MV to refer to empty vesicles, for example right side-out and inside-out MV from Escherichia coli applied for studying mechanism of transport in different b acteria[8]
Summary
Bacteria and Eukarya produce and release extracellular vesicles (EVs) that are membrane-contained structures playing a relevant role in the inter-communications established among these tree domains of life; there are still many unsolved questions on this field[1]. Bioengineering bacterial EVs produced by commensal bacteria has been proposed as a putative method for the delivery of biotherapeutics to the intestinal tract[22] Another open question in this field, related to the purification of bacterial EVs within a pool of eukaryotic EVs from human body fluids has been recently a ddressed[23], which will open an avenue for research on the application of these cargo vehicles. It has been reported the isolation and proteomic characterization of Lactobacillus-derived EVs vesicles from three strains of the probiotic species L. casei, L. acidophilus and L. reuteri[24]. The aim in the current work is to achieve the characterization of the EVs released by the potential probiotic L. plantarum BGAN8, an exopolysaccharide (EPS) producing strain, as well as, given the scarce information about the internalization pathways in Gram-positives, to gain insight about the putative mechanism of probiotic EVs uptake by intestinal epithelial cells
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