Download Biogenesis and multifaceted roles of outer membrane

Microbiology (2014), 160, 2109–2121
Review
DOI 10.1099/mic.0.079400-0
Biogenesis and multifaceted roles of outer
membrane vesicles from Gram-negative bacteria
Heramb M. Kulkarni and Medicharla V. Jagannadham
Correspondence
CSIR – Centre for Cellular and Molecular Biology, Tarnaka, Hyderabad-500007, India
Medicharla V. Jagannadham
[email protected]
Received 24 March 2014
Accepted 25 July 2014
Outer membrane vesicles (OMVs) released from Gram-negative bacteria consist of lipids,
proteins, lipopolysaccharides and other molecules. OMVs are associated with several biological
functions such as horizontal gene transfer, intracellular and intercellular communication, transfer of
contents to host cells, and eliciting an immune response in host cells. Although hypotheses have
been made concerning the mechanism of biogenesis of these vesicles, research on OMV
formation is far from complete. The roles of outer membrane components, bacterial quorum
sensing molecules and some specific proteins in OMV biogenesis have been studied. This review
discusses the different models that have been proposed for OMV biogenesis, along with details of
the biological functions of OMVs and the likely scope of future research.
Introduction
The perception that prokaryotic organisms exhibit a simple
structure as compared with eukaryotes prevailed for a long
time. The prokaryotes, including bacteria, were thought to
be a collection of independently surviving autonomous
cells with few interactions, and almost no compartmentalization (Manning & Kuehn, 2013). Various new findings
have shifted the paradigm, and prokaryotes are now known
to possess a very ordered structure and organization, finely
tuned physiology, and numerous social interactions which
give rise to collective solutions for their survival (Raymond
& Bonsall, 2013; Spitzer & Poolman, 2013). Different
classes of prokaryotes have evolved different organelles and
strategies, of which many are analogous to those in eukaryotic cells. The Gram-negative bacteria are known to release
extracellular membrane-bound vesicles, which have been
compared with the exosomes produced by unicellular and
multicellular eukaryotes. They are named outer membrane
vesicles (OMVs) and are the subject of this review.
OMVs
The reasons for the term OMV are mainly historical. It was
first seen that a cell-free filtrate of Vibrio cholerae was able
to elicit an immune response in rabbits (De, 1959). As the
outer membrane (OM) components of Gram-negative
bacteria were known to be immunogenic, it was suspected
that the components of the OM were somehow leaching
into the medium. Later it was shown that Escherichia coli
cultures grown on lysine-limiting medium showed secretion of LPS in the form of spherical ‘bags’ (Bishop & Work,
1965; Work et al., 1966). Soon after that, V. cholerae was
Abbreviations: OM, outer membrane; OMV, outer membrane vesicle;
PQS, 2-heptyl-3-hydroxy-4-quinolone.
079400 G 2014 The Authors
found to release spherical membrane-bound vesicles which
were produced by pinching off of the OM (Chatterjee &
Das, 1967). It was also observed that the density of some
granular material was higher near the site where the
‘pinching off’ event was taking place on the OM, and some
granules were entering into the OMVs, suggesting possible
sorting of cytosolic contents to these vesicles. Later, many
different Gram-negative bacteria were shown to release
similar vesicles, ranging in size from 20 to 250 nm (Kulp &
Kuehn, 2010). Studies of the chemical composition of the
OMVs showed the presence of components from the OM
as well as other cellular compartments. Therefore, these
structures were called ‘membrane vesicles’, ‘extracellular
vesicles’, ‘outer membrane fragments’ or ‘blebs’ (Mayrand
& Grenier, 1989), although the historical term ‘outer
membrane vesicles’ is now generally accepted for Gramnegative bacteria and is used throughout this review.
Gram-positive bacteria and Archaea are also reported to
exhibit the presence of membrane vesicles produced from
cell surfaces (Manning & Kuehn, 2013), but this review
mainly focuses on OMVs produced by Gram-negative
bacteria.
Functional studies have shown that OMVs have a multifaceted role in the physiology of bacteria. OMVs were
found to be released in all growth phases of the bacterial
culture (Manning & Kuehn, 2011) although their amount
and composition may be dependent upon the growth
conditions. Attempts to isolate a non-vesiculated mutant
have been unsuccessful so far, although many genes have
been identified whose alteration changes the number of
OMVs released (McBroom et al., 2006). As the OMVs are
known to provoke the immune system, they have been
recognized as promising agents to be used as vaccines.
Many efforts in this direction are being carried out (Aoki
et al., 2007), one successful example being a vaccine for
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2109
H. M. Kulkarni and M. V. Jagannadham
meningitidis caused by Neisseria meningitidis (Findlow et al.,
2006; Boutriau et al., 2007; Williams et al., 2007). Efforts to
prepare a vaccine for other infectious diseases continue
(Keenan et al., 2003; Zhu et al., 2005; Schild et al., 2009;
Nieves et al., 2014), and OMVs hold great promise to
combat these diseases. Besides direct medical applications,
the study of OMVs is also shedding new light on the
physiology of Gram-negative bacteria.
increases their efficacy. Furthermore, if multiple enzymes
are needed for a distant activity, for example degradation
of complex molecules from a solid surface, then OMVs can
co-transport proteins so that they reach the remote site
simultaneously. Thus, the energy expenditure of the cell in
making OMVs is justified by their action on remote targets
with high efficiency.
The Gram-negative bacteria possess sophisticated machineries for secretion of various biologically important molecules.
Secretion systems designated type I to type VI have been
identified and studied in Gram-negative bacteria. OMVs are
proposed to be a different secretion system, ubiquitously
present in all Gram-negative bacteria, which are independent of the conventional systems (Kadurugamuwa &
Beveridge, 1995; Kuehn & Kesty, 2005). For example, the
cytotoxic protein cytolysin A (ClyA) is secreted via OMVs in
E. coli (Bendtsen et al., 2005). Secretion via OMVs has many
important advantages over the other secretion systems.
Structural components of OMVs
Exploration of the chemical components of OMVs showed
that they contain phospholipids, lipopolysaccharides, proteins and in rare cases nucleic acids. They are also known to
transport metabolites, quorum sensing signals and other
small molecules. The structural studies of OMVs invariably
showed that they were spherical and enclosed with a
membrane bilayer, and had a strain-specific characteristic
size distribution.
Proteins
Characteristics of OMV-mediated secretion
OMVs facilitate the secretion of insoluble or hydrophobic
materials such as lipids, membrane proteins and signalling
molecules. The success of pathogenic bacteria lies in the
successful colonization of host tissue. Integral membrane
proteins called adhesins mediate co-aggregation of bacterial cells and attachment to host surfaces. In Porphyromonas gingivalis, OMVs contain multivalent complexes
of adhesins which cause cellular aggregation and the
formation of dental plaque biofilms (Grenier & Mayrand,
1987; Inagaki et al., 2006). The role of OMVs in the
formation of biofilms will be discussed in greater detail
later. OMVs were also found to transport the hydrophobic
quorum sensing molecule 2-heptyl-3-hydroxy-4-quinolone
(PQS) from Pseudomonas aeruginosa, and it has been shown
that PQS causes the biogenesis of OMVs (MashburnWarren et al., 2008).
In other secretion systems, soluble proteins are secreted into
the medium and so are exposed to the potentially harsh
conditions of the extracellular environment. OMVs may be a
means by which soluble proteins and nucleic acids can be
encapsulated in a bilayer membrane and released in a
protective structure. Soluble proteins are transported in the
OMV lumen (Lee et al., 2007). Proteins inside the OMV
lumen, as well as soluble proteins externally associated with
the surface of OMVs, are highly resistant to external
proteases (Kesty & Kuehn, 2004). Nucleic acids encapsulated
into OMVs are similarly protected from external nucleases.
So, OMV-mediated transport can allow less stable soluble
molecules, such as protease-susceptible toxins, and nucleic
acids, to reach their destinations undamaged. OMVs may
even behave like time-release capsules, which provide
beneficial activity a period of time after they were released.
OMVs allow proteins to be delivered at high local
concentrations and in proximity of the target site, which
2110
The protein content of OMVs has been studied thoroughly
by many groups using different bacterial strains. The presence of virulence factors was shown in the OMVs
produced by different pathogenic bacteria (Kadurugamuwa
& Beveridge, 1997; Chatterjee & Chaudhury, 2011). Wensink
& Witholt (1981) showed that the protein profile of OMVs
of E. coli is different from that of other subcellular fractions,
which was later confirmed in other organisms. Advances in
the MS-based proteomics have given momentum to the
high-throughput profiling of OMV proteins (Lee et al.,
2007). Many protein constituents have also been identified
by classical Western blotting and functional experiments.
The proteins found in OMVs belong to the OM and periplasm. Some researchers have used the presence of outermembrane proteins (e.g. OmpW) as markers of the OMVs
(McBroom & Kuehn, 2007; Manabe et al., 2013). Many
cytoplasmic and inner-membrane proteins were also found
in OMVs, and these were thought to be contaminants from
lysed cells in the cultures (Berlanda Scorza et al., 2008; Kulp
& Kuehn, 2010). To resolve this issue, a density-gradient
centrifugation step was added to the protocol for preparation of OMVs, to separate OMVs from proteins which
were free or loosely associated with OMVs. Even after this
stringent preparation, the OMVs from multiple bacterial
strains showed the presence of cytosolic and inner
membrane proteins (Lee et al., 2007). So it appears that
there must be a machinery that sorts cytosolic proteins into
the OMVs. Several functional studies also supported this
view. In some strains, DNA fragments were found in OMVs,
as we discuss in a subsequent section. The presence of DNA
in OMVs proved that cytosolic contents can, in principle, be
sorted into OMVs. The proteins from these OMVs belonged
to diverse functional categories, including OM structural
proteins, porins, ion channels, transporters for different
molecules, periplasmic and cytoplasmic enzymes, and
proteins related to stress responses (Lee et al., 2007, 2008).
Not all outer membrane proteins from a given strain were
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Microbiology 160
Studies on outer membrane vesicles
Table 1. Proteomics studies on OMVs of different Gram-negative bacteria
Study
no.
Source for OMVs
Sample
description
1
Neisseria meningitidis serogroup B
Wild-type
2
Neisseria meningitidis MC58
Dgna33
3
Neisseria meningitidis NZ98/254
Dgna33
4
5
6
7
8
9
10
11
12
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Vegetative cell
Developmental cell
Wild-type
Wild-type
13
14
15
16
17
18
19
20
21
Escherichia coli DH5a
Legionella pneumophila
Acinetobacter baumannii DU202
Helicobacter pylori J99
Helicobacter pylori NCTC 11637
Myxococcus xanthus DK1622
Myxococcus xanthus DK1622
Francisella philomiragia ATCC 25015
Francisella tularensis subsp. novicida
ATCC 15482
Edwardsiella tarda ED45
Pseudomonas aeruginosa PAO1
Staphylococcus aureus 06ST1048*
Acinetobacter baumannii ATCC 19606T
Klebsiella pneumoniae ATCC 13883
Pseudomonas aeruginosa PAO1
Pseudomonas aeruginosa PAO1
Pseudomonas aeruginosa PAO1
Pseudomonas aeruginosa PAO1
22
Acinetobacter baumannii AbH12O-A2
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
DlexAN
Wild-type biofilm
Wild-type
planktonic culture
Wild-type
23
Pseudomonas syringae pv. tomato T1
Wild-type
24
Francisella novicida ATCC 15482
25
Francisella novicida ATCC 15482
26
Neisseria meningitidis H44/76
(serogroup B)
Neisseria gonorrhoeae strains FA1090,
F62, MS11, 1291
Wild-type
exponential phase
Wild-type early
stationary phase
Wild-type
27
28
29
30
31
32
33
34
Escherichia coli Nissle 1917 (EcN)
Porphyromonas gingivalis
Campylobacter jejuni 11168
Bacteroides fragilis
Bacteroides thetaiotaomicron
Pseudomonas syringae Lz4W
Vibrio cholerae
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
sorted to OMVs (Lee et al., 2008), although membrane
proteins make up a significant portion of the OMV proteome. Table 1 summarizes proteomic analyses of OMVs
and the number of proteins that were identified in each case.
Enzymes present in OMVs include proteases, peptidases,
nucleases and b-lactamases (Lee et al., 2007; Schaar et al.,
http://mic.sgmjournals.org
Proteomics strategy
No. of
identified
proteins
Reference
(1) 1DE-MALDI-MS/MS,
(2) 1DE-LC-ESI-MS/MS
(1) 1DE-MALDI-MS/MS,
(2) 2DE-MALDI-MS/MS
(1) 1DE-MALDI-MS/MS,
(2) 2DE-MALDI-MS/MS
LC-ESI-MS/MS
2DE-MALDI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-MALDI-MS/MS
1DE-LC-MALDI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
50
Post et al. (2005)
63
Ferrari et al. (2006)
63
Ferrari et al. (2006)
132
181
131
86
61
429
218
238
415
Lee et al. (2007)
Galka et al. (2008)
Kwon et al. (2009)
Mullaney et al. (2009)
Mullaney et al. (2009)
Kahnt et al. (2010)
Kahnt et al. (2010)
Pierson et al. (2011)
Pierson et al. (2011)
1DE-LC-ESI-MS/MS
LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
72
314
143
113
159
105
71
76
193
Park et al. (2011)
Choi et al. (2011)
Gurung et al. (2011)
Jin et al. (2011)
Lee et al. (2012)
Maredia et al. (2012)
Maredia et al. (2012)
Toyofuku et al. (2012)
Toyofuku et al. (2012)
(1) 1DE-LC-MALDI-MS/MS,
(2) LC-MALDI-MS/MS
1DE-LC-ESI-MS/MS
175
Mendez et al. (2012)
139
LC-ESI-MS/MS
99
Chowdhury &
Jagannadham (2012)
McCaig et al. (2013)
LC-ESI-MS/MS
286
McCaig et al. (2013)
LC-ESI-MS/MS
140
LC-MALDI-MS/MS,
quantitative study by
isobaric tagging (iTRAQ)
1DE-LC-ESI-MS/MS
1DE-LC-ESI-MS/MS
LC-ESI-MS/MS
1DE-Q-TOF
1DE-Q-TOF
1DE-LC-ESI-MS/MS
In-solution digestion-LCESI-MS/MS
308
van de Waterbeemd
et al. (2013)
Zielke et al. (2014)
192
151
134
115
58
429
90
Aguilera et al. (2014)
Veith et al. (2014)
Jang et al. (2014)
Elhenawy et al. (2014)
Elhenawy et al. (2014)
Kulkarni et al. (2014)
Altindis et al. (2014)
2011, 2013, 2014). OMVs may also contain toxins and other
virulence factors (Kulp & Kuehn, 2010). The makeup of the
OMV proteome may help to shed light on the physiological
functions served by OMVs. The exact process by which
proteins are sorted to OMVs is not yet known. Protein
profiles of OMVs harvested at different growth phases or
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H. M. Kulkarni and M. V. Jagannadham
after imposition of different stresses were found to be
different (Berlanda Scorza et al., 2012). OMVs have been
used successfully as delivery vehicles for recombinant
proteins (Chen et al., 2010).
Phospholipids
Similar to the OM, the outer leaflet of OMVs is predominantly composed of LPSs in addition to lipids. In an
early study, the OMVs of E. coli were found to have a
similar phospholipid profile to the OM (Hoekstra et al.,
1976). Various studies have subsequently been carried out
to compare the phospholipids from the OM and OMVs,
mostly using classic techniques such as TLC (Horstman
& Kuehn, 2000; Kato et al., 2002; Nevot et al., 2006).
Apparently, OM phospholipids are present in the membranes of OMVs (Tashiro et al., 2011; Chowdhury &
Jagannadham, 2013; Kulkarni et al., 2014), and there is
evidence that OMVs contain some lipids that are not
detected in the OM (Kato et al., 2002). There are some
reports of the fine structural characterization of the
phospholipids present in OMVs using MS (Chowdhury
& Jagannadham, 2013; Kulkarni et al., 2014), and fatty acid
analysis by GC-MS (Tashiro et al., 2011; Fulsundar et al.,
2014). However, a quantitative lipidomic analysis of the
different kinds of phospholipids present has not yet been
done using modern high-throughput MS-based techniques. The curvature of the OMV membrane is far higher
than that of the parent cell and therefore it is likely
to be characterized by a different composition of various
phospholipids. The phospholipid composition of the OM
and OMVs from Pseudomonas aeruginosa was found to be
different. In the OM, phosphatidylethanolamine was the
abundant species while in OMVs it was phosphatidylglycerol (Tashiro et al., 2011). Furthermore, the relative
amount of saturated fatty acyl chains in OMVs was higher
as compared with the OM (Tashiro et al., 2011) making the
OMVs more rigid than the OM. Tashiro et al. (2012) later
proposed that the release of OMVs leaves the bacterial OM
more fluid, and cells may shed OMVs to maintain the
fluidity optimum for the prevailing growth conditions.
LPSs
LPSs found in the OM are a characteristic feature of the
Gram-negative cell envelope. They are also known to have
endotoxin and pyrogenic activity in mammalian systems
(Gu & Tsai, 1991) and they are important adhesins for
biofilm formation. LPS molecules were also found in
OMVs (Bishop & Work, 1965; Work et al., 1966). As for
proteins, not all LPSs of the parent cell appear in OMVs;
rather only a fraction of the LPS complement seems to be
sorted into OMVs (Li et al., 1996; Chowdhury &
Jagannadham 2013). OMVs from Pseudomonas aeruginosa
are mainly composed of the negatively charged B-band
of LPS instead of the more neutral A-band
(Kadurugamuwa & Beveridge, 1995). LPS may be an
important player in the biogenesis of OMVs (Tashiro
2112
et al., 2012), and this point is further clarified below. MS
studies on the sorting of the Lipid A component of LPS
from the OM to OMVs have been carried out by
Feldman’s group. They showed that in the case of the
dental pathogen Porphyromonas gingivalis, deacetylated
Lipid A accumulated in OMVs (Haurat et al., 2011) but
in the related species Bacteroides fragilis the Lipid A
profiles of the OM and OMVs were not different
(Elhenawy et al., 2014).
Nucleic acids
The presence of plasmid DNA was shown for the first time
in the OMVs of Haemophilus parainfluenzae (Kahn et al.,
1982). Later, other OMVs were found to contain RNA and
DNA (Dorward et al., 1989; Mayrand & Grenier, 1989);
they belonged to the genera Escherichia, Pseudomonas,
Haemophilus, Neisseria and others. DNA fragments from
lysed cells could potentially bind to the positively charged
surface of OMVs. Treatment of OMVs with DNase proved
that some DNA fragments were trapped in the lumen of
the OMVs (Renelli et al., 2004). A detailed study of the
presence of nuclease-resistant DNA in membrane vesicles
and OMVs from Gram-positive and Gram-negative bacteria, respectively, was carried out by Dorward & Garon
(1990). They observed that DNA in the form of circular
plasmids, linear plasmids and chromosomal fragments is
likely to be present in Gram-negative OMVs but not in
Gram-positive membrane vesicles. Very little is known
regarding the sorting of DNA to the OMVs. It has been
shown that OMVs can pick up free DNA fragments present
in their environment, and that DNA can enter through the
periplasm during their biogenesis (Renelli et al., 2004). The
nucleic acids from an infecting bacteriophage might be
thrown out from the cell by means of OMVs (Loeb &
Kilner, 1978). The DNA fragments found in the OMVs
were of plasmid, phage or chromosomal origins (Yaron
et al., 2000). They can be linear or circular. The OMVs of
Neisseria gonorrhoeae were found to contain RNA in
addition to DNA (Dorward et al., 1989). DNA was not
found in the OMVs of many other strains (Zhou et al.,
1998). Three different strains of Porphyromonas gingivalis
were found to produce OMVs that did not contain any
DNA (Zhou et al., 1998). Although DNA was present in
some OMVs, not all of them could transform other cells
(Yaron et al., 2000; Renelli et al., 2004).
Other molecules
OMVs also transport a variety of ions, quorum sensing
signals and metabolites (Schertzer & Whiteley, 2012; Biller
et al., 2014). The appearance of these molecules in OMVs has
not been studied extensively to date. There is speculation that
many other types of bioactive molecules will continue to be
found in OMVs. There have been some reports describing
the presence of cell wall components such as peptidoglycan
and muramic acid in OMVs (Kadurugamuwa & Beveridge,
1995), but this aspect has not since been revisited.
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Biogenesis of OMVs
The formation of OMVs was initially thought to be a
physical process associated with the routine wear and tear
of the OM of the cell (Haurat et al., 2011). Although this
idea is still held by some, recent discoveries show that there
is likely to be an elaborate mechanism behind the biogenesis of OMVs. Also, the formation of OMVs is an active
resource-depleting process, which suggests that OMVs are
not products of simple material shearing of the membrane.
Although there is no direct evidence of an energy requirement for OMV biogenesis, there is likely to be energy
expenditure required for the sorting of biomolecules
to OMVs (Kulp & Kuehn, 2010). The exact pathway of
OMV biogenesis is not currently known. However, many
mechanisms have been proposed, and it may be possible
that all of them work together for the formation of OMVs.
OMVs as a product of cell wall turnover
The earliest attempts to decipher the mechanism of biogenesis of OMVs were those of Burdett & Murray (1974)
and Hoekstra et al. (1976). It was noted that OMVs are
shed from the OM, despite the presence of covalent linkages
between the peptidoglycan layer and OM. Later, OMVs of E.
coli were found to contain a relatively low amount of
lipoproteins, as compared with the OM (Wensink & Witholt
1981). It was further proposed that OMVs form due to the
faster growth of the OM compared with the cell wall and
associated lipoproteins. Zhou et al. (1998) studied the
biogenesis of OMVs from Porphyromonas gingivalis and
proposed a model that suggested that OMV production was
due to turnover of the cell wall. This model was further
revisited by showing that autolysin mutants of Porphyromonas gingivalis produced fewer OMVs than wild-type
strains (Hayashi et al., 2002). During cell wall synthesis,
peptidoglycan and muramic acid exert a turgor pressure
onto the OM. The blebs thus produced ultimately pinch off
from the membrane and relieve the cell of the pressure caused
by excess wall materials. Biochemical methods showed that
the OMVs contain muramic acid, but not nucleic acids or
cytoplasmic proteins. The OM protein profile was comparable with that of OMVs (Zhou et al., 1998). This model does
not explain how cytosolic molecules may be packaged into
OMVs. In later experiments with other organisms, nucleic
acids were found in OMVs, and the presence and possibly
selective sorting of proteins from other compartments of the
cell were demonstrated (Haurat et al., 2011).
OMV production as a stress response
It is expected that any external physical and chemical stress
would be felt on the OM of the Gram-negative cell. It has
been found in a number of studies that the presence of
stress increases the production of OMVs (Kulp & Kuehn,
2010). Careful experiments using lysed cells as a control
showed that the observed increase in OMV production
during stress occurred without any increase in cell lysis
(MacDonald & Kuehn, 2013; Schwechheimer et al., 2013).
http://mic.sgmjournals.org
Therefore, it has been thought that the biogenesis of OMVs
from bacteria is primarily a stress response. It was seen in
the case of OMVs of Pseudomonas putida DOT-T1E that
chemical stresses such as toxic concentrations of long chain
alcohols and EDTA (which chelates ions needed for the
normal growth of bacterial cells), as well as physical stresses
such as osmotic pressure and heat shock, caused the cells to
release OMVs (Baumgarten et al., 2012). Antibiotic stress
has also been studied in this context (Maredia et al., 2012).
The antibiotic ciprofloxacin disrupts DNA replication and
leads to the SOS response. The SOS response was found to
be involved in increasing OMV production. It was also
observed that the shedding of OMVs makes the cells more
prone to form biofilms, possibly as they increase the
hydrophobic nature of the bacterial cell surface (Tashiro
et al., 2012). However, the fact that OMVs are released even
when cells grow without external stress makes it difficult to
accept that OMV biogenesis is exclusively a stress response.
McBroom et al. (2006) showed that membrane instability
cannot be correlated with OMV biogenesis. In their study,
many of the genes responsible for the overproduction of
OMVs were found to be related to peptidoglycan synthesis,
OM proteins and the sigma E stress response pathway.
However, instability of the OM was probably not the cause
of the production of OMVs (McBroom et al., 2006).
McBroom & Kuehn (2007) found that temperature stress
in E. coli leads to accumulation of misfolded proteins that
ultimately are thrown out by being packaged in OMVs.
Although they did not discuss it, the increased fluidity of
the OM at higher temperatures may also have partially
contributed to their observations. They recognize OMV
production as a novel stress response, but also assert that it
is a vital physiological process found in all Gram-negative
bacteria. The most interesting observation, however, is that
mutants producing higher amounts of OMVs could better
resist temperature stress. Considering all of these observations, we conclude that OMV biogenesis occurs and is
presumably important in non-stressed cells, but is also a
component of multiple stress responses.
A bilayer-couple model for biogenesis
The process of making of OMVs, which leaves the integrity
and functions of the bacterial OM intact, hints that there
must be some well-defined process with many steps. The
membrane curvature of the OMVs is far higher than that of
the OM. The cell wall determines the size and shape of
bacteria. As OMVs are devoid of cell wall components
or any similar structure, the curvature required for their
structure calls for a different explanation. Mashburn &
Whiteley (2005) found that, in Pseudomonas aeruginosa,
PQS is needed for OMV production. They further showed
that PQS induces membrane curvature by introducing
itself into the membrane. The extent of curvature was
found to be in proportion to the local concentration of
PQS. They proposed a ‘bilayer-couple model’, in which
the secreted PQS becomes inserted into the OM of the
bacterial cell, inducing curvature that ultimately leads to
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the formation of OMVs (Mashburn-Warren et al., 2009;
Schertzer & Whiteley, 2012). This model has not yet been
extrapolated to other organisms. However, it accepts
the production of OMVs to be a fundamental physical
process, and identifies a direct cause which marks the start
of OMV biogenesis. PQS may also be involved in the
destabilization of Mg2+ and Ca2+ salt bridges in the OM,
which leads to the negative charge repulsion between LPS
molecules. As quinolones are known to sequester positive
charges (Marshall & Piddock, 1994), and chelating agents
such as EDTA are known to increase OMV production
(Eagon & Carson, 1965), a specific role for PQS becomes
possible. The observation that external supplementation
of Mg2+ ions in the growth medium suppressed the formation of OMVs in Pseudomonas aeruginosa (MashburnWarren & Whiteley, 2006) also supports this hypothesis.
In further research, Tashiro et al. (2010) showed that the
production of PQS itself is further controlled by bicyclic
compounds such as 1-naphthol, 2-naphthol, 2, 3-dihydroxynaphthalene, 1-aminonaphthalene and 8-quinolinol.
These compounds inhibited the production of PQS,
which further repressed the production of OMVs. This
effect is significant as it shows that certain chemicals
can directly affect OMV production and therefore the
pathogenicity of bacteria, which may be applicable in
medicine.
Proteins playing a role in OMV biogenesis
The OMVs of Salmonella enterica contain the proteins
PagC and OmpX, whose overexpression is known to
accelerate the production of OMVs (Kitagawa et al., 2010).
Similarly, the E. coli inner membrane protein NlpA was
found to regulate the biogenesis of OMVs (Bodero et al.,
2007). The presence of curvature-inducing proteins has
been found to be important in vesicle formation in many
eukaryotic cells (Graham & Kozlov, 2010), although similar
observations have not yet been made for prokaryotes.
Previously, it was shown that lipoproteins are specifically
depleted in OMV membranes (Hoekstra et al., 1976).
Subsequently, Wensink & Witholt (1981) also showed a
decreased amount of lipoproteins in the OMVs of E. coli.
Since then, disruptions in several other tethering proteins (e.g. Braun’s lipoprotein, OmpA and the Tol–PAL
complex) have also been shown to be associated with
increased OMV production. Note that Pseudomonas aeruginosa possesses fewer lipoproteins than E. coli (Martin
et al., 1972), which may provide an explanation for the
observation that P. aeruginosa usually produces significantly higher levels of OMVs than E. coli (MashburnWarren & Whiteley, 2006). Some OM proteins such as
Omps, proteins related to Tol–Pal systems and YbgF are
suspected to have a role in the bulging of the bacterial OM
which marks the onset of OMV production (Bernadac et al.,
1998). Very recently it has been shown that in V. cholerae
the DegP protein influences OMV contents, and the
abundance of several other functionally significant proteins
(Altindis et al., 2014).
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OMV production through an interplay of peptidoglycan,
LPS and the OM
Some early studies performed with Salmonella and Pseudomonas aeruginosa showed that strains containing truncated LPS exhibited enhanced membrane blebbing (Smit
et al., 1975; Meadow et al., 1978). A model for Pseudomonas aeruginosa OMV formation has been proposed in
which the electronegative charge of the B-band LPS causes
charge-to-charge repulsion and membrane instability, which
results in outward membrane blebbing and a random trapping
of periplasmic components within OMVs (Kadurugamuwa &
Beveridge, 1995). This model is supported by the observation
that growth under conditions that enrich the B-band of LPS
enhances OMV formation by Pseudomonas aeruginosa
(Sabra et al., 2003). Deatherage et al. (2009) showed that
some of the connections between the peptidoglycan layer and
the OM (OM-PG) and those which connect OM–peptidoglycan–inner membrane (OM-PG-IM) are associated with
the release of OMVs from Salmonella. They also suggested
that, during various physiological processes, whenever the
density of these connections reaches some threshold, OMVs
pinch out as a result. Based on these results, Kuehn & Kesty
(2005) have proposed a model of OMV biogenesis.
Nanopods for OMV biogenesis
A strikingly different phenomenon was seen to work for
OMV production and delivery in a soil bacterium, Delftia
sp. Cs1-4. Cells of this strain were found to form long tubes
projecting from the cell surface and OMVs were siphoned
out of these tubes (Shetty et al., 2011). The tubes have been
termed ‘nanopods’ by the authors. The tube-like structures
are also found associated with the OMVs of Francisella
novicida (McCaig et al., 2013), but their connection with the
biogenesis of OMVs has not been studied. A mechanism of
this kind is suspected to be operative only in strains living in
habitats such as soil where the availability of water is scarce.
In other habitats, secreted OMVs quickly spread over long
distances through water. So, the long-distance transfer of
OMVs requires physical transfer to the site of delivery
(Sanchez, 2011).
Functions of OMVs
Many different functions have been demonstrated for
OMVs, making them an important player in the physiology
of Gram-negative bacteria. The prevalent idea is that OMVs
are indispensable for the survival of these bacteria. Over
many decades, different roles played by the OMVs were
found; some of the prominent functions are discussed below.
Assistance in biofilm formation
Biofilms are surface-adhering structures made from a
matrix consisting of exopolysaccharides, DNA, proteins
and many other molecules, in which bacterial cells remain
embedded. In general, bacteria form biofilms as a response
to stress. OMVs were found to be associated with the
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Studies on outer membrane vesicles
biofilms of Pseudomonas aeruginosa (Beveridge et al.,
1997). The number of OMVs released from bacteria is
found to increase during stress, which suggests a relationship between OMVs and biofilm formation (Baumgarten
et al., 2012; Fulsundar et al., 2014). OMVs and the proteins
they contain have been shown to play an important role in
the biofilm formation of Helicobacter pylori (Yonezawa
et al., 2009, 2011). Biofilm formation is characterized by
the expression of genes responsible for exopolysaccharide
production and co-aggregation of cells. OMVs were found
to be involved in co-aggregation of cells (Grenier & Mayrand,
1987; Whitchurch et al., 2002; Kamaguchi et al., 2003;
Inagaki et al., 2006; Ellis et al., 2010). Also, it has been
suggested that OMVs provide a platform for the interactions
of exopolysaccharides, DNA, proteins and the attachment
surface, along with the bacterial cells (Schooling &
Beveridge, 2006; Schooling et al., 2009). Although OMVs
showed a role in biofilm formation, the detailed mechanism
of their mode of action remains to be investigated.
Delivery of biomolecules between cells
OMVs, as described earlier in this review, encapsulate a
variety of biomolecules. Horizontal transfer of genes among
bacterial species has been attributed to OMVs (Yaron et al.,
2000; Biller et al., 2014). The OMVs from Acinetobacter
baylyi were found to transfer small DNA fragments to E. coli
(Fulsundar et al., 2014). Transfer of autolysins to competing
bacterial species (Li et al., 1998), and virulence factors or
cytotoxins to host eukaryotic cells (Furuta et al., 2009a, b;
Alaniz et al., 2007) have also been described. Haurat et al.
(2011) showed that virulence factors are specifically sorted
into OMVs. The first described function of OMVs is to
provoke the mammalian immune system, as they deliver the
antigenic OM components (De, 1959; Kondo et al., 1993),
and LPS, which is pyrogenic, along with other endotoxins.
Cell-free preparations of OMVs create infection-like symptoms in the host (Bauman & Kuehn, 2006; Ellis et al., 2010).
Thus, OMVs seem to spearhead the infection process.
The soluble luminal part of the OMV cargo is delivered to
target cells by two mechanisms. First, the OMVs burst open
or lyse in the vicinity of the target cells, shedding their
contents at very high local concentrations. Second, the
OMVs bind to the surface of target cells and deliver the
content via proximal lysis, phagocytic internalization or
membrane fusion (Kadurugamuwa & Beveridge, 1996).
OMVs are known to be stable (Kadurugamuwa &
Beveridge, 1999; Post et al., 2005), and there is almost no
evidence for spontaneous lysis. Renelli et al. (2004) proposed that OMVs can open transiently, internalize DNA
fragments from the medium and reseal. Although this does
not give direct evidence for the spontaneous lysis theory of
cargo transport, it shows that OMVs can open and reseal
without external stimulus. Lysis of OMVs near the target
site triggered by external stimuli has been demonstrated (Li
et al., 1998). OMVs were found to lyse near Gram-positive
cells, supposedly triggered by interaction of the OMVs with
http://mic.sgmjournals.org
the positively charged peptidoglycan, releasing the autolysins that kill these cells.
In the same study, the OMVs were also found to kill Gramnegative cells, where they were found to transfer their
contents to the cells by membrane fusion, highlighting the
second proposed mechanism of transport. OMVs were
also shown to transfer their contents to eukaryotic cells via
membrane fusion (Kadurugamuwa & Beveridge, 1999).
The particular fusion of these OMVs suggests a possible
self–non-self distinguishing mechanism at work. OMVs are
also known to take advantage of a natural process of
endocytosis to deliver cargo to eukaryotic cells (Furuta
et al., 2009a, b; Bomberger et al., 2009). This pathway does
not depend upon the fusion of heterotypic membranes of
OMVs and eukaryotic cells; rather, the OMVs enter into
the target cells as a whole entity. In immune cells, the
delivery of bacterial antigens to the antigen-presenting cells
resulted in a display of a cohort of bacterial epitopes to the
host immune system (Alaniz et al., 2007). The fusion of the
OMVs with the eukaryotic cell membranes has also been
shown by Bomberger et al. (2009), who used microscopic
studies using fluorescently labelled OMVs. OMVs from
enterotoxicogenic E. coli are known to elicit enterotoxic
and proinflammatory responses (Kesty & Kuehn, 2004).
The OMVs are known to transport cell surface attachment
protein and haemolysin, which greatly increases the
efficiency of haemolysis (Aldick et al., 2009).
Killing of competing microbial cells
OMVs produced from one bacterium can kill other
competing microbes in the vicinity. The OMVs of various
Gram-negative strains were found to kill many Grampositive and Gram-negative bacteria (Li et al., 1998). Their
study investigated the effectiveness of OMVs in killing
target bacteria with differing peptidoglycan chemotypes.
Killing was most effective when the target possessed
peptidoglycan that was similar to the OMV donor. The
self–non-self recognition of cells by the OMVs may even be
simpler. If the peptidoglycan hydrolases present in the
OMVs are the same as those of the target strain then they
cannot cleave the peptidoglycan layer. However, if they fuse
with cells of a non-self strain, then they degrade the cell
wall, killing the target cell (Beveridge, 1999). In a more
recent study, Vasilyeva et al. (2008) found that Lysobacter
sp. XL1 secreted bacteriolytic enzymes in OMVs. This
activity of OMVs shows that they might be capable of
distinguishing between self and non-self cells in a mixed
community. The idea of using OMVs as a new antimicrobial agent has been explored. However, the process
does not seem to be commercially feasible. It was found
that OMVs can package antibiotics and deliver them at a
very high concentration at the target (Kadurugamuwa &
Beveridge, 1995). This phenomenon can be exploited to kill
intracellular pathogens that are not reached by the
antibiotics in external body fluids (Kadurugamuwa et al.,
1998).
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H. M. Kulkarni and M. V. Jagannadham
Response to physical and chemical stress
As discussed previously, bacteria tend to produce more
OMVs as a response to stress (McBroom et al., 2006). It
was also shown that hyper-vesiculating mutants were better
able to adapt to stress conditions (Kulp & Kuehn, 2010;
Manning & Kuehn, 2011). During temperature stress, OMVs
were found to remove the misfolded proteins (Baumgarten
et al., 2012). In cells exposed to antibiotics, OMVs were
found to sequester (Manning & Kuehn, 2011) or to degrade
(Ciofu et al., 2000) the antibiotics. When stress is induced by
antibodies or bacteriophages, OMVs can act as decoy targets
protecting cells by titration of the antibodies or phages
(Manning & Kuehn, 2011). The production of OMVs is also
found to increase with nutrient stress, which shows that
OMVs have a role in bacterial nutrition (further explained in
the next subsection). OMV production increased when the
genes for the envelope stress response were mutated, showing
that OMVs can compensate for the decreased stress handling
by other pathways (McBroom et al., 2006).
Nutrition of bacterial cells
In natural bacterial environments, metal ions are typically
scarce, leading to interspecies and intraspecies competition
for them. OMVs are found to be enriched with these ions,
and therefore are thought to collect and concentrate the
rare ions for the consumption of bacterial cells (Kulp &
Kuehn, 2010). Proteomics studies on different OMVs have
shown the presence of different metal ion binding proteins,
and the machinery for ATP synthesis (Lee et al., 2007).
Enzymes found in OMVs may be involved in degrading
complex biomolecules in the medium to make nutrients
available. Biller et al. (2014) showed that, in marine
bacteria, OMVs may be a significant player in the carbon
flux between several species of bacteria and cyanobacteria.
Thus, OMVs perform a role in intraspecies nutrient
transfer.
Defence and resistance
A role for OMVs in the defence and resistance of bacteria is
a recently discovered phenomenon. The production of
OMVs requires resources, and the expenditure of biomolecules by bacteria would be justified if OMVs play a role in
stress resistance. OMVs are used to remove misfolded
proteins and toxins (McBroom & Kuehn, 2007). External
stress factors initially impact the cell envelope. OMVs are
able to remove agents that potentially harm the cell surface.
For example, OMV production increases the survival of
bacterial cells treated with lytic bacteriophages (Manning &
Kuehn, 2011). It has been shown that, during phage
adhesion, vesiculation increases and OMVs act as decoy
targets for the phages (Loeb & Kilner, 1978; Manning
& Kuehn, 2011). It was also shown recently that OMVs can
alleviate the stress caused by membrane-targeted peptide
antibiotics, such as polymyxin B, by acting as decoy targets,
and transporting these molecules away from the cell
(Manning & Kuehn, 2011). The absorption by OMVs of
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antibiotics and other molecules such as complement has
also been shown (Tan et al., 2007). Inactivation of antibiotics by OMVs has been studied by Ciofu et al. (2000).
The presence of various proteases, peptidases and enzymes
such as b-lactamases has also been widely demonstrated in
OMVs (Lee et al., 2007; Schaar et al., 2011, 2013). The
OMVs produced by one bacterial species were found to
protect other bacterial species from the antibiotic stress,
emphasizing the ecological role of OMVs in microbial
niches (Mashburn & Whiteley, 2005; Schaar et al., 2014;
our unpublished data, see Fig. 1). Manning & Kuehn (2011)
called OMVs the ‘innate defence’ of bacterial cells. The
offensive and defensive roles of these ‘innate defence’ players
have been described by MacDonald & Kuehn (2012).
Conclusion and future directions
OMVs seem to be fundamental for the survival of Gramnegative bacteria. Understanding the biogenesis of OMVs
will allow us to engineer them for potential applications.
To understand the genetic basis and the exact pathway of
biogenesis of OMVs, a high-throughput approach should
be applied. As described above, many genes are known to
play an important role in increasing or decreasing the yield
of OMVs. In a high-throughput approach, all the viable
single gene mutants of a bacterial species should be
examined for the production and yield of OMVs. Gramnegative bacteria may have 4000–6000 genes, and the
biggest hurdle is the development of techniques that will
enable the researcher to prepare and quantify OMVs from
a large number of bacterial cultures. With the availability of
the technologies required for investigation of genes one by
one in a planned fashion in a short time, global study may
be possible. The information obtained from such experiments will hopefully help to elucidate the genetic basis of
OMV biogenesis.
The organization and the quantity of OMVs are suspected to
be responsive to the growth conditions, stress factors and
growth phases of bacterial cultures. How some proteins
are selectively sorted into OMVs is currently unknown.
Quantitative proteomics studies of OMVs prepared from
cultures grown under different conditions or of different
mutants have not been undertaken. Information from such
experiments would help to show how dynamic the contents
of OMVs are, and, more importantly, what are the common
minimum components of OMVs. Comparative proteomics
of OMVs from mutants may provide hints about the genes
and pathways that affect sorting of certain classes of proteins
to OMVs. This would help in preparing engineered OMVs.
Quantitative lipidomics is still an emerging field; it may
prove useful in the study of OMVs as a complement to
proteomic studies. The curvatures of the surface of OMVs,
the properties of the OMV membrane and the factors which
determine the size of OMVs have been studied only
sporadically.
The function of OMVs in consortia of different bacterial
species is an emerging area of investigation. In a natural
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Microbiology 160
Studies on outer membrane vesicles
Outer leaflet (lipopolysaccharides)
Inner leaflet (glycerophospholipids)
OMVs interacting with:
Membrane active peptides
(a)
(b)
Membrane
active
peptide
β -Lactam antibiotics
β -Lactam
antibiotic
molecules
(c)
Bacteriophages
β -Lactamasedegrading antibiotic
molecules
Bacteriophage
Protease/
peptidase
Nuclease
Outer membrane
porin
Fig. 1. Schematic depiction of the role of OMVs as an innate defence of bacterial cells against antibacterials and other toxins.
The mode of action of OMVs is depicted. (a) The interaction of membrane active peptides with OMVs. The OMVs can sequester
or degrade membrane active peptides. (b) The degradation of the b-lactam antibiotics by b-lactamase in the lumen of OMVs. (c)
The fate of bacteriophages, as they are either titrated away from bacteria or possibly inactivated by OMVs.
niche, numerous bacterial species generally coexist and
individuals are probably exposed to a pool of OMVs from a
variety of other bacterial cells. OMVs have been described to
kill competing microbes, as well as to protect members of the
producer species. These contradictory roles of OMVs imply
their multifaceted influence in bacterial ecology. Knowledge
of the ecological role of OMVs may help the design of
probiotic drugs to combat various bacterial pathogens.
The preparation of vaccines using OMVs has been well
studied. High-yield OMVs with consistent composition
is a key factor in developing OMV-based vaccines.
Some bacterial genera, such as Pseudomonas, are known
to naturally produce higher yields of OMVs than others.
Expressing antigenic proteins of interest in a species with
higher yields of OMVs may be advantageous, provided the
antigen is sorted into the OMVs. Engineered OMVs
containing human proteins that act as sites for virus
attachment can be used as decoy targets for human viral
diseases. The viral particles may inject their genetic
material into these decoy targets, which would degrade it
by using bacterial nucleases in their lumen.
There is a resurgence of classical bacterial diseases with a
steep decline in the efficacy of antibiotics. New bacterial
and viral diseases are emerging, for which rapid methods
for vaccine development would be important. The application of OMVs holds some promise in this context. The
availability of high-throughput ‘omics (genomics, proteomics,
lipidomics) technologies, automation in microbiological
http://mic.sgmjournals.org
techniques and support from bioinformatics makes the
exploration of this promise more practical. Apart from
potential applications, the study of OMVs will also improve
our overall understanding of bacterial physiology.
Acknowledgements
We thank DST, New Delhi, for the financial support to carry out
the research work. H. M. K is a recipient of a CSIR senior research
fellowship.
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