Isolation and Characterization of Extracellular Vesicles Secreted In Vitro by Porcine Microbiota

The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic cells for intercellular communication, survival, and pathogenesis. Previous studies have illustrated the presence of EVs in supernatants from pure cultures of bacteria, including Gram-positive and Gram-negative glycan-degrading gut commensals. However, the isolation and characterization of EVs secreted by a complex microbial community have not been clearly reported. In a recent paper, we showed that wood-derived, complex beta-mannan, which shares a structural similarity with conventional dietary fibers, can be used to modulate the porcine gut microbiota composition and activity. In this paper, we investigated the production, size, composition, and proteome of EVs secreted by pig fecal microbiota after 24 h enrichment on complex beta-mannan. Using transmission electron microscopy and nanoparticle tracking analysis, we identified EVs with an average size of 165 nm. We utilized mass spectrometry-based metaproteomic profiling of EV proteins against a database of 355 metagenome-assembled genomes (MAGs) from the porcine colon and thereby identified 303 proteins. For EVs isolated from the culture grown on beta-mannan, most proteins mapped to two MAGs, MAG53 and MAG272, belonging to the ordersClostridialesandBacilli, respectively. Furthermore, the MAG with the third-most-detected protein was MAG 343, belonging to the orderEnterobacteriales. The most abundant proteins detected in the beta-mannan EVs proteome were involved in translation, energy production, amino acid, and carbohydrate transport, as well as metabolism. Overall, this proof-of-concept study demonstrates the successful isolation of EVs released from a complex microbial community; furthermore, the protein content of the EVs reflects the response of specific microbes to the available carbohydrate source.