Figure - available from: Applied and Environmental Microbiology
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Proteome profiles of cell-free supernatant (CFS) and extracellular vesicles (EVs). (A) Venn diagram showing the number of identified proteins in CFS and EVs, (B) volcano plot showing the difference in abundance of common proteins between CFS and EVs, (C) GO term enrichment analysis of detected proteins for biological function, and (D) localization of detected proteins in cellular components.
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Campylobacter jejuni (C. jejuni) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BA...
Citations
... The cell-free supernatant was prepared from cell culture and divided into three portions for testing with three different methods: ultracentrifugation, Optiprep density gradient, and size exclusion chromatography. EVs were prepared using the following three methods as previously described [16,44,56], with some modifications. Based on our results on EV purity, size exclusion chromatography was selected for EV extraction in subsequent experiments. ...
... The supernatants were centrifuged at 10,000 rpm for 30 min to remove cell debris. Next, EVs were precipitated from the supernatant by ultracentrifugation at 40,000 × g for 70 min at 4 °C (Beckman Optima L-100 XP Ultracentrifuge Centrifuge, Rotor: 90 Ti Rotor, k-factor of 25, Beckman Coulter, Inc., Brea, CA, USA) and washed three times with PBS using the same centrifugation setting [16]. After pelleting, the EVs were resuspended in PBS (final volume of 1.5 mL) and stored at − 80 °C until use. ...
... Our findings indicated that the majority of the tested virulence factors associated with the invasion and adhesion of selected pathogens were downregulated. In our previous study, we observed that the adhesion and invasion abilities of S. Typhimurium and C. jejuni were reduced in the presence of cell-free supernatants from selected probiotic strains [16,47]. The results of this study confirmed the suppression of most virulence genes in both pathogens (avrA, invA, and hilA in S. Typhimurium and CdtA, CdtB, CdtC, ciaB, and cadF in C. jejuni). ...
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.
