Proteomic characterization of extracellular vesicles from 12 commensal bacterial species

Bacterial extracellular vesicles (bEVs) produced by intestinal commensal bacteria mediate host-microbe interactions, but their proteomes have been explored in only a small number of species. We characterized bEVs from in vitro cultures of 12 species of Actinomycetota, Bacillota (Firmicutes), Bacteroidota, Fusobacteria, Pseudomonadota, and Verrucomicrobia. This is the first report of bEVs from Veillonella magna , an exceptional gram-negative Bacillota, and the gram-positives Peptostreptococcus russellii and Turicibacter sanguinis . The morphology and protein subcellular localization patterns of bEVs reflected the envelope structures of their parent bacteria. Notably, V. magna may be able to produce both outer membrane and cytoplasmic membrane vesicles. The proteome compositions were dictated by phylogeny, suggesting largely non-selective packaging of proteins. Annotation of protein functions indicated roles in nutrient metabolism and transport, and in host immune system modulation. Peptidoglycan modifying enzymes and the abundant bacteriophage proteins in Enterobacter cloacae and Limosilactobacillus reuteri bEVs may be involved in vesicle biogenesis. The functions of many abundant bEV proteins are unknown. The most abundant bEV proteins were largely species-specific, but we identified several conserved proteins that may be used as markers to distinguish commensal bEVs from host EVs. Comparison to previous in vitro and fecal metaproteomics data indicates that bEV proteome compositions are reproducible.