Extraction and Evaluation of Outer Membrane Vesicles from Two Important Gut Microbiota Members, Bacteroides fragilis and Bacteroides thetaiotaomicron.

OBJECTIVE: The gastrointestinal tract (GI) is colonized by a complex microbial community of gut microbiota. Bacteroides spp. have significant roles in gut microbiota and they host interactions by various mechanisms, including outer membrane vesicle (OMVs) production. In the present study, we extracted and assessed Bacteroides fragilis (B. fragilis) and Bacteroides thetaiotaomicron (B. thetaiotaomicron) OMVs in order to evaluate their possible utility for in vivo studies. MATERIALS AND METHODS: In this experimental study, OMVs extraction was performed using multiple centrifugations and tris-ethylenediaminetetraacetic acid (EDTA)-sodium deoxycholate buffers. Morphology, diameter, protein content, profile, and lipopolysaccharide (LPS) concentrations of the OMVs were assessed by scanning electron microscopy (SEM), nanodrop, Bradford assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the Limulus Amoebocyte Lysate (LAL) test, respectively. Zeta potential (ζ-P) was also assessed. The viability effect of OMVs was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in Caco-2 cells. RESULTS: Spherical OMVs with diameters of 30-110 nm were produced. The OMVs had different protein profiles. The LPS concentrations of the B. fragilis and B. thetaiotaomicron OMVs were 1.80 and 1.68 EU/mL, respectively. ζ-P of the B. fragilis OMVs was -34.2 mV and, for B. thetaiotaomicron. it was -44.7 mV. The viability of Caco-2 cells treated with OMVs was more than 95%. CONCLUSION: The endotoxin concentrations of the spherical OMVs from B. fragilis and B. thetaiotaomicron were within the safe limits. Both OMVs had suitable stability in sucrose solution and did not have any cytotoxic effects on human intestinal cells. Based on our results and previous studies, further molecular evaluations can be undertaken to design OMVs as possible agents that promote health properties.

Induction effects of Faecalibacterium prausnitzii and its extracellular vesicles on toll-like receptor signaling pathway gene expression and cytokine level in human intestinal epithelial cells.

A single layer of epithelial cells creates an interface between the host and microorganisms colonizing the gastrointestinal tract. In a healthy intestine, commensal bacteria and their metabolites can interact with epithelial cells as they are identified by Toll-like receptors (TLRs); This interaction results in homeostasis and immune responses. The present study aimed at evaluating Faecalibacterium prausnitzii- and extracellular vesicles (EVs)-induced expression of involved genes in TLRs signaling pathway and cytokines production in Caco-2 cell line. In this study, Caco-2 cell line was treated with F. prausitzii and its EVs. Using the protein levels of 12 cytokines were also evaluated by ELISA assay. F. prausnitzii induced upregulation in FOS, JUN, TNF-α, NFKB1, TLR3, IKBKB and CD86 genes. Furthermore, stimulation of Caco-2 cells with EVs derived from F. prausnitzii induced upregulation of CXCL8, CCL2, FOS, MAP2K4, TLR7, TLR3, IRF1, NFKBIA and TNF-α genes. Based on ELISA assay, Caco-2 cells treated with F. prausnitzii and its EVs showed a significant increase in TNF-α, IL-4, IL-8, and IL-10 expression and significant decreased in IL-1, IL-2, IL-6, IL-12, IL-17a, IFN-γ compared to the control group (P < 0.05). In conclusion, EVs derived from F. prausnitzii showed greater efficacy in decreasing the inflammatory cytokines and increasing the anti-inflammatory cytokines, compared to F. prausnitzii. Our findings can be used as a theoretical model for EVs application in the potential treatment of inflammation.