Ligilactobacillus-Derived Extracellular Vesicles Inhibit Growth and Virulence of Enteric Pathogens

Bacterial intra-kingdom communication involves the secretion of outer membrane vesicles as signaling carriers to the target cells. However, limited research exists on extracellular vesicles (EVs) from Gram-positive gut bacteria, their interactions with enteric pathogens, and potential inhibitory effects. In this study, we characterized the structure, protein content, and inhibitory effects of EVs from three new potential probiotic gut symbionts, Ligilactobacillus salivarius UO.C109, Ligilactobacillus saerimneri UO.C121, and Ligilactobacillus salivarius UO.C249. EVs were isolated and characterized using three different methods (ultracentrifugation, density gradient purification, and size exclusion chromatography). The purity, dose-dependency, structure, and proteome profiles of the purified EVs were evaluated. Antibacterial and anti-virulence activities of EV subpopulations were assessed against Salmonella enterica serovar Typhimurium and Campylobacter jejuni. EVs from Lg. salivarius UO.C109 and Lg. saerimneri UO.C121 showed inhibitory activity against S. Typhimurium, whereas EVs from Lg. salivarius UO.C249 inhibited the growth of C. jejuni. Notably, purified F3 fraction exhibited the highest inhibitory activity and was enriched in lysin motif (LysM)-containing proteins, peptidoglycan hydrolases, peptidoglycan recognition proteins (PGRPs), and metallopeptidases, which have been shown to play a prominent role in antimicrobial activities against pathogens. F3 had the highest concentration (73.8%) in the 80-90 nm size compared to the other fractions. Gene expression analysis revealed that EVs from Lg. salivarius UO.C109 and Lg. saerimneri UO.C121 downregulated adhesion and invasion factors in S. Typhimurium. Likewise, EVs from Lg. salivarius UO.C249 reduced pathogenicity gene expression in C. jejuni. This study highlighted the potential of gut bacterial EVs as therapeutic agents against enteric pathogens.