Extraction and Evaluation of Outer Membrane Vesicles from Two
Important Gut Microbiota Members, Bacteroides fragilis and
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.
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
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.
Spherical OMVs with diameters of 30-110 nm were produced. The OMVs had different protein profiles. The
LPS concentrations of the
The endotoxin concentrations of the spherical OMVs from
Gut microbiota are a diverse and complicated
microbial community that colonize the gastrointestinal
tract (GI) (1). The gut microbiota have beneficial
roles in the host that include colonization resistance,
assist with digestion, harvest energy from the diet,
metabolism of nutrients, and immune system regulation
(2, 3). This microbial community consists of bacteria,
archea, viruses, fungi, and protozoa (4). Bacteria are
the dominant microbial population. Bacteroidetes
and Firmicutes constitute two major bacterial phyla in
gut microbiota (5). Bacteroidetes are gram-negative
bacteria are abundant and diverse in gut microbiota
(6). These bacteria are found at high frequencies (up
to 1011 cells/g) in intestinal material (7). Bacteroidetes
are important in host metabolism since they degrade
proteins and complex carbohydrates (8). Moreover, the
OMVs are nano-sized vesicles (20 to 250 nm)
secreted by gram-negative bacteria under various
conditions during all growth phases (13). The
bacterial OMVs were first reported in the 1970s when
In the normal state, the gut microbiota-host interactions are balanced due to desirable functions of the gut barrier. Many factors such as intestinal epithelial cell integrity, tight junction proteins, and the mucus layer maintain proper gut barrier functions that control gut microbiota-host interactions (8, 21). In recent studies, it was demonstrated that beneficial intestinal commensal bacteria might have adverse effects on the host, while their OMVs maintain beneficial effects on the host functions in leaky gut syndrome, which is characterized by disruption of gut barrier integrity and increased intestinal epithelial cell permeability (21, 22).
The roles of OMVs in gut microbiota homeostasis and
host functions are under investigation. In this regard,
the study of OMVs production from key gut microbiota
members and their properties could contribute to an
understanding of the gut microbiota-host interactions.
Accordingly, in the present study, the OMVs from
two important gut microbiota members,
Materials and Methods
Bacterial strains and growth conditions
In this experimental study,
Outer membrane vesicle purification
After an overnight incubation under anaerobic conditions, OMVs were isolated as described previously (23). Briefly, 500 mL of the bacterial cultures were centrifuged at 6000 g at 4˚C. The cell pellets were washed twice in phosphatebuffered solution (PBS). Then, the cell pellets were resuspended in a 9% sodium chloride solution. The cell suspensions were homogenized and concentrated by centrifugation at 2900 g for 1 hour at 4˚C. The total wet weight of cell pellets was calculated and resuspended in 7.5 times the wet weight of 0.1 M tris-10 mM ethylenediaminetetraacetic acid (EDTA) buffer (Sigma-Aldrich, USA). The vesicles were extracted by the addition of 1/20th the volume of 0.1 M Tris, 10 mM EDTA, and sodium deoxycholate (100 g/L) buffer (Merck, Germany). OMVs were separated from cell debris at 20 000 g for 60 minutes at 4˚C. The supernatant that contained the vesicles was centrifuged at 20 000 g for 120 minutes at 4˚C in order to concentrate the vesicles. The pellet was resuspended in 10 mM EDTA, 0.1 M Tris, and sodium deoxycholate (5 g/L) buffer, and the suspension was centrifuged again at 20000 g for 120 minutes at 4˚C. The concentrated OMVs were resuspended in a 3% sucrose solution. Finally, the suspension was filtered through a 0.22-µm polyvinylidene difluoride filter (Millipore, Billerica, MA, USA).
Scanning electron microscopy
The OMVs were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in PBS (Sigma-Aldrich, USA). After washing with PBS, dried samples were coated with gold by a sputter coater (SBC-12, KYKY, China) using a physical vapor deposition method. The prepared samples were examined by SEM (KYKYEM3200, KYKY, China) (24).
Determination of the outer membrane vesicle protein content and pattern
To estimate the amount of total proteins,
purified OMVs were analyzed using a NanoDrop
spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the Bradford assay with coomassie
brilliant blue, at 590 nm. The protein contents of B.
Quantification of outer membrane vesicle endotoxins
The content and biological activity of the OMVs endotoxins was measured using the Pierce™ LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, USA) according to the manufacturer’s instructions. Briefly, the microplate was incubated for 10 minutes at 37˚C. We dispensed 50 μL each of the samples and standards into the microplate wells and allowed them to incubate for 5 minutes at 37˚C. Then, 50 μL of LAL was added to each well. The plate was incubated at 37˚C for 10 minutes. We added 100 μL of substrate solution to the microplate and incubated it for 6 minutes at 37˚C. Finally, 50 μL of stop reagent (25% acetic acid) was added to each well and we measured the optical density (OD) the wells at 405-410 nm on a plate reader. The amount of endotoxin in the samples was calculated using the standard curve (25).
Zeta (ζ-P) potential measurement
The OMVs were prepared by sonication in 35 kHz for 3 minutes (Bandelin ultrasonic bath). The zeta (ζ-P) potential of the OMVs was assessed using a Malvern Zetasizer Nano ZEN3600 (Malvern Instruments, United Kingdom).
The human epithelial cell line, IBRC C10094 Caco- 2 (Iranian Biological Resource Centre) was cultured in Dulbecco’s modified eagle medium (DMEM/high glucose, Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Gibco USA), 1% non-essential amino acids (Gibco, USA), and 1% penicillin/ streptomycin (Gibco USA) and incubated at 37˚C in a 5% CO2 atmosphere. Caco-2 cells were seeded at a density of 2×104 cells/well in a 96-well culture plate and incubated overnight before the OMVs treatment. The cells were treated with OMVs (50 µg/ml) and incubated for 24 hours. The cell culture medium was discarded and replaced by fresh medium. After 4 hours of incubation, the cells were incubated with 100 µl medium with MTT for 4 hours. After incubation, the medium was removed and 100 µl dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals that formed in the living cells. The absorbance was measured at 570 nm (26).
|- 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).|
Our results based on SDS-PAGE showed that OMVs
The LAL test was performed to detect and quantify
the amount of endotoxin from the OMVs. By using
the standard curve (Fig .3,), we determined that the LPS
concentration of OMVs from
ζ-P of these vesicles was measured by electrophoretic
light scattering (ELS). Both
|- 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).|
|- The zeta potential (ζ-P) distribution of Bacteroides fragilis (B. fragilis) derived-outer membrane vesicles (OMVs).|
|- The zeta potential (ζ-P) distribution of Bacteroides thetaiotaomicron (B. thetaiotaomicron) derived-outer membrane vesicles (OMVs).|
The human intestinal epithelial cell line, Caco-2, was used
to study the effects of
It is well documented that gut microbiota has a profound
effect on host health and diseases. The communication
between gut microbiota and the host is mainly dependent
on the microbial released factors, which could access
intestinal epithelial cells (27). OMVs have considerable
roles in putative communication since they interact
with host cells through their various components, such
as bacterial outer membrane determinants, hydrolytic
enzymes, and signaling molecules (11). Among numerous
microbial species that colonize the GI, the Bacteroidetes
comprise the most gram-negative bacteria. Both
OMVs originate from the outer membrane of gramnegative bacteria and are released to the extracellular
milieu as small particles by bilayer spherical shaped
vesicles. Several pathogenic and non-pathogenic bacteria
are proposed to produce OMVs, such as
The evaluation of the physicochemical properties
of OMVs is a characteristic examination marker.
Morphology, size, and protein content of
Recently, several studies have reported the potential
applications of OMVs as novel vaccine adjuvants and
cancer immunotherapeutic agents (33, 34). As mentioned,
a previous work and our studies (unpublished data) have
noted potent roles for
Bacteroidetes phylum is the major LPS-producing
bacteria in gut microbiota. In this study, the LPS
concentration of OMVs was assessed by the LAL test.
This test, which is known as the bacterial endotoxin test
(BET), is performed for over 90% of pyrogenic tests.
This measurement is significant for OMVs application
as therapeutic agents. To our knowledge, this is the first
report of the LPS concentration of
Surface charges of vesicles are measured and reported
as ζ-P potentials. The particle charge has a determinative
role in the physical stability of suspensions. Generally,
particles which have ζ-P potentials more positive than
+30 mV or more negative than -30 mV are stable (36).
Measurement of ζ-P potential could provide information
about the aggregation and stability of OMVs in sucrose
solution. In this study, the ζ-P potentials of
Intestinal epithelial cells are the interface between gut
microbiota and host interactions. Therefore, the effects
According to our results,
This research was funded by Iran Biotech Fund grant 94/10243 and Pasteur Institute of Iran. The authors would like to thank our colleagues at the Pasteur Institute of Iran. There is no conflict of interest in this study.
S.A.B., A.M., F.E.M., V.K., M.M., S.D.S.; Contributed to conception and design of all experimental work, data and statistical analysis, and interpretation of data. S.D.S., V.K.; Were responsible for overall supervision. All authors read and approved the final manuscript.