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. 2020 Oct;22(3):344-349.
doi: 10.22074/cellj.2020.6499. Epub 2019 Dec 15.

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

Sara Ahmadi Badi  1 , Arfa Moshiri  2   3 , Fatemeh Ettehad Marvasti  1   4 , Mojtaba Mojtahedzadeh  5 , Vida Kazemi  6 , Seyed Davar Siadat  4   7
Affiliations

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

Sara Ahmadi Badi et al. Cell J. 2020 Oct.

Abstract

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.

Keywords: Bacteroides fragilis; Bacteroides thetaiotaomicron; Gut Microbiota.

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Conflict of interest statement

There is no conflict of interest in this study.

Figures

Fig 1
Fig 1
Scanning electron microscopy (SEM) of outer membrane vesicles (OMVs). A. Bacteroides fragilis (B. fragilis) and B. Bacteroides thetaiotaomicron (B. thetaiotaomicron) derived OMVs (SEM at magnification: ×40 KX).
Fig 2
Fig 2
Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) showing the protein profile of outer membrane vesicles. The protein bands of A. Bacteroides fragilis (B. fragilis) and B. Bacteroides thetaiotaomicron (B. thetaiotaomicron) derived- outer membrane vesicles (OMVs) at 0.35 mg/ ml and 0.45 mg/ml protein concentrations, respectively.
Fig 3
Fig 3
The standard curve of the limulus amoebocyte lysate (LAL) test to determine the endotoxin levels of Bacteroides fragilis (B. fragilis) and Bacteroides thetaiotaomicron (B. thetaiotaomicron) derived-outer membrane vesicles (OMVs).
Fig 4
Fig 4
The zeta potential (ζ-P) distribution of Bacteroides fragilis (B. fragilis) derived-outer membrane vesicles (OMVs).
Fig 5
Fig 5
The zeta potential (ζ-P) distribution of Bacteroides thetaiotaomicron (B. thetaiotaomicron) derived-outer membrane vesicles (OMVs).
See this image and copyright information in PMC

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