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

(Pages: 344-349)
Sara Ahmadi Badi, Ph.D, 1Arfa Moshiri, Ph.D, 2,3Fatemeh Ettehad Marvasti, M.Sc, 1,4Mojtaba Mojtahedzadeh, Ph.D, 5Vida Kazemi, Ph.D., 6,*Seyed Davar Siadat, Ph.D., 4,7,*
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
Cancer Department, Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Laboratory of Experimental Therapy in Oncology, G. Gaslini Children’s Hospital, Genoa, Italy
Microbiology Research Centre, Pasteur Institute of Iran, Tehran, Iran
Department of Pharmacotherapy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Medicinal Plants Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran, Tehran, Iran
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
Cancer Department, Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Laboratory of Experimental Therapy in Oncology, G. Gaslini Children’s Hospital, Genoa, Italy
Microbiology Research Centre, Pasteur Institute of Iran, Tehran, Iran
Department of Pharmacotherapy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Medicinal Plants Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran, Tehran, Iran
*Corresponding Addresses: P.O.Box: 14155-6451 Medicinal Plants Research Centre Faculty of Pharmacy Tehran University of Medical Sciences Tehran Iran P.O.Box: 113169-43551 Mycobacteriology and Pulmonary Research Department Pasteur Institute of Iran Tehran Iran Emails:dv.kazemi@gmail.com,d.siadat@gmail.com
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Ahmadi Badi S, Moshiri A, Ettehad Marvasti F, Mojtahedzadeh M, Kazemi V, Siadat SD. Extraction and evaluation of outer membrane vesicles from two important gut microbiota members, bacteroides fragilis and bacteroides thetaiotaomicron. Cell J. 2020; 22(3): 344-349. doi: 10.22074/cellj.2020.6499.

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.