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ORIGINAL RESEARCH
published: 10 September 2019
doi: 10.3389/fcimb.2019.00324
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1September 2019 | Volume 9 | Article 324
Edited by:
Jyl S. Matson,
University of Toledo, United States
Reviewed by:
Medicharla Venkata Jagannadham,
Centre for Cellular Molecular Biology
(CCMB), India
Bo Peng,
Sun Yat-sen University, China
*Correspondence:
Sigrun Lange
s.lange@westminster.ac.uk
Specialty section:
This article was submitted to
Molecular Bacterial Pathogenesis,
a section of the journal
Frontiers in Cellular and Infection
Microbiology
Received: 04 April 2019
Accepted: 28 August 2019
Published: 10 September 2019
Citation:
Kosgodage US, Matewele P,
Awamaria B, Kraev I, Warde P,
Mastroianni G, Nunn AV, Guy GW,
Bell JD, Inal JM and Lange S (2019)
Cannabidiol Is a Novel Modulator of
Bacterial Membrane Vesicles.
Front. Cell. Infect. Microbiol. 9:324.
doi: 10.3389/fcimb.2019.00324
Cannabidiol Is a Novel Modulator of
Bacterial Membrane Vesicles
Uchini S. Kosgodage 1, Paul Matewele 1, Brigitte Awamaria 1, Igor Kraev 2, Purva Warde 3,
Giulia Mastroianni 4, Alistair V. Nunn 5, Geoffrey W. Guy6, Jimmy D. Bell 5, Jameel M. Inal 3
and Sigrun Lange 7
*
1Cellular and Molecular Immunology Research Centre, School of Human Sciences, London Metropolitan University, London,
United Kingdom, 2School of Life, Health and Chemical Sciences, The Open University, Milton Keynes, United Kingdom,
3Bioscience Research Group, Extracellular Vesicle Research Unit, School of Life and Medical Sciences, University of
Hertfordshire, Hatfield, United Kingdom, 4School of Biological and Chemical Sciences, Queen Mary University of London,
London, United Kingdom, 5Research Centre for Optimal Health, School of Life Sciences, University of Westminster, London,
United Kingdom, 6GW Pharmaceuticals Research, Cambridge, United Kingdom, 7Tissue Architecture and Regeneration
Research Group, School of Life Sciences, University of Westminster, London, United Kingdom
Membrane vesicles (MVs) released from bacteria participate in cell communication and
host-pathogen interactions. Roles for MVs in antibiotic resistance are gaining increased
attention and in this study we investigated if known anti-bacterial effects of cannabidiol
(CBD), a phytocannabinoid from Cannabis sativa, could be in part attributed to effects
on bacterial MV profile and MV release. We found that CBD is a strong inhibitor
of MV release from Gram-negative bacteria (E. coli VCS257), while inhibitory effect
on MV release from Gram-positive bacteria (S. aureus subsp. aureus Rosenbach)
was negligible. When used in combination with selected antibiotics, CBD significantly
increased the bactericidal action of several antibiotics in the Gram-negative bacteria. In
addition, CBD increased antibiotic effects of kanamycin in the Gram-positive bacteria,
without affecting MV release. CBD furthermore changed protein profiles of MVs released
from E. coli after 1 h CBD treatment. Our findings indicate that CBD may pose as a
putative adjuvant agent for tailored co-application with selected antibiotics, depending
on bacterial species, to increase antibiotic activity, including via MV inhibition, and help
reduce antibiotic resistance.
Keywords: bacterial membrane vesicles (MVs), cannabidiol (CBD), antibiotic resistance, gram-negative,
gram-positive, E. coli VCS257, S. aureus subsp. aureus Rosenbach
INTRODUCTION
Outer membrane vesicles (OMVs) and membrane vesicles (MVs) are released from Gram-negative
and Gram-positive bacteria and participate in inter-bacterial communication, including
via transfer of cargo molecules (Dorward and Garon, 1990; Li et al., 1998; Fulsundar
et al., 2014; Jan, 2017; Toyofuku et al., 2019). MVs are released in greater abundance
from Gram-negative, than Gram-positive bacteria and their production seems crucial for
bacterial survival and forms part of the stress response (McBroom and Kuehn, 2007;
Macdonald and Kuehn, 2013; Jan, 2017). Gram-negative bacteria generate, in addition to
common one-bilayer vesicles (OMV), also double-bilayer vesicles (O-IMVs), and in some
stress conditions other types of MVs (Pérez-Cruz et al., 2016) and therefore we will use
the umbrella term “membrane vesicles” (MVs) hereafter. MVs are important in biofilm
formation and dissemination of toxins in the host (Wang et al., 2015; Cooke et al., 2019).
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
MVs participate in host-pathogen interactions (Gurung et al.,
2011; Koeppen et al., 2016; Bitto et al., 2017, 2018; Codemo
et al., 2018; Turner et al., 2018; Cecil et al., 2019) and may also
be involved in antibiotic resistance, for instance by protecting
biofilms from antibiotics via increased vesiculation (Manning
and Kuehn, 2011). Furthermore, MVs from Porphyromonas
gingivalis have been linked to metabolic remodeling in the host
(Fleetwood et al., 2017), while MVs from Neisseria gonorrhoeae
have been shown to target host mitochondria and to induce
macrophage death (Deo et al., 2018). Besides roles for cellular
and bacterial communication, the use of MVs as nano-carriers
for various compounds, including for antibiotic and vaccine
delivery, has also raised considerable interest in the research
community (Gnopo et al., 2017; Rüter et al., 2018; Tan et al., 2018;
Wang et al., 2018).
The regulation of bacterial MV biogenesis and release may
therefore be of great importance, both in relation to inter-
bacterial communication, including biofilm formation, their
host interactions as commensals, as well as in host-pathogen
interactions and in antibiotic resistance.
Cannabidiol (CBD) is a phytocannabinoid from Cannabis
sativa with anti-inflammatory (Martin-Moreno et al., 2011), anti-
cancerous (Pisanti et al., 2017; Kosgodage et al., 2018) and
anti-bacterial activity (Hernández-Cervantes et al., 2017). While
immunoregulatory roles for cannabinoids have been reported
in infectious disease (reviewed in Hernández-Cervantes et al.,
2017), and C. sativa has been identified as a natural product
with a capability of controlling bacterial infections, including a
strong anti-bacterial activity against antibiotic resistant strains
(Appendino et al., 2008), a link between CBD and bacterial MV
release has hitherto not been investigated.
As our recent work identified CBD as a potent inhibitor of
extracellular vesicle (EV) release in eukaryotes (Kosgodage et al.,
2018; Gavinho et al., 2019), we sought to investigate whether CBD
may work via phylogenetically conserved pathways, involving
bacterial MV release from bacteria. As we, and other groups,
have previously shown that cancer cells can be sensitized
to chemotherapeutic agents via various EV-inhibitors (Jorfi
et al., 2015; Koch et al., 2016; Muralidharan-Chari et al., 2016;
Kosgodage et al., 2017), including CBD (Kosgodage et al., 2018,
2019), we sought to establish whether in bacteria, similar putative
MV modulatory effects could be utilized to sensitize bacteria
to antibiotics.
MATERIALS AND METHODS
MV Isolation From E. coli VCS257 and
S. aureus subsp. aureus Rosenbach
E. coli (VCS257, Agilent, La Jolla, CA) and S. aureus subsp.
aureus Rosenbach (ATCC 29247, USA) static cultures were
grown in Luria-Bertani (LB) broth for 24 h at 37◦C. The growth
phase before vesicle isolation was exponential; the volume of
the cultures was 20 ml. For MV isolation, ultracentrifugation
and nanoparticle tracking analysis (NTA) were used based on
previously established methods by other groups (McCaig et al.,
2013; Klimentova and Stulik, 2015; Roier et al., 2016).
E. coli and S. aureus cultures were maintained by plating
on Mueller-Hinton agar plates and weekly sub-culturing was
performed according to previously established methods (Iqbal
et al., 2013).
Before MV isolation, all bacterial growth medium (LB broth)
was pre-treated before use by ultracentrifugation at 100,000 g
for 24 h to ensure minimum contamination with extracellular
vesicles (EVs) from the medium (Kosgodage et al., 2017).
For MV isolation, bacteria were grown in EV-free medium
(as described above) for 24 h at 37◦C, the culture medium
was collected and centrifuged once at 400 g for 10 min for
removal of cells, followed by centrifugation at 4,000 g for 1 h
at 4◦C to remove cell debris. The resultant supernatant was
then centrifuged for 1 h at 100,000 g at 4◦C and the isolated
MV pellet was resuspended in Dulbecco’s phosphate buffered
saline (DPBS; ultracentrifuged and sterile filtered using a 0.22 µm
filter) and centrifuged again at 100,000 gfor 1 h at 4◦C. The
resulting MV pellet was sterile filtered (0.45 µm) once and then
resuspended in sterile filtered DPBS. The quantitative yield of
vesicles was ∼6.5 ×109MVs per liter of culture. The isolated MV
pellets were then either used immediately, or stored at −80◦C for
further experiments.
Transmission Electron Microscopy (TEM)
Imaging of Bacterial MVs
A suspension of isolated MVs (1.4 ×108MVs/ml) was used for
TEM imaging. MV samples (10 µL) were applied to mesh copper
grids, prepared with glow discharged carbon support films, and
incubated for 2 min. The grids were then washed five times with
50 µl of 1 % aqueous uranyl acetate. Grids were left to dry
for 5 min before being viewed. Micrographs were taken with a
JEOL JEM 1230 transmission electron microscope (JEOL, Japan)
operated at 80 kV at a magnification of 80,000 to 100,000. Digital
images were recorded using a Morada CCD camera (EMSIS,
Germany) and processed via iTEM (EMSIS).
Western Blotting
Protein was isolated from MV pellets using Bacterial Protein
Extraction Reagent (B-PER, ThermoFisher Scientific, U.K.),
pipetting gently and shaking the pellets on ice for 2 h, where
after samples were centrifuged at 16,000 g at 4◦C for 20 min and
the resulting supernatant collected for protein analysis. Samples
were prepared in 2x Laemmli buffer, boiled at 95◦C for 5 min,
electrophoresed by SDS-PAGE on 4–20 % TGX gels (BioRad,
U.K.), followed by semi-dry Western blotting. Approximately
10 µg of protein was loaded per lane and even protein transfer
was assessed by Ponceau S staining (Sigma, U.K.). Blocking of
membranes was performed for 1 h at room temperature (RT)
in 5 % BSA in TBS-T. The membranes were then incubated
with the anti-OmpC (Outer-membrane protein C antibody;
orb6940, Biorbyt, U.K.; diluted 1/1000 in TBS-T) overnight at
4◦C, followed by washing in TBS-T and incubation for 1 h in anti-
rabbit-HRP conjugated secondary antibody at RT. Visualization
was performed using ECL (Amersham, U.K.) and the UVP
BioDoc-ITTM System (U.K.).
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2September 2019 | Volume 9 | Article 324
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
Nanoparticle Tracking Analysis for
Assessment of MV Release From E. coli
VCS257 and S. aureus subsp. aureus
Rosenbach
MVs were isolated from control and CBD-treated bacterial
cultures as described above. Nanoparticle tracking analysis
(NTA) was performed using the Nanosight LM10 (Malvern,
U.K.), equipped with a 405 nm diode laser and a sCMOS camera.
MV pellets were resuspended in equal volumes (100 µl) of
DPBS before NTA analysis to ensure comparable analysis of
quantification. Before application, samples were diluted 1:50 in
sterile-filtered EV-free DPBS and applied at a constant flow
rate, maintaining the number of particles in the field of view in
the range of 20–40 with a minimum concentration of samples
at 5 ×107particles/ml. Camera settings were according to
the manufacturer’s instructions (Malvern), five 60 s videos per
sample were recorded and replicate histograms averaged. Each
experiment was repeated three times.
CBD-Mediated MV Release Inhibition in
E. coli VCS257 and S. aureus subsp.
aureus Rosenbach
E. coli and S. aureus cultures were cultivated using EV-free
Müeller-Hinton broth for 24 h. An inoculate of 0.1ml of bacteria,
in a 20 ml culture volume of bacterial growth medium (Luria-
Bertani (LB) broth), were grown at exponential phase overnight,
as assessed by OD600. The bacterial cells were then washed using
DPBS at 4,000 g for 10 min and seeded in 1.5 ml triplicates in
micro centrifuge tubes. For treatment with CBD, CBD (GW
research Ltd) was applied at concentrations of 1 or 5 µM and
incubated with the bacterial cultures for 1 h at 37◦C. Treatments
were performed in triplicates, including DMSO as a control.
MV isolation following CBD and control treatment was carried
out using step-wise centrifugation and ultracentrifugation as
before. Changes in MV release were assessed by quantifying
numbers of MVs by NTA analysis as described above, with
each experiment repeated three times. Cell viability was assessed
before the start of every experiment and after treatment with
CBD compared to controls determined by colony forming unit
(CFU) measurement.
Disc Diffusion Test for Assessment of
CBD-Mediated Enhancement of Antibiotic
Treatment
Discs were impregnated with the following antibiotics (all from
Sigma-Aldrich): colistin (10 µg/ml), rifampicin (15 µg/ml),
erythromycin (50 µg/ml), kanamycin (1,000 µg/ml) and
vancomycin (5 µg/ml). Concentration of the antibiotics used
was based on previously published and established MIC values
(Maclayton et al., 2006; Moskowitz et al., 2010; Kshetry et al.,
2016; Rojas et al., 2017; Goldstein et al., 2018). E. coli and S.
aureus agar plates were prepared for the disc diffusion test (Iqbal
et al., 2013) by soaking a sterile paper disc in 5 µM CBD and
placing it in the middle of the agar plate, while the impregnated
antibiotic discs were placed equidistant to the CBD disc. Zones
of inhibition were assessed after 24 h using the Kirby-Bauer test.
Proteomic Analysis of MVs Released From
CBD Treated and Control Untreated E. coli
VCS257
To assess differences in E. coli VCS257 MV protein composition
in response to CBD treatment, MVs were isolated as before,
after 1 h treatment with 1 µM or 5 µM CBD treatment or
control untreated, respectively. MVs were assessed by SDS-PAGE
(using 4–20 % gradient TGX gels, BioRad, U.K.) and silver
staining using the BioRad Silver Stain Plus Kit (1610449, BioRad,
U.K.), according to the manufacturer’s instructions (BioRad). For
assessment of proteomic changes, MVs were subjected to liquid
chromatography-mass spectrometry (LC-MS/MS) analysis. MVs
from CBD treated, vs. non-treated E. coli were run 1 cm into a
SDS-PAGE gel and the whole protein lysate cut out as one band,
whereafter it was processed for proteomic analysis (carried out by
Cambridge Proteomics, U.K.). Peak list files were submitted to
Mascot (in-house, Cambridge Center for Proteomics) using the
following database: Uniprot_Escherichia_coli_20180613 (4324
sequences; 1357163 residues).
Statistical Analysis
Histograms and graphs were prepared and statistical analysis was
performed using GraphPad Prism version 8 (GraphPad Software,
San Diego, U.S.A.). One-way ANOVA and Student’s t-test
analysis were performed, followed by Tukey’s post-hoc analysis.
Histograms represent mean of data, with error bars representing
standard error of mean (SEM). Significant differences were
considered as p≤0.05.
RESULTS
Characterization of MVs From E. coli
VCS257 and S. aureus subsp. aureus
Rosenbach
Isolated MVs were assessed by morphology using transmission
electron microscopy (TEM), revealing a poly-dispersed
population in the size range of mainly 20–230 nm in
diameter for E. coli, including MVs showing inner and
outer membranes (Figure 1A.1), and characteristic one layer
membranes for S. aureus MVs, which were in the 37–300 nm
range (Figure 1A.2). Nanoparticle tracking analysis (NTA)
verified that the majority of the vesicle population fell in a
similar size range under standard culture conditions (mode
143.3 nm; SD ±72.3 nm for E. coli (Figure 1A.1) and 141.4 nm;
SD ±7.3 nm for S. aureus (Figure 1A.2). Furthermore,
Western blotting showed positive for the MV specific marker
OmpC (Figure 1A).
Effects of CBD on Membrane Vesicle
Release From E. coli VCS257 and S. aureus
subsp. aureus Rosenbach
CBD changed the MV release profile from E. coli compared to
control treatment (Figures 1B–D). Modal size of MVs released
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 3September 2019 | Volume 9 | Article 324
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
FIGURE 1 | Bacterial MV profile under standard conditions and after CBD treatment. (A) MVs released from untreated E. coli VCS257 (A.1) and S. aureus subsp.
aureus Rosenbach (A.2), shown by NTA analysis (Nanosight); Transmission electron microscopy (TEM, scale bar =200 nm) and Western blotting with the MV-specific
marker OmpC. (B) NTA analysis showing MV release from E. coli after 1 h CBD treatment (1 µM). (C) NTA analysis showing MV release from E. coli after 1 h CBD
treatment (5 µM). (D) Modal size of MVs released from E. coli under normal culture conditions compared to CBD treatment. Error bars indicate SEM; *prepresents
p-values compared to control (ctrl) while #prepresents p-values compared to 1 µM CBD treatment.
from E. coli was significantly increased (p=0.01) after 1 µM CBD
treatment, compared to control treated cells, while 5 µM CBD
treatment did not have statistically significant effects on MV size
(p=0.0685). Effects on modal size of vesicles released from E. coli
between the two doses of CBD was also not statistically significant
(p=0.0643; Figure 1D).
CBD had a significant inhibitory effect (p<0.0001) on
total MV release from E. coli VCS257 at both concentrations
tested (1 and 5 µM, respectively; Figure 2A). In addition,
the lower dose of CBD (1 µM) had stronger MV-inhibitory
effects (73 % reduction, p<0.0001) than 5 µM CBD (54 %
reduction, p<0.0001; Figure 2A) and resulted in a markedly
increased peak at 500 nm (Figure 1B), which otherwise was
negligible in the control (Figure 1A) and 5 µM CBD (Figure 1C)
treated E. coli.
Effects of CBD on E. coli VCS257 MVs was furthermore
assessed by TEM, verifying the presence of fewer vesicles per field
and showing some change in vesicle size and morphology after
CBD (Supplementary Figures 2A–C).
FIGURE 2 | CBD affects MV-release from the Gram-negative bacteria E. coli
VCS257 but not Gram-positive S. aureus subsp. aureus Rosenbach. (A) MV
release from E. coli was significantly reduced after CBD treatment, with lower
dose of CBD being more effective (p=0.0063). (B) MV release from S. aureus
was not significantly affected by CBD treatment. Exact p-values are shown.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 4September 2019 | Volume 9 | Article 324
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
Contrary to what was observed for the Gram-negative E. coli,
CBD treatment (5 µM) had no significant effect on MV release
from the Gram-positive bacterium S. aureus subsp. Aureus
Rosenbach (p>0.1; Figure 2B).
Effects of CBD on Bacterial Viability of
E. coli VCS257 and S. aureus subsp.
aureus Rosenbach
CBD had negligible effect on E. coli cell viability after 24 h
incubation with the lower 1 µM dose, while an 11 % (p=
0.0161) reduction in cell viability was observed in response
to 5 µM CBD, but no significant effect was observed on
S. aureus cell viability, as assessed by disk diffusion test
(Supplementary Figure 1).
CBD Treatment Affects Antibiotic
Sensitivity in E. coli VCS257
CBD treatment (5 µM), when applied in combination with
a range of antibiotics tested, was found to sensitize E. coli
VCS257 to selected antibiotics, as assessed by an increase
in the radius of zone of inhibition, using the disk diffusion
test (Figure 3). Significantly enhanced antibacterial effects were
found for erythromycin (35 % increase; p=0.006), rifampicin
(50 % increase; p=0.0007) and vancomycin (100 % increase;
p<0.0001), when combined with CBD treatment (5 µM),
compared to antibiotic treatment alone. Notably, vancomycin
alone did not have bactericidal effects on E. coli, but only in
the presence of CBD. Antibacterial effects of kanamycin were
increased by 18 % but this was not statistically significant
compared to antibiotic alone (p=0.09). Zone of inhibition
with CBD treatment only was also observed in the E. coli
plates (Figure 3), but this was significantly lower than when
CBD was combined with antibiotics, except for vancomycin.
The zone of inhibition for E. coli caused by antibiotic treatment
only, vs. CBD alone, differed also significantly for erythromycin
(p=0.0010), vancomycin (p=0.0158), rifampicin (p=
0.0003) and kanamycin (p=0.0008), but not for colistin (p
=0.224). Therefore, while CBD showed some anti-bacterial
activity against E. coli when applied in isolation, this was
significantly lower than observed for the antibiotics alone
(except for vancomycin which did not show antibacterial activity
while CBD did). However, when applied in combination, CBD
increased bactericidal effects of all antibiotics tested, except
for colistin.
CBD-Mediated Effects on Antibiotic
Sensitivity in S. aureus subsp. aureus
Rosenbach
When added to S. aureus subsp. aureus Rosenbach, 5 µM
CBD increased the antibiotic activity of kanamycin (30
%; p=0.0028), as assessed by increased radius of zone
around the diffusion disk (Figure 4). CBD did not enhance
anti-bacterial activity for the other antibiotics tested and
reduced antibacterial effects of both erythromycin and
rifampicin (p=0.0325 and p=0.0001, respectively).
Importantly, there was no halo observed around the
FIGURE 3 | CBD sensitizes Gram-negative bacteria E. coli VCS257 to
selected antibiotics. Combinatory treatment of CBD with a range of antibiotics
(24 h treatment) showed enhanced CBD-mediated antibacterial effects on E.
coli VCS257, as assessed by increased radius of zone around the diffusion
disks. CBD was most effective in combination with rifampicin (p=0.0007),
vancomycin (p≤0.0001) and erythromycin (p=0.006). CBD in isolation also
had bactericidal effects on E. coli, while combinatory treatment with the
antibiotics was most effective. Exact p-values are shown.
FIGURE 4 | CBD sensitizes Gram-positive bacteria S. aureus subsp. aureus
Rosenbach to kanamycin. Combinatory treatment of CBD with a range of
antibiotics showed enhanced antibacterial effects of kanamycin only on S.
aureus, as assessed by an increased radius of zone around the diffusion disk
(p=0.0028). CBD did not enhance bactericidal activity for the other
antibiotics tested and reduced bactericidal effects of both erythromycin (p=
0.0325) and rifampicin (p=0.0001). CBD application in isolation did not form
a halo around the diffusion disk in the S. aureus plates, opposed as to what
was observed in E. coli, and CBD treatment in isolation is therefore not
included in the histogram. Exact p-values are shown.
diffusion disk containing CBD alone in the S. aureus plates,
indicating no bactericidal effects of CBD on this strain
of S. aureus.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 5September 2019 | Volume 9 | Article 324
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
Effects of CBD Treatment on Protein
Profiles of MVs Released From E. coli
VCS257
Protein composition of MVs was assessed in MVs isolated from
E. coli VCS257 after 1 h treatment with 1 µM and 5 µM CBD,
respectively, compared to non-treated E. coli MVs, using SDS-
PAGE silver stained gels and LC-MS/MS analysis. Silver stained
gels revealed some band differences between the three conditions
(Figure 5A). Proteins were further analyzed by LC-MS/MS and
peak list files submitted to Mascot (in-house, Cambridge Center
for Proteomics, Uniprot_Escherichia_coli_20180613). Hits are
listed in Tables 1–3. Compared to untreated MVs, five protein
hits were absent in MVs released from the 1 µM CBD treated E.
coli and four protein hits were absent in MVs released from the
5µM CBD treated E. coli, respectively (Table 1 and Figure 5B).
When comparing the two CBD treatments, 26 protein hits were
specific to the E. coli MVs following 1 µM CBD treatment
(Table 2 and Figure 5B) while 68 protein hits were unique to the
MVs released from E. coli treated with 5 µM CBD (Table 3 and
Figure 5B).
DISCUSSION
To our knowledge this is the first study to evaluate the putative
effects of CBD on the release of membrane vesicles (MVs)
from bacteria and effects of CBD on MV profile, including
protein composition. In eukaryotic cells, CBD was recently
identified as an effective inhibitor of extracellular vesicle (EV)
release both in human cancer cells (Kosgodage et al., 2018,
2019) as well as in the intestinal parasite Giardia intestinalis
(Gavinho et al., 2019). Therefore, our present findings may
indicate phylogenetically conserved pathways of membrane
vesicle release from bacteria to mammals that can be modulated
via CBD. Moreover, CBD could enhance the anti-bacterial effect
of certain antibiotics in some bacterial types, but also inhibit it
in others. This indicates that inhibition of MV release and anti-
bacterial action are likely linked, as previously suggested (Tashiro
et al., 2010). Indeed, a recent study using indole derivatives has
revealed a role for MVs in antibiotic resistance/persistence,
in particular in Gram-negative bacteria tested
(Agarwal et al., 2019).
Here we report that CBD significantly reduced MV release in
E. coli VCS257, a Gram-negative bacterium, but had negligible
effects on membrane vesicle release in S. aureus subsp. aureus
Rosenbach, a Gram-positive bacterium, as assessed here by in
vitro analysis. In addition, we also found that lower doses of
CBD had a stronger MV inhibitory effect in E. coli VCS257
than a higher 5 µM dose (p=0.0063), and such an effect
has also previously been observed for EVs in certain cancer
cell types (Kosgodage et al., 2018). Biphasic effects of CBD
are indeed recognized (Bergamaschi et al., 2011) and may be
reminiscent of “hormesis,” an effect we have suggested could
explain its more general medical benefits as well as effects on
mitochondrial dynamics (Nunn et al., 2013). Interestingly, at
the lower 1 µM concentration, CBD significantly increased the
release of a 500 nm peak of MVs, as observed by NTA analysis,
while this peak was negligible both in the control treated bacteria
and those treated with 5 µM CBD. Such an effect of CBD on
MV profile, and protein MV profile as observed by proteomic
analysis here, may be relevant in the light of recent recognition
of the importance of MV size for cellular entry and uptake
(Turner et al., 2018) and in line with an increased interest in the
research community for the identification and characterization
of MV sub-populations (Pérez-Cruz et al., 2016; Turner et al.,
2018; Cooke et al., 2019; Toyofuku et al., 2019; Zavan et al.,
FIGURE 5 | CBD affects protein composition of E. coli VCS257 MVs. (A) A SDS-PAGE silver stained gel reveals banding differences between the CBD treated and
non-treated E. coli derived MVs (see arrows highlighting some present and absent bands). (B). Venn diagram showing protein changes in MVs released from CBD
treated compared to untreated control E. coli VCS257. Plus (“+”) indicates hits unique to MVs following CBD 1 or 5 µM treatment, respectively; minus (“–“) indicates
number of proteins absent in the respective CBD treated MVs, compared to control untreated MVs. For specific protein hits see Tables 1–3.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 6September 2019 | Volume 9 | Article 324
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
TABLE 1 | Proteins identified as present in E. coli VCS257 control untreated MVs only and absent in MVs from CBD treated E. coli.
Protein name Symbol Score (p<0.05)‡CBD 1 µM CBD 5 µM
Glutamate decarboxylase alpha P69908|DCEA_ECOLI 37 –+
2-oxoglutarate dehydrogenase E1 component P0AFG3|ODO1_ECOLI 36 +–
RNA chaperone ProQ P45577|PROQ_ECOLI 32 –+
Uncharacterized protein YffS P76550|YFFS_ECOLI 29 –+
Serine transporter P0AAD6|SDAC_ECOLI 26 +–
Fumarate and nitrate reduction regulatory protein P0A9E5|FNR_ECOLI 26 – –
Uncharacterized protein YcaQ P75843|YCAQ_ECOLI 22 – –
Proteins were isolated from E. coli derived MVs and analyzed by LC-MS/MS. Peak list files were submitted to Mascot (in-house, Cambridge Center for Proteomics,
Uniprot_Escherichia_coli_20180613; 4324 sequences; 1357163 residues).
‡Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores >19 indicated identity or extensive homology (p <0.05). Protein
scores were derived from ions scores as a non-probabilistic basis for ranking protein hits.
2019). The approximately 6.5-fold and 2.5-fold decreases in
MV release observed after CBD (1 and 5 µm, respectively)
treatment from E. coli, compared to non-treated controls, also
correlated with a trend in shift toward proportionally larger
vesicles released according to NTA analysis and change in
protein profile. The exact mechanism for packaging proteins and
other reagents in MVs is not fully understood and given the
plethora of targets for CBD (Ibeas Bih et al., 2015; Hernández-
Cervantes et al., 2017; Pisanti et al., 2017), the exact mechanism
of this cannabinoid on MV formation remains subject to further
extensive studies. In the current study we have indeed identified
a range proteins, including proteins involved in metabolism and
antibiotic metabolic processing, which differ in MVs released
from E. coli VCS257 treated with CBD, compared to MVs
released from non-treated E. coli. Previous studies have discussed
the use of MVs for example as drug delivery vehicles (Ellis
and Kuehn, 2010; Gujrati et al., 2014; Gerritzen et al., 2017;
Jain and Pillai, 2017; Jan, 2017; Wang et al., 2018), while
MVs have also been tested as delivery vehicles for targeted
gene silencing using siRNA-packaged MVs (Alves et al., 2016).
Whether CBD may be utilized for combinatory application with
such approaches may also be of putative interest, in addition
to its observed effects in this study, in effectively reducing
MV release.
In relation to antibiotic activity, cannabinoids including
CBD, have been widely studied for their anti-bacterial activity
(Wasim et al., 1995; Bass et al., 1996; Appendino et al., 2008;
Hernández-Cervantes et al., 2017). For example, C. sativa
extracts have previously been shown to have microbicidal
activity on various Gram-positive bacteria, including several
strains of S. aureus, as well as some Gram-negative bacteria
(Wasim et al., 1995; Elphick, 2007; Nissen et al., 2010), with
the minimum inhibitory concentrations (MIC) for the main
phytocannabinoids, such as CBD, being in the 0.5–5 µM range,
which is similar to many modern antibiotics (Van Klingeren and
Ten Ham, 1976; Appendino et al., 2008). How precisely CBD
may be working as an anti-bacterial agent is still not entirely
clear (Appendino et al., 2008), particularly in the light of a
plethora of targets for CBD (Ibeas Bih et al., 2015; Hernández-
Cervantes et al., 2017), while structure-activity studies indicate
that the ability of plant-derived phenolic compounds to interact
with membranes and the existence of electrophilic functional
groups are important (Miklasinska-Majdanik et al., 2018).
Hitherto though, no association has been made into a putative
regulatory effect of cannabinoids on bacterial membrane vesicle
release. Furthermore, as the current study has revealed changes
in proteomic profile of MVs released from E. coli VCS257
following CBD treatment, such findings may inform anti-
bacterial effects of CBD. Using LC-MS/MS analysis to assess
changes in protein profile of MVs from CBD treated and
untreated E. coli, respectively, five proteins were found to be
absent in the 1 µM CBD treated MVs and 4 proteins were
absent in the 5 µM CBD treated MVs, compared to control
untreated E. coli MVs. Out of these, 2 proteins overlapped
between the two CBD treatments. In addition, comparing 1 and
5µM CBD treated E. coli MVs, 26 protein hits were unique
to MVs released following the 1 µM CBD treatment and 68
protein hits to MVs released following the 5 µM CBD treatment.
Using STRING analysis, PPI enrichment p-value was found to
be p=0.0204 for proteins identified as unique to MVs from
the 1 µM CBD treatment and p=1.56 ×10−6for proteins
identified as unique to MVs from the 5 µM CBD. This indicates
that for both treatments these proteins have significantly more
interactions among themselves, than what would be expected
for a random set of proteins of similar size, drawn from
the genome. Such enrichment indicates that the proteins are
at least partially biologically connected, as a group. Protein
networks are represented showing biological GO pathways and
KEGG pathways, respectively, in Supplementary Figures 3,4
for proteins specific to EVs from E. coli after 1 and 5 µM
CBD treatment, respectively. Proteins identified are related to
metabolic processes, cellular respiration and antibiotic functions
(Supplementary Figures 3A,B,4A,B).
When assessing the effectivity of CBD to enhance
susceptibility of Gram-positive and Gram-negative bacterial
species to a range of antibiotics, CBD-mediated MV inhibition
rendered E. coli VCS257 significantly more sensitive to
erythromycin, vancomycin and rifampicin and somewhat
to kanamycin, but did not augment the bactericidal effects
observed for colistin. This was somewhat unexpected, given
a previous study showing that MVs isolated from the E. coli
strain MG1655 could protect bacteria against membrane-active
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 7September 2019 | Volume 9 | Article 324
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
TABLE 2 | Proteins identified as present only in MVs released from E. coli VCS257 following 1 h treatment with 1 µM CBD.
Protein name Symbol Score (p<0.05)‡
Glutamate decarboxylase beta P69910|DCEB_ECOLI 230
Tryptophan synthase alpha chain P0A877|TRPA_ECOLI 85
2-oxoglutarate dehydrogenase E1 component P0AFG3|ODO1_ECOLI 70
Uncharacterized protein YgaU P0ADE6|YGAU_ECOLI 67
Spermidine/putrescine-binding periplasmic protein P0AFK9|POTD_ECOLI 67
Serine transporter P0AAD6|SDAC_ECOLI 57
Inorganic pyrophosphatase P0A7A9|IPYR_ECOLI 56
Succinate dehydrogenase flavoprotein subunit P0AC41|SDHA_ECOLI 54
NADH-quinone oxidoreductase subunit A P0AFC3|NUOA_ECOLI 53
Periplasmic dipeptide transport protein P23847|DPPA_ECOLI 49
Uncharacterized protein YqiC Q46868|YQIC_ECOLI 48
Formate dehydrogenase, nitrate-inducible, major subunit P24183|FDNG_ECOLI 47
Acyl carrier protein P0A6A8|ACP_ECOLI 45
Maltose/maltodextrin-binding periplasmic protein P0AEX9|MALE_ECOLI 44
Septum site-determining protein MinD P0AEZ3|MIND_ECOLI 42
Phosphate-specific transport system accessory protein PhoU P0A9K7|PHOU_ECOLI 40
Ribosome-associated inhibitor A P0AD49|YFIA_ECOLI 36
DNA-binding protein H-NS P0ACF8|HNS_ECOLI 35
RNA-binding protein Hfq P0A6X3|HFQ_ECOLI 33
Phosphate transport system permease protein PstA P07654|PSTA_ECOLI 32
Galactoside transport system permease protein MglC P23200|MGLC_ECOLI 32
Sec translocon accessory complex subunit YajC P0ADZ7|YAJC_ECOLI 31
Isoform Beta of Translation initiation factor IF-2 P0A705-2|IF2_ECOLI 30
2,3-bisphosphoglycerate-dependent phosphoglycerate mutase P62707|GPMA_ECOLI 30
Peptidoglycan D,D-transpeptidase FtsI P0AD68|FTSI_ECOLI 28
Inner membrane protein YjcH P0AF54|YJCH_ECOLI 27
HTH-type transcriptional regulator GntR P0ACP5|GNTR_ECOLI 27
Histidinol-phosphate aminotransferase P06986|HIS8_ECOLI 26
SsrA-binding protein P0A832|SSRP_ECOLI 25
2-dehydro-3-deoxyphosphooctonate aldolase P0A715|KDSA_ECOLI 25
Deoxyribose-phosphate aldolase P0A6L0|DEOC_ECOLI 25
Ribosome hibernation promoting factor P0AFX0|HPF_ECOLI 24
Ribokinase P0A9J6|RBSK_ECOLI 24
Probable ATP-dependent helicase l hr P30015|LHR_ECOLI 22
Membrane-bound lytic murein transglycosylase B P41052|MLTB_ECOLI 21
Uncharacterized protein YjaA P09162|YJAA_ECOLI 21
Adenylate kinase P69441|KAD_ECOLI 21
Fructose-1,6-bisphosphatase 2 class 2 P21437|GLPX2_ECOLI 20
Transcription termination/antitermination protein NusA P0AFF6|NUSA_ECOLI 20
Proteins were isolated from CBD treated (1 µM) E. coli MVs and analyzed by LC-MS/MS. Peak list files were submitted to Mascot (in-house, Cambridge Center for Proteomics,
Uniprot_Escherichia_coli_20180613; 4324 sequences; 1357163 residues).
‡Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores >18 indicated identity or extensive homology (p <0.05). Protein
scores were derived from ions scores as a non-probabilistic basis for ranking protein hits.
antibiotics such as colistin (Kulkarni et al., 2015). Our finding,
that CBD did not sensitize E. coli further to colistin, when
applied in combination with this antibiotic, may arise from
the fact that a different strain of E. coli (VCS257) was used in
the current study, compared to in the study by Kulkarni et al.
(2015). It has also been previously shown that the presence
of calcium decreases the bactericidal effect of colistin on
Paenibacillus polymyxa, suggesting a role for Ca2+in generating
a protective barrier against colistin (Yu et al., 2015). As CBD
is known to modulate calcium (Rimmerman et al., 2013) it
can be postulated that this may interfere with the mode of
action of colistin. Our findings also indicate that combinatory
application of CBD is not effective for all antibiotics, which
may possibly be explained by their different modes of action.
Importantly, zones of inhibition were observed in the plates
which were only treated with the CBD discs in the presence
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Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
TABLE 3 | Proteins identified as present only in E. coli VCS257 derived MVs following 1 h treatment with 5 µM CBD.
Protein name Symbol Score (p<0.05)‡
Glutamate decarboxylase alpha P69908|DCEA_ECOLI 189
ATP-dependent zinc metalloprotease FtsH P0AAI3|FTSH_ECOLI 128
Rod shape-determining protein MreB P0A9X4|MREB_ECOLI 109
Uncharacterized protein YibN P0AG27|YIBN_ECOLI 101
Outer membrane protein X P0A917|OMPX_ECOLI 99
Galactitol 1-phosphate 5-dehydrogenase P0A9S3|GATD_ECOLI 91
UPF0381 protein YfcZ P0AD33|YFCZ_ECOLI 85
50S ribosomal protein L31 P0A7M9|RL31_ECOLI 83
Biotin carboxylase P24182|ACCC_ECOLI 83
GMP synthase [glutamine-hydrolyzing] P04079|GUAA_ECOLI 82
Cytochrome bd-I ubiquinol oxidase subunit 1 P0ABJ9|CYDA_ECOLI 74
Galactokinase P0A6T3|GAL1_ECOLI 74
RNA chaperone ProQ P45577|PROQ_ECOLI 71
Protein GrpE P09372|GRPE_ECOLI 68
Purine nucleoside phosphorylase P0ABP8|DEOD_ECOLI 61
50S ribosomal protein L21 P0AG48|RL21_ECOLI 59
Dihydrolipoyllysine-residue succinyltransferase component of
2-oxoglutarate dehydrogenase complex
P0AFG6|ODO2_ECOLI 58
Sec-independent protein translocase protein TatA P69428|TATA_ECOLI 56
Bifunctional protein GlmU P0ACC7|GLMU_ECOLI 56
PTS system mannose-specific EIIAB component P69797|PTNAB_ECOLI 55
Anaerobic glycerol-3-phosphate dehydrogenase subunit C P0A996|GLPC_ECOLI 54
Proline/betaine transporter P0C0L7|PROP_ECOLI 52
Pyruvate formate-lyase 1-activating enzyme P0A9N4|PFLA_ECOLI 52
Pyruvate/proton symporter BtsT P39396|BTST_ECOLI 52
Protein translocase subunit SecY P0AGA2|SECY_ECOLI 49
Penicillin-binding protein activator LpoB P0AB38|LPOB_ECOLI 49
Signal peptidase I P00803|LEP_ECOLI 45
Thiol peroxidase P0A862|TPX_ECOLI 45
UPF0307 protein YjgA P0A8X0|YJGA_ECOLI 45
Peptidyl-prolyl cis-trans isomerase D P0ADY1|PPID_ECOLI 44
3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase P0A6Q3|FABA_ECOLI 44
ATP-dependent protease subunit HslV P0A7B8|HSLV_ECOLI 43
Inosine-5’-monophosphate dehydrogenase P0ADG7|IMDH_ECOLI 42
Peptide chain release factor RF2 P07012|RF2_ECOLI 41
Nucleoside diphosphate kinase P0A763|NDK_ECOLI 40
Inositol-1-monophosphatase P0ADG4|SUHB_ECOLI 40
Respiratory nitrate reductase 1 gamma chain P11350|NARI_ECOLI 40
Succinate dehydrogenase hydrophobic membrane anchor subunit P0AC44|DHSD_ECOLI 39
Outer membrane protein assembly factor BamB P77774|BAMB_ECOLI 36
Signal recognition particle receptor FtsY P10121|FTSY_ECOLI 36
Anaerobic C4-dicarboxylate transporter DcuB P0ABN9|DCUB_ECOLI 34
Glucans biosynthesis protein P33136|OPGG_ECOLI 34
Adenine phosphoribosyltransferase P69503|APT_ECOLI 34
Maltoporin P02943|LAMB_ECOLI 34
NADH-quinone oxidoreductase subunit C/D P33599|NUOCD_ECOLI 32
ATP-dependent protease ATPase subunit HslU P0A6H5|HSLU_ECOLI 32
CDP-diacylglycerol–serine O-phosphatidyltransferase P23830|PSS_ECOLI 32
PTS system trehalose-specific EIIBC component P36672|PTTBC_ECOLI 31
Transcription termination/antitermination protein NusG P0AFG0|NUSG_ECOLI 31
(Continued)
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9September 2019 | Volume 9 | Article 324
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
TABLE 3 | Continued
Protein name Symbol Score (p<0.05)‡
Protein translocase subunit SecF P0AG93|SECF_ECOLI 30
Oligopeptide transport system permease protein OppB P0AFH2|OPPB_ECOLI 30
Uncharacterized protein YffS P76550|YFFS_ECOLI 29
NADH-quinone oxidoreductase subunit J P0AFE0|NUOJ_ECOLI 29
Glucosamine-6-phosphate deaminase P0A759|NAGB_ECOLI 29
Uncharacterized protein YiaF P0ADK0|YIAF_ECOLI 28
Tol-Pal system protein TolQ P0ABU9|TOLQ_ECOLI 28
Multidrug export protein EmrA P27303|EMRA_ECOLI 27
UPF0246 protein YaaA P0A8I3|YAAA_ECOLI 25
DNA-directed RNA polymerase subunit omega P0A800|RPOZ_ECOLI 24
ATP-binding/permease protein CydD P29018|CYDD_ECOLI 24
Glycine betaine-binding protein YehZ P33362|YEHZ_ECOLI 23
NADP-dependent malic enzyme P76558|MAO2_ECOLI 23
Multiphosphoryl transfer protein P69811|PTFAH_ECOLI 23
Ribose-5-phosphate isomerase A P0A7Z0|RPIA_ECOLI 22
Disulfide bond formation protein B P0A6M2|DSBB_ECOLI 22
Uncharacterized protein YbjD P75828|YBJD_ECOLI 22
NADH-quinone oxidoreductase subunit L P33607|NUOL_ECOLI 21
Pyridoxine 5’-phosphate synthase P0A794|PDXJ_ECOLI 21
Proteins were isolated from CBD treated (5 µM) E. coli MVs and analyzed by LC-MS/MS. Peak list files were submitted to Mascot (in-house, Cambridge Center for Proteomics,
Uniprot_Escherichia_coli_20180613; 4324 sequences; 1357163 residues).
‡Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores >19 indicated identity or extensive homology (p <0.05). Protein
scores were derived from ions scores as a non-probabilistic basis for ranking protein hits. Cut-off was set at Ions score 20.
of E. coli, and this clearly revealed the antibacterial property
of CBD.
Interestingly, CBD did increase antibacterial effects of
vancomycin on E. coli, in spite of vancomycin’s limited
effectiveness on Gram-negative species, also seen here by the
fact that vancomycin alone did not result in a halo around the
diffusion disk for E. coli. Therefore, CBD seems to overcome
previously established resistance of E. coli to vancomycin, which
has reported to partly be due to its inability to significantly
penetrate the outer membrane (Zhou et al., 2015). It may also be
important to note that erythromycin, rifampicin and kanamycin
inhibit protein synthesis, whereas vancomycin is a glycopeptide
that inhibits cell biosynthesis in Gram-positive bacteria, while
colistin binds to the outer membrane of Gram-negative bacteria,
disrupting it. Thus, these antibiotics display very different modes
of action.
In the Gram-positive bacterium S. aureus subsp. aureus
Rosenbach, CBD increased bactericidal activity of kanamycin
only. The reduced ability of CBD to sensitize this Gram-positive
bacterium to antibiotics, compared to the significantly higher
effects in the Gram-negative bacterium, tallied in with CBD’s
ability to regulate MV-release, indicating a relevant contribution
of MVs to antibiotic resistance. Roles for MVs in protecting
biofilms via adsorption of antimicrobial agents have indeed
been previously recognized (Schooling and Beveridge, 2006;
Manning and Kuehn, 2011; Toyofuku et al., 2019). This also
indicates that MV-inhibitors that target membrane vesicles from
specific bacteria species, such as CBD here, could be applied
in combination with selected antibiotics for tailored antibiotic
treatment to tackle antibiotic resistance.
CONCLUSIONS
CBD effectively inhibited MV release from the Gram-negative
bacterium E. coli VCS257, exhibiting a stronger MV-inhibiting
effect at lower dose. In addition, CBD modulated MV protein
profiles of E. coli following 1 h treatment. CBD did not
have significant effects on MV release in the Gram-positive
bacterium S. aureus subsp. aureus Rosenbach. When applied in
combination with a range of antibiotics, CBD increased anti-
bacterial effects of selected antibiotics, depending on bacteria
type. CBD, in combination with specific antibiotics, may
thus possibly be used as an adjuvant to selectively target
bacteria to sensitize them to antibiotic treatment and reduce
antibiotic resistance.
DATA AVAILABILITY
All datasets generated for this study are included in the
manuscript and/or the Supplementary Files.
AUTHOR CONTRIBUTIONS
UK, PM, BA, IK, PW, and SL performed the experiments. UK,
JB, AN, JI, and SL analyzed the data. PM, GM, GG, IK, SL, and JI
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 10 September 2019 | Volume 9 | Article 324
Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs
provided resources. UK, SL, and JI designed the study. SL, UK,
and AN wrote the manuscript. All authors critically reviewed
the manuscript.
ACKNOWLEDGMENTS
This work was supported in parts by a University of Westminster
Start-up Grant to SL and an unrestricted grant from GW
Pharmaceuticals. Thanks are due to the Guy Foundation for
funding the purchase of equipment utilized in this study. The
funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fcimb.
2019.00324/full#supplementary-material
Supplementary Figure 1 | Effects of CBD on bacterial growth in (A) E. coli
VCS257 and (B) S. aureus subsp. aureus Rosenbach, after 24 h incubation, as
assessed by disk diffusion test. Exact p-values are shown.
Supplementary Figure 2 | TEM of MV released from E. coli VCS257 following 1
or 5 µM CBD treated for 1 h, compared to MVs isolated from control, untreated E.
coli.(A) A composite image showing MVs released from control, untreated E. coli.
(B) A composite image showing MVs released from E. coli treated with 1 µM CBD
for 1 h. (C) A composite image showing MVs released from E. coli treated with
5µM CBD for 1 h. Scale bars indicate 100 nm, respectively, and are included in
the individual figures.
Supplementary Figure 3 | Protein-protein interaction networks of protein hits
identified in MVs from 1 µM CBD treated E. coli VCS257. Reconstruction of
protein-protein interactions based on known and predicted interactions using
STRING analysis. Colored nodes represent query proteins and first shell of
interactors; white nodes are second shell of interactors. (A) Biological GO
processes are highlighted as follows: red, citrate metabolic process; green,
antibiotic metabolic process; yellow, regulation of cellular amide metabolic
process; purple, carboxylic acid metabolic process; dark green, regulation of
phosphate metabolic process; light blue, cellular respiration; orange, small
molecule metabolic process; dark red, negative regulation of translational
elongation; dark blue, generation of precursor metabolites and energy. (B) KEGG
pathways are highlighted as follows: dark green, oxidative phosphorylation; dark
red, citrate cycle (TCA cycle); red, biosynthesis of antibiotics; purple, butanoate
metabolism; dark blue, biosynthesis of secondary metabolites; light blue, carbon
metabolism; orange, phenylalanine, tyrosine and tryptophan biosynthesis; light
green, Microbial metabolism in diverse environments; yellow, Metabolic pathways;
violet, glycine, serine, and threonine metabolism. Colored lines indicate whether
protein interactions are identified via known interactions (curated databases,
experimentally determined), predicted interactions (gene neighborhood, gene
fusion, gene co-occurrence) or via text mining, co-expression or protein homology
(see color key for connective lines).
Supplementary Figure 4 | Protein-protein interaction networks of protein hits
identified in MVs from 5 µM CBD treated E. coli VCS257. Reconstruction of
protein-protein interactions based on known and predicted interactions using
STRING analysis. Colored nodes represent query proteins and first shell of
interactors; white nodes are second shell of interactors. (A) Biological GO
processes are highlighted as follows: red, cellular respiration; green,
purine-containing compound metabolic process; yellow, electron transport chain;
purple, ribose phosphate metabolic process; dark green, purine ribonucleotide
metabolic process; light blue, generation of precursor metabolites and energy;
orange, nucleobase-containing small molecule metabolic process; dark red,
purine ribonucleoside metabolic process; dark blue, organophosphate metabolic
process. (B) KEGG pathways are highlighted as follows: red, bacterial secretion
system; light green, metabolic pathways; yellow, oxidative phosphorylation;
purple, butanoate metabolism; dark green, quorum sensing; light blue, amino
sugar and nucelotide sugar metabolism; dark blue, protein export; violet, purine
metabolism. Colored lines indicate whether protein interactions are identified via
known interactions (curated databases, experimentally determined), predicted
interactions (gene neighborhood, gene fusion, gene co-occurrence) or via text
mining, co-expression or protein homology (see color key for connective lines).
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Conflict of Interest Statement: GG is founder and chairman of GW
Pharmaceuticals. AN is a scientific advisor to GW Pharmaceuticals.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2019 Kosgodage, Matewele, Awamaria, Kraev, Warde, Mastroianni,
Nunn, Guy, Bell, Inal and Lange. This is an open-access article distributed under the
terms of the Creative Commons Attribution License (CC BY). The use, distribution
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