1
Production of outer membrane vesicles by the human pathogen Burkholderia
cenocepacia: purification and preliminary characterization
Rui Filipe Brás Teles Martins
Abstract
Cystic Fibrosis (CF) is an autossomal recessive disease resulting from mutations in the cystic
fibrosis transmembrane conductance regulator (CFTR) gene, leaving affected patients more
susceptible to infection by bacteria. One group of bacteria, the Burkholderia cepacia complex (Bcc), is
responsible for dangerous infections in these patients, due to their resistance to multiple antibiotics
being therefore necessary to develop alternative treatments. Outer membrane vesicles (OMV) have
been used for the development of vaccines against pathogens such as Neisseria meningitidis and
Burkholderia pseudomallei, with an OMV vaccine against N. meningitidis already available in the
market. This study aimed at the development of a scalable production method of OMVs, in order to
allow their use in vaccine development studies. OMVs from B. cenocepacia K56-2 were isolated and
purified by size exclusion chromatography (SEC), however the final purity was not high, with OMVs
still contaminated with pili and flagella. This prompted the development of an alternative strategy to
obtain purified OMVs in which the strain used for OMV production was changed to B. cenocepacia
AU1054 non piliated strain. With this strain it was possible to obtain contaminant-free OMVs after the
isolation process, which was confirmed by transmission electron microscopy (TEM). OMVs obtained
from B. cenocepacia AU1054 were used to perform cytotoxic assays in 16HBE14o- human bronchial
epithelial cells and in Galleria melonella. 16HBE14o- cells suffered reduced survival upon exposure to
AU1054-derived OMVs, however this result was not reproduced in G. melonella, with all injected
caterpillars surviving the assay.
Keywords: Outer membrane vesicles, Cystic fibrosis, purification, Burkholderia cepacia complex, Size
exclusion chromatography, cytotoxicity
Introduction
Cystic Fibrosis (CF) is an autossomal recessive
disease resulting from mutations in the cystic
fibrosis transmembrane conductance regulator
(CFTR) [53], among other problems CF patients
suffer from thicker mucus in the lungs [3; 62],
which leaves these patients more susceptible to
bacterial infections [62; 66]. Among the bacterial
infections these patients may suffer it is included
infection by species belonging to the
Burkholderia cepacia complex (Bcc), which
despite the small percentage of infections in CF
patients being caused by these bacteria [36],
leads to the development of problematic
infections [21].
Bcc is a group of 20 closely related species of
bacteria, to which Burkholderia cenocepacia,
among other species, belongs [15].
Infection with Bcc bacteria usually causes
reduced lung function, and in a subset of patients
it may cause necrotizing pneumonia and a rapidly
fatal septicaemia termed “Cepacia Syndrome”
[21]. The severity of Bcc infection is explained by
several characteristics of this group of bacteria,
such as the ability of resisting exposure to a wide
range of antibiotics [16; 37], ability to form
biofilms [12], among others [13; 46; 64], which
2
facilitate the infection with Bcc strains and difficult
eradication of the bacteria. Furthermore, these
species are easily spread by contact between
infected CF patients [25; 36], saline solutions
[14], contaminated hospital equipment [32] and
even from the environment [38].
Due to the severity of infections caused in CF
patients and the poor outcome of infected
patients, as well as the resistance to multiple
antibiotics, it becomes interesting to investigate
possible alternative treatments and mechanisms
to avoid the infection.
There have been several attempts for the
development of a vaccine against species of the
Bcc, several strategies have been employed in
order to achieve this objective [9; 13], with the
most promising results being achieved with outer
membrane proteins (OMP) [5; 43]. However
strategies employed were not able to achieve the
desired efficiency.
In the last decade there has been an increasing
interest in the use of outer membrane vesicles
(OMV) for the development of vaccines, since
these vesicles exhibit some characteristics which
make them interesting candidates for vaccine
development, such as the presence of multiple
antigens, being acellular and stability at 4ºC [19;
51]. Studies in vaccine development using OMVs
have been performed in several bacteria species
[2; 27; 50], with one OMV-based vaccine against
N. meningitids already in the market [24] and
studies for a vaccine against B. pseudomallei
well advanced [47; 49]. However there are no
studies regarding development of OMV vaccines
against Bcc species.
OMVs are formed by the blebbing of the outer
membrane of bacterial cell wall, they do not result
from cell death or lysis, and are produced during
all phases of growth [7; 44; 69]. OMVs are
spherical with a bilayered membrane, with sizes
ranging from 50 to 250nm and produced by
organisms from the three branches of the tree of
life. They are produced by Gram-negative and
Gram-positive bacteria [28; 50], Fungi [48],
archaea [18], and even in parasites [55], in which
seems to be an evolutionary conserved
mechanism [35]. These vesicles have several
biological functions which provide an advantage
to OMV-producing bacteria, they provide
resistance to antibiotics [42; 52], provide
protection from environmental stresses [44],
deliver virulence factors [28; 65], among others
[6; 34].
OMVs may be isolated from in vitro cultures.
For the isolation of OMVs the most usual process
is to perform a bacterial culture until late
logarithmic phase, followed by a series of
centrifugations and filtrations in order to remove
bacteria and concentrate the vesicles [28].
However in order to obtain purified OMVs it is
necessary to apply a purification step, usually
OMVs are purified by density gradient
centrifugation [28]. In order to evaluate the purity
of the OMV preparation the method most
commonly used is Transmission Electron
Microscopy (TEM), which allows to visualize the
vesicles and other contaminants, if they are
present [28; 41; 44].
This study will evaluate the purity of a B.
cenocepacia OMV preparation. Followed by
optimization of OMVs production and purification
methods. Purified OMVs will be used for
cytotoxicity assays, in order to perform a
preliminary evaluation of OMV cytotoxicity.
Materials and methods
2.1 Culture conditions and bacterial strains- 10
different bacterial strains were used in this work (Table
1), the strains were maintained frozen in glycerol at -
80ºC. When necessary strains were defrosted to Luria-
Bertani (LB) plates.
3
OMV production and purification- Vesicles were
purified by a method adapted from Huang et al. [29]
and Allan et al. [1]. Bacteria were grown in 2 L of LB
medium, they were incubated with an initial OD640nm of
0.1 and the culture was stopped when OD640nm
reached 1.5. Cells were removed by pelleting (10,000
x g, 10 min.). Supernatants were filtered through a
0.45µm cellulose membrane (Milipore) and
concentrated via a 100-kDa tangential filtration
concentration unit (GE Healthcare) to approximately 70
mL. The OMVS present in the retentate were pelleted
by ultracentrifugation (150,000 x g, 3h), resuspended
in 1.5 mL of PBS (pH 7.4). Ressuspended vesicles
were filtered through a 0.2 µm polyethersulfone
membrane (Whatman, GE Healthcare).
Size exclusion chromatography (SEC)- Sephacryl
S-1000 Superfine chromatography (GE Healthcare
Life Sciences ) was performed using an Äkta™ Purifier
10 system, using Tricorn™ 5/100 or Tricorn™ 10/600
columns. 1.5mL of OMV solution were loaded using a
1 mL loop with a flow rate of 0.5 mL/min. The flow-
through and elute were continuously collected in 0.5
mL fractions in a FRAC 950 Fraction collector (GE
Healthcare).
Optimization of growth parameters, conditions
and strains used- In order to optimize the OMVs
production process the OMVs production was
evaluated in different Bcc isolates. The selected
isolates were cultured in 150 mL of LB medium. The
initial OD640nm was 0.1 and the culture was maintained
until OD640nm reached 1.5. Analysis of OMV production
in different points of the growth was performed using
the same protocol, differing only in the volume used
(500 mL) and the length of the culture (4h, 10h, 16h
and OD640nm 1.5).
OMVs production under different growth conditions
and mediums was performed using a volume of 500
mL of LB. Two different mediums were used, regular
LB medium and LB medium with 300 mM NaCl.
Culture was performed until OD640nm reached 1.5,
initial OD640nm was 0.1.
Microaerophilic conditions were achieved using an
anaerobic box (Mitsubishi Gas Chemical Company)
and GENbox microaerophilic generator (BioMérieux),
bacteria were incubated over-night, until a OD640nm 1.5
was reached. Cells were removed by centrifugation
(10,000 x g, 10 min). Supernatants were filtered
through a 0.45µm cellulose membrane (Milipore) and
concentrated using centrifugal filter Amicon® (Merck
Millipore) with 100,000 Da NMWL.
Proteomic analysis- OMVs were analyzed by 2-DE
(CBMA- Centre of Molecular Environmental Biology,
Department of Biology, University of Minho, Braga,
Portugal) using the immobilized pH gradient technique
(IPG). OMV samples were resuspended in 7 M urea, 2
M thiourea, 1% (w/v) dodecylmaltoside prior to loading
in the first dimension of IPG. IPG strips used were 24
cm non-linear, with pH range from 3 to 10. Isoelectric
focusing (IEF) was performed during 3 hours at 7,000
V. The completed first-dimensional strip was subjected
to a second dimension using 12% dodecyl sulfate
polyacrylamide gel (SDS-PAGE), separation was
performed at 110V. Gels were stained with silver
nitrate and scanned on a Typhoon Trio laser scanner
(GE Healthcare).
Proteins spots were excised manually and identified
by mass spectrometry (MS) (Proteomic and Analytical
Biochemistry Laboratory, Department of Biology,
University of York, York, UK ). Proteins in gel spots
were digested with trypsin, tryptic peptides analyzed by
matrix-assisted laser desorption ionization (MALDI)-
MS and MS/MS using a Bruker ultraflex III (Bruker-
Daltonic, USA) tandem time of flight (TOF/TOF) mode.
For identification MS spectra were searched using
MASCOT software (Matrix Science) using data from
the NCBI non-redundant protein database.
Classification into functional categories was performed
using the Burkholderia genome database
(http://www.burkholderia.com), TIGR database
(http://www.tigr.org/), the Role Category Lists, KEGG
database (http://www.genome.jp/kegg/) and NCBI
(http://www.ncbi.nlm.nih.gov/). Subcellular prediction
was performed using the bioinformatic tool PSORTb
version 2.0.4 [23].
OMVs quantification (BCA assay)- In this study
Total protein quantification was used as an indicator of
the OMV present in the analyzed sample. Total protein
quantification was performed by a bicinchoninic acid
(BCA) assay kit (Pierce). Standard solutions of bovine
albumin serum (BSA) ranging from 25 to 450 µg/mL
4
Table 1- Characteristics of the Bcc isolates used in this work.
Strain name Other name Source, location Relevant information Source
Burkholderia cenocepacia
K56-2 LMG 18863 CF patient,
Canada
ET-12 lineage BCESM+;
cblA+; recA-A
J. J. LiPuma
J2315 LMG 16656 CF patient, UK ET-12 lineage BCESM+;
cblA+; recA-A
G. Doring
AU1054 LMG 24506 CF patient, USA recA-B PHDC lineage BCCM™/LMG
MCO-3 LMG 24308 Maize
rhizosphere, USA
recA-B BCCM™/LMG
HI2424 LMG 24507 Soil, USA recA-B PHDC lineage BCCM™/LMG
Burkholderia multivorans
ATCC17616 Soil, USA ATCC
D2095 CF patient,
Canada
D. P. Speert
HI2229 LMG 17588 CF patient, USA BCESM-; cblA
- J. J. LiPuma
Burkholderia cepacia
PC783 LMG 1222 Onion, USA J. J. LiPuma
Burkholderia dolosa
AU0654 LMG 18943 CF patient, USA J. J. LiPuma
BCESM: Burkholderia cenocepacia epidemic strain marker – cblA: cable pilus gene – recA: recombinase A gene – PHDC: Philadelphia-District of
Columbia clone – ET: Electrophoretic type
were used to establish a calibration curve. The assays
were performed in 96 wells microplates (Orange
Scientific) with 200 µL of BCA reagent and 10 µL of
sample. Absorbance was read at 562nm in a
Spectrostarnano
(BMG Labtech) microplate reader.
MTT assay- 16HBE14o- human bronchial epithelial
cells were maintained in fibronectin/vitrogen coated
flasks in Minimum Essential Medium with Earle’s salt
(MEM) (Gibco) supplemented with 10% fetal bovine
serum (FBS) (Lonza), 0.292 g/L L-glutamine (Sigma
Aldrich) and Penicilin/Streptomycin 100 U/mL (Gibco),
in a humified atmosphere at 37ºC and 5% CO2. Cells
were seeded in a 96 well microplate (Orange
Scientific) at a density of 7.5x103 cells per well. 0.5 µg,
1 µg and 2 µg of B. cenocepacia AU1054-derived
OMVs were added to 100 µL of MEM medium and
incubated in the same conditions for 24 hours, after
which 20 µL of 5 mg/mL [3-(4,5 dimethylthiazol-2-yl-2,5
tetrazolium bromide)] (MTT) were added in each well
and incubated for 3 hours in the same conditions.
Following the incubation MTT was removed and 150
µL of 40 mM HCl in isopropanol were added and
incubated for 15 min., the crystals were resuspended
and plate’s absorbance was read at 590nm in a
SpectroStarnano
(BMG Labtech) microplate reader.
Galleria melonella killing assays- Galleria
melonella killing assays were based on the methods
previously described [45]. A micrometer was adapted
to control the volume of a mycrosyringe and inject 3.5
µL (0.2 µg) or 10 µL (1µg) of purified OMVs in each
caterpillar, via the hindmost left proleg, previously
sanitized with 70% (v/v) ethanol. Following injection
larvae were placed in petri dishes and incubated in the
dark, at 37ºC. Survival was followed during a 96 hour
period, larvae were considered dead when no
response to touch was displayed. Control larvae were
injected with PBS (pH 7.4).
Transmission Electron Microscopy (TEM)- 10µL
of purified OMV preparation were negatively stained by
freshly prepared 2% (v/v) uranyl acetate and applied to
200-mesh carbon-coated cooper grids (Electron
Microscopy Sciences), being visualized with a JEOL
1200-EX transmission electron microscope.
5
Results
B. cenocepacia K56-2-derived OMVs purity
analysis
OMVs were isolated from cultures of B.
cenocepacia K-56-2 in order to evaluate the
purity of the OMVs by TEM. From a 2 L culture of
B. cenocepacia, it was possible to obtain,
following isolation of OMVs, a yield of 0.1 ±0.03
mg of OMVs/L of initial culture. The OMVs
obtained were analyzed by TEM, allowing to
confirm the presence of OMVs with diameters
ranging from 10 nm to 300 nm, with OMVs with
50 nm being more commonly observed (Fig.1). It
is also possible to observe the presence of
contaminants such as flagella and cable pili
(Fig.1).
Proteomic analysis of B. cenocepacia K56-2-
derived OMVs
Three different samples of purified OMVs were
subjected to a proteomic analysis. 33 different
proteins were identified from 73 spots (Fig.2).
The proteins identified were divided into 8
different functional groups with the most
abundant groups being cell envelope biogenesis
and outer membrane, energy conversion and
amino acid transport and metabolism. It was
possible to observe the presence of a large
number of cytoplasmic proteins which is
expected since OMVs incorporate proteins from
multiple bacterial compartments. It is also
possible to observe the presence of cable pilus
and flagella, which corroborates TEM results.
A set of proteins identified in the proteomic
analysis is of particular interest. This group is
composed by proteins which are immunogenic. In
this study 6 of 15 immunogenic proteins were
identified [54].
Fig. 1- TEM analysis of OMV samples obtained from B.
cenocepacia K56-2 in vitro growth
Optimization of OMVs production
The process of OMVs production was
optimized, by analyzing OMVs production by
different strains of the Bcc, under different
incubation periods, and under different growth
conditions. The strains analyzed were chosen
from the four clinically most relevant Bcc species
[8; 16]. From the strains tested B. cenocepacia
K56-2 achieved the best yields (Fig.3).
Fig. 2- Proteome reference 2-DE gel of B cenocepacia K56-2-
derived OMVs. On the left is represented the scale of the
molecular weight (higher on top to lower on bottom), and on
the top is represented the pH scale (from 3 to 10). The circles
and numbers indicate excised protein spots which were
analyzed by MALDI-TOF
6
For the optimization of culture time both B.
cenocepacia K56-2 and B. multivorans HI2229
were tested. The OMVs produced in cultures
stopped at different points suggested that longer
incubation times lead to increased OMVs
production, however the increase in longer
incubation times may be due to cell lysis, which
was not analyzed in this study.
The final optimization step was the growth
conditions. Two stresses, which are present in
CF lung, were tested, namely osmotic stress [33]
represented by LB medium with 300 mM NaCl,
and reduced oxygen concentration [68]
represented by microaerophilic conditions.
The growth conditions tested yielded different
results, while the use of LB with 300 mM of NaCl
resulted in reduced OMVs production compared
with LB medium, the use of microaerophilic
conditions resulted in an increase of OMV
production.
Despite the results obtained in the optimization
of OMVs production process, the conditions were
maintained. The strains being used was the best
producer. The increase in the total protein
produced in longer incubation periods may be
caused by cell lysis.
Table 2 OMV yields (mg/L of initial culture) obtained following culture of B. cenocepacia K56-2, under different growth conditions.
Growth conditions OMV yield (mg/L)
Broth Stiring Oxygen
LB 250 aerobic 0.9
0.3M NaCl LB
250 aerobic 0.7
LB 60 microaerophilic 1.5
Size exclusion chromatography of B.
cenocepacia K56-2-derived OMVs
The results obtained in TEM and proteomic
analysis of B. cenocepacia K56-2-derived OMVs
suggested that further purification of the samples
was required.
Fig. 3- Graphic representation of the OMVs yield (mg of OMVs/L of initial culture) produced by the different strains used in the strain screening
To further purify the OMVs a SEC step was
applied to the samples, to increase purity of the
preparation.
The OMVs were loaded into a Tricorn™ 5/100
with 30 cm of length. The use of this column did
not allow complete separation of the OMVs and
the contaminants present in the sample, with only
a partial separation being achieved.
In order to increase the separation between the
OMVs and the contaminants in the sample, a
longer column was used. The reasoning behind
the employment of a longer column was that the
increased retention time in the column would
allow for a more complete separation.
The longer column used was a Tricorn™
10/600 with 60 cm of length. OMVs were also
loaded into the column. The separation of the
samples was greater using the longer column,
however complete separation was not achieved,
with an small overlap between the OMVs
fractions and contaminants fraction, thus not
achieving complete separation between the
samples and contaminants.
0
0,1
0,2
0,3
0,4
0,5
B. c
eno
cep
acia
K5
6-2
B. c
eno
cep
acia
J2
31
5
B. c
eno
cep
acia
AU
10
54
B. c
eno
cep
acia
MC
O-3
B. c
eno
cep
acia
HI2
42
4
B. m
ult
ivo
ran
s A
TCC
…
B. m
ult
ivo
ran
s D
20
95
B. m
ult
ivo
ran
s H
I22
29
B. c
epac
ia P
C7
83
B. d
olo
sa A
U0
65
4
OM
V y
ield
(m
g/L)
Strains tested
7
Production of B. cenocepacia AU 1054-
derived OMVs
Since the use of SEC for the purification of B.
cenocepacia K56-2-derived OMVs did not
achieved the desired results, a different strategy
was employed for the achievement of purified
OMVs.
Taking into consideration the observations from
the TEM analysis of B. cenocepacia K56-2-
derived OMVs, in which Cable pili and flagella
were the main contaminants, we reasoned that
the use of a strain not capable of producing cable
pili would reduce the contamination of the OMV
preparation.
To achieve this objective the strain used for
OMVs production was switched to B.
cenocepacia AU 1054, which is CblA-, thus not
being able to produce cable pili. This strain was
used for production of OMVs, the protocol used
for production and isolation of B. cenocepacia
AU1054-derived OMVs was the same as the one
used for B. cenocepacia K56-2. From the 2 L of
initial culture and following the isolation of OMVs
from B. cenocepacia AU 1054-derived OMVs, it
was possible to obtain 0.04 ±0.003 mg of OMV/L
of initial culture.
Transmission Electron Microscopy of B.
cenocepacia AU1054-derived OMVs
B. cenocepacia AU1054-derived OMVs were
analyzed by TEM to confirm the presence of
vesicles and evaluate the purity of the OMVs.
TEM analysis of OMVs isolated from B.
cenocepacia AU1054 revealed the presence of
OMVs with diameters between 100 and 150 nm.
It was also possible to observe vesicles with
smaller diameters (75 to 100nm) and large
diameters (175 to 225 nm) were less commonly
observed (Fig. 3).
It is also important to notice that cable pilus or
flagella were not observed in these preparations
(Fig.4).
Fig. 4- Transmission electron microscopy image from a sample of B. cenocepacia AU1054-derived OMVs.
In vitro cytotoxic effects of B. cenocepacia
AU1054-derived OMVs
Following the isolation of purified OMVs from
B. cenocepacia AU1054 it became possible to
perform a preliminary evaluation of cytotoxic
potential of OMVs. Initial cytotoxic assays were
performed in vitro by performing MTT assays in
16HBE14o- human bronchial epithelial cells.
Exposure to OMVs revealed a reduction in cell
survival, which increased with an increase in the
amount of OMVs to which cells were exposed,
reaching almost 50% in the highest quantity of
OMVs (Fig. 5).
Fig. 5- Cell survival after exposure to purified B. cenocepacia AU1054-derived OMVs during a 24 hour period.
0
20
40
60
80
100
0,5 1 2
Surv
ival
(%
)
OMVs (ug)
8
In vivo cytotoxic effects of B. cenocepacia
AU1054-derived OMVs
Following the in vitro cytotoxic assays
performed with OMVs, the cytotoxic effects of
OMVs were also tested in Galleria melonella. The
results obtained from the in vivo testing of
cytotoxic effects were different from the MTT
assays. Exposure to purified OMVs in G.
melonella did not result in reduced survival, in
fact all larvae injected with OMVs survived the
testing period (Table 3).
Table 3- Number of Galleria mellonella caterpillars alive after a 96 hours period after being injected with a OMV sample isolated from the strain B. cenocepacia AU1054
Sample Caterpillars
inoculated
Caterpillars
alive after 96
hours
Control 10 10
AU1054
(0.2µg)
10
10
AU1054
(1µg)
10
10
Discussion The development of a vaccine against Bcc
species has been subject to various studies,
however they were not able to achieve enough
protection [13].
Recently OMVs have been employed in the
development of vaccines due to their
characteristics [51].
Using B. cenocepacia K56-2 we were able to
produce and isolate OMVs. Assessment of purity
of isolated OMVs by TEM and proteomic analysis
revealed the presence of contaminants, requiring
further purification. Several authors use density
gradients for OMV purification [4; 63], however
this purification process is not easily scalable.
Taking those observations into consideration
we employed a size exclusion chromatography
step for the purification of isolated OMVs based
on a previously described protocol for OMVs
purification in N. meningitidis [59]. Testing of SEC
efficiency in the purification of OMVs revealed
that the process was not capable of achieving the
desired results, therefore a different strategy was
employed.
As said before the proteomic analysis of
B.cenocepacia K56-2-derived OMVs revealed the
presence of 6 of 15 immunoproteins across B.
cenocepacia and B. multivorans species, which
are good candidates for vaccine development,
namely chaperonin GroEL, elongation factor Tu,
OMPA/MotB domain-containing protein, porin,
alkyl hidroperoxide reductase/Thiol specific
antioxidant and phospopyruvate hydratase [54].
Besides, the 33 proteins identified in the
proteomic analysis may also be encountered in
OMVs derived from other organisms, such as P.
aeruginosa [11], N. lactamica [60] and N.
meningitidis [67].
In the optimization of the OMVs production
process, when optimizing culture length a B.
multivorans strain was used, this strain is a
clinical isolate, and was used in order to have a
representative of the two clinically relevant
species. The results suggested that using longer
incubation periods lead to increased OMVs
yields, however the increase in OMVs production
may be caused by cell lysis. Since the cell lysis
was not evaluated, the incubation length was
maintained, this incubation period is similar to the
period used by other authors, which stop the
cultures at lat log phase/early stationary [4; 28].
Growth conditions have an important effect in
vesicle production [40; 41]. Taking that
observation into consideration it was necessary
to evaluate the effect of growth conditions in
OMV production. Two different conditions were
tested, osmotic stress and microaerophilic
9
conditions, which, as previously stated, represent
the conditions encountered by bacteria in host’s
lungs. Literature reports regarding OMV
production under osmotic stress and
microaerophilic conditions are scarce, however
some reports may be found. Regarding OMV
production under osmotic stress Fulsundar et al.
have studied the effect of different stresses in
vesicle production in Acineobacter bayly,
concluding that LB medium supplemented with
500 mM of NaCl for the culture of A. bayly,
resulted in a small increase in vesiculation [22].
The results obtained in the present study are not
in agreement with the results obtained by
Fulsundar et al. In respect to reduced oxygen
availability, Tran et al., have evaluated the
release of Shiga toxin by a strain of E. coli. They
evaluated the release of Shiga toxin in
microaerobic conditions, and concluded that the
release of Shiga toxin was reduced under these
conditions [56]. Since Shiga toxin is released by
OMVs [17], it may be possible that in E. coli
strain used also releases Shiga toxin via OMVs.
The results obtained in the present study, once
again, are not in agreement with the literature,
having achieved higher OMV yields in
microaerophilic conditions.
For the development of an alternative
purification strategy, and taking into account the
results obtained in TEM and proteomic analysis
of B. cenocepacia K56-2, the strain used for
OMVs production was replaced with B.
cenocepacia AU 1054, which does not produce
cable pilus or flagella, facilitating the purification
of OMVs.
The yields obtained by B. cenocepacia AU
1054 are lower than the yields obtained by B.
cenocepacia K56-2, however TEM analysis
revealed the presence of OMVs in B.
cenocepacia AU 1054 OMVs preparations,
without the presence of contaminants, thus
achieving the objective of producing purified
OMV preparations using an alternative strategy.
The evaluation of cytotoxic effects both in vitro
and in vivo revealed some differences, while the
in vitro model exhibited reduced cell survival, the
in vivo model did not exhibited reduced survival.
This discrepancy between the models used may
be explained by the lipopolyssacharide (LPS)
toxicity, which may cause some toxicity [51].
Work performed by Elmi et al., revealed that
Campylobacter jejuni 11168H-derived OMVs
revealed dose-dependent cytotoxic effects in
Caco-2 intestinal epithelial cells and in G.
mellonella [20], while in this work only in
16HBE14o- human bronchial epithelial cells was
possible to observe cytotoxic effects derived from
OMVs exposure, which may be explained by the
higher OMVs dose used in the work performed
by Elmi et al [20].
Conclusions and future work Some aspects of this work should be further
investigated. The effect of osmotic stress and
microaerophilic conditions should be further
studied, repeating the assays performed, in order
to achieve a more solid conclusion, since
literature reports are not in agreement with the
results obtained.
The cytotoxic effects of OMVs should also be
subjected to a more in-depth analysis, evaluating
the effect of exposure to higher amounts of
OMVs.
One interesting strategy which could be used in
the future to overcome the low OMVs yields
obtained is the use of genetic engineering
techniques to cause a mutation in the rmpM
gene, which would lead to a mutant with
increased vesiculation [57], thus increasing the
OMVs yields.
10
This work lead to the development of a strategy
to obtain purified OMVs from B. cenocepacia.
This strategy allows the production of OMVs
suitable for the development of future studies in
the development of OMVs-based vaccines.
Acknowledgements We would like to acknowledge Dr. Pedro M.
Santos at CBMA- Centre of Molecular and
Environmental Biology, Department of Biology,
University of Minho, Braga, Portugal, for
performing the proteomic analysis.
We would also like to acknowledge Dr. António
Pedro Alves de Matos, at Centro de Investigação
Interdisciplinar Egas Moniz (CiiEM), Egas Moniz–
Cooperativa de Ensino Superior, Caparica,
Portugal, for performing the Transmission
Electron Microscopy.
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