ISSN 00036838, Applied Biochemistry and Microbiology, 2015, Vol. 51, No. 5, pp. 485493. Pleiades Publishing, Inc.

., 2015.
Original Russian Text K.A. Lusta, 2015, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2015, Vol. 51, No. 5, pp. 443452.

Bacterial Outer Membrane Nanovesicles: Structure, Biogenesis,

Functions, and Application in Biotechnology and Medicine (Review)
K. A. Lusta
Research Institute of Human Morphology, Russian Academy of Medical Sciences, Moscow, 117418 Russia
Institute for Atherosclerosis Research (Skolkovo), Moscow, 121609 Russia
email: k_lusta@rambler.ru
Received November 7, 2014

AbstractThe review summarizes the comprehensive biochemical and physicochemical characteristics of

extracellular membrane nanovesicles (EMN) derived from different kinds of bacteria. The EMN structure,
composition, biogenesis, secretion mechanisms, formation conditions, functions, involvement in pathogen
esis, and application in biotechnology and medicine are discussed.
Keywords: extracellular membrane nanovesicles, bacterial outer membrane, secretion, biogenesis, interspe
cies communication, intercellular signals, bacterial toxins, immunity modulation, vaccine
DOI: 10.1134/S0003683815040092

In the past two decades, some bacteria have been

shown to separate from their surface into the medium
the extracellular membrane nanovesicles (EMN),
which are bounded by the bacterial membrane and
contain periplasmic components [14]. EMN were
discovered for the first time when it was noticed that
the cellfree supernatant of E. coli cultured under the
conditions of growth limitation by lysine contained
soluble lipopolysaccharides (LPS). The authors sup
posed that peptidoglycan synthesis in the bacteria is
limited under these conditions, without affecting the
outer membrane (OM) synthesis. As a result, excessive
OM cannot remain bound to the cell [5]. Electron
microscopic study of these bacteria showed that,
indeed, small spherical structures with an electron
dense center were surrounded by a single membrane
separated from the external cell envelope [6, 7]. How
ever, subsequent studies showed that EMN were
formed also under normal conditions of bacterial
growth, both in vitro and in vivo in the infected host
tissues, and that EMN are produced by many species
of Gramnegative bacteria, indicating the natural
ness of the process and its widespread occurrence
among microorganisms [8, 9]. All of the Gramnega
tive bacteria that have been studied up to now (both
pathogenic and nonpathogenic) naturally produce
EMN at all stages of their growth [8, 10]. The method
of the quantification of radiolabeled protein and tox
ins has shown that the native EMN produced by bac
terial cells make up a considerable fraction of cell
material: up to 12% [11]. EMN are composed mainly
by the outer membrane components of Gramnega
tive bacteria, including LPS, glycerophospholipids,
bacterial OM proteins, periplasmic and cytoplasmic

elements [1, 12, 13]. In addition, it has been shown

that EMN contain plasmid and chromosomal DNA
and the DNA of bacteriophages [1417]. It was previ
ously established that these vesicles possess quite a
number of biological properties, including the ability
to adhere to pro and eukaryotic cell surfaces [18].
Reliable experimental evidence demonstrates that the
EMN released by pathogenic bacteria contain viru
lence factors. They are supposed to be the means of
delivery of pathogenic factors to eukaryotic tissues.
Studies of LPS density and composition, as well as
protein analysis of isolated EMN, have shown that
they lack or may contain trace amounts of compo
nents of the bacterial inner membrane (IM) and cyto
plasm [19]. This fact indicates that EMN formation is
not a result of cell lysis, during which vesicles appear
that contain a mixture of the proteins and lipids
released from all cell fractions. The bacterially pro
duced EMN contain biologically active proteins and
perform various functions. In contrast to other secre
tory mechanisms, EMN allow the bacteria to release
complexes of insoluble and soluble compounds. The
secreted enzymes can be used after reaching distant
objects in a concentrated and protected form. EMN
are produced by bacteria as a stress response and play
a key role in their survival and nutrition, biofilm for
mation, and pathogenesis [9].
EMN are used as vaccine carriers for immuniza
tion of humans and animals against many infectious
diseases. The possibility of using these nanoparticles in
other fields of biotechnology is under discussion.
EMN biogenesis. During evolution, microorgan
isms adapted to environmental conditions (water, soil,
host organism), including extreme habitats such as hot

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springs. Microbes develop specific features ensuring

their interaction with the environment. One of them is
EMN release by prokaryotes [20]. Although there is a
wide range of bacteria that have been shown to have
EMN and these vesicles are involved in many impor
tant biological processes, there are not enough data on
the molecular mechanism of their formation [9].
The cell wall of Gramnegative bacteria consists of
the inner and outer membranes, which are separated
by the periplasm and a peptidoglycan layer. The OM is
tethered to the underlying peptidoglycan layer via
lipoprotein. The OM comprises phospholipids, LPS,
integral membrane proteins, and lipoproteins. The
major component is a lipid bilayer. The internal and
outer OM layers are represented by phospholipids and
LPS, respectively. The IM and OM differ in the lipid
and protein compositions [21]. The thickness of peri
plasmic space between the two membranes (~13 nm)
is 710% of the total cell volume [22]. The OM con
tains a lot of proteins and performs the following func
tions: supply of nutrients to cells, cell adhesion on sur
faces, secretion, signaling, and protection from the
environment [23, 24]. The release of EMN by bacteria
is one more type of secretion, in addition to the six
known and studied types of secretion of multiprotein
complexes from the cells [25].
The biogenesis of EMN includes the protrusion of
bacterial OM into areas where there is no membrane
tethering to peptidoglycans, the regulation of vesicle
formation without the loss of OM integrity, the satura
tion of EMN with various components, or the exclu
sion of certain proteins and lipids, as well as mem
brane division without the energy of ATP/GTP
hydrolysis [9, 26, 27].
The mechanism of EMN formation and separation
from the cell is still not quite clear; however, genetic
and biochemical studies have shown that EMN for
mation is one of the main growth characteristics of
Gramnegative bacteria, and this process involves a lot
of factors [2831].
Three models of formation of these vesicles have
been proposed, and five hypothetical factors contribut
ing to the biogenesis of extracellular vesicles have been
distinguished [32, 33]. EMN are formed in places
where there is no tethering between the outer mem
brane and peptidoglycans [31]. EMN formation is
facilitated by the repulsion of anionically charged
neighboring LPS molecules on the cell surface [33, 34].
EMN formation induces the accumulation of foreign
compounds in the periplasm [29, 35, 36]. EMN are
formed in regions with enhanced membrane rigidity
[34, 37]. EMN are secreted in the regions with
enhanced membrane curvature [31].
All bacteria have developed a complex communi
cation strategy of quorum sensing (QS) for the coordi
nation of their activity. This mechanism is used during
pathogenesis. The signaling molecule of 2heptyl3
hydroxy4quinolone (Pseudomonas quinolone sig
nal, PQS), which is transported to neighboring cells

by EMN, plays the key role in this system. These mol

ecules both act as signals and participate in EMN for
mation. PQS have been shown to interact with the acyl
chains and 4'phosphate of bacterial LPS and to stim
ulate the formation of liposomelike structures (puri
fied LPS). These studies show that quorum signals not
only perform the signaling function but also partici
pate in EMN biogenesis [34, 38].
The effects of external factors and stress on EMN
production. It is known that EMN are more intensively
produced by bacteria under the influence of different
factors [39], including temperature [19], antibiotics
[36, 40], and oxygen stress [41]. Thus, it has been
shown that vesicle production by the bacterium Serra
tia marcescens is thermally regulated. EMN were
formed in appreciable quantities at 2230C, but
many of them were formed at 37C [42]. There is a
hypothesis that EMN production considerably
increases in response to stress (SOS signals). Such
conclusions were based, in particular, on studies show
ing that bacteria induce a SOS signal and produce
EMN when bacterial infections are suppressed by
antibiotics. We have verified this hypothesis by study
ing EMN production in the wildtype bacterium
Pseudomonas aerugonisa and its mutant strain lexAN,
in which the SOS response was suppressed by ciprof
loxacin. The protein and lipid content and EMN cyto
toxicity for host cells increased after antibiotic treat
ment in both strains. A further increase in EMN pro
duction by the lexAN strain was suppressed,
suggesting the involvement of SOS in this process.
Thus, stress response plays a key role in the stimula
tion of EMN production and contributes to bacterial
cytotoxicity [43]. The presence of trace elements in
bacterial habitat also substantially influences EMN
formation [44, 45].
EMN composition. EMN were shown to consist
mostly of the typical widespread OM proteins [46] and
periplasmic proteins [47]. In addition, EMN are
enriched in certain proteins and lipids, while others
are completely excluded [12, 46, 48, 49]. For example,
EMN of the bacterium P. aeruginosa contained only
the LPS of group B, which were present in the OM of
this bacterium in the minimum amount [47, 49].
These studies indicate that EMN production is a result
of biological process.
Proteomic analysis of EMN released by different
bacterial species has shown that isolated vesicles con
tain more than one hundred various proteins. They
include the bacterial OM proteins such as lipoprotein
AcfD precursor, outer membrane receptor protein
TolC, ferrichrome protein precursor, OM antigen,
phospholipase A1 precursor, channelforming protein
Tsx, OM porin precursor, colicin I receptor, vitamin
B12 receptor precursor, cytolethal distending toxins,
etc., and periplasmic proteins such as protease,
glutaminebinding periplasmic protein, glucosi
dase, lipoprotein carrier protein, sulfatase precursor
(YdeN), cytotoxic metalloprotease, etc. The proteins

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of bacterial cytoplasm include chaperone HtpG, alkyl

hydroperoxide reductase, elongation factor (Tu), the
flavoprotein subunit of succinate dehydrogenase, etc.
[37, 5055].
The EMN from Mannheimia haemolytica was ana
lyzed by a combination of liquid chromatography meth
ods and tandem mass spectrometry (LCMS/MS). As a
result, 226 proteins were identified; 58% of them were
from OM and periplasm and one (0.44%) was extracel
lular [56]. The study of EMN from Shewanella vesicu
losa by the improved method (nano LCMS/MS)
showed that vesicular proteins were present mainly in
the outer membrane (69.57%); most of them were
TonBdependent receptors and porins involved in inor
ganic ion transport and metabolism. The revealed peri
plasmic proteins (4.35%) were from the family of pro
teases. Among the cytoplasmic membrane proteins
(6.5%), there were cytochrome c oxidases and trans
ferases; the cytoplasmic proteins included F0F1 ATP
synthase and Natranslocating NADquinone reduc
tase (4.3%).
EMN of Grampositive bacteria. For a long time it
had been considered that EMN secretion is character
istic only of Gramnegative bacteria constitutively
secreting membrane vesicles as a mechanism of con
tactless cellcell communication, and this cellular
process was not examined in Grampositive bacteria.
However, 5 years ago it was shown for the first time
that Grampositive bacteria naturally release EMN
into the external medium. Further studies showed that
the density and size of EMN produced, e.g., by the
bacterium Staphylococcus aureus, are analogous to
those of EMN from Gramnegative bacteria. It was
shown by proteomics methods that EMN of these bac
teria contained altogether more than 90 protein com
ponents. The character of the identified proteins dem
onstrates that the EMN of Grampositive bacteria
have a specific sorting mechanism. The proteins iden
tified within these EMN promote the transport of var
ious components to other bacteria, are necessary for
the elimination of competing organisms, provide anti
biotic resistance, perform pathological functions
when infecting other organisms, and are necessary for
EMN biogenesis [58, 59].
EMN functions. Although EMN were discovered
more than 40 years ago, their biological role has been
studied only in the past decade. The mechanisms of
the formation and functions of EMN have only
recently been elucidated, because nanoparticle analy
sis is rather difficult. At present, considerable progress
is being made in EMN studies. However, many biolog
ical functions of EMN that allow bacteria to survive in
complex microbial communities have not yet been
determined. Studies of the extracellular vesicles of dif
ferent bacterial origin made it possible to define their
common function as a provider of interaction with
prokaryotic [49] and eukaryotic cells [10, 60].
Bacteria must use a means for the lysis of other
cells to win the competition in polymicrobial com
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487

munities. It has been shown that EMN possess an

antimicrobial activity against other Gramnegative
and Grampositive bacteria [32, 49, 61]. The lytic
activity of EMN from many bacteria is determined by
the presence of murein hydrolase [47] and antibacte
rial quinolones [32]. Murein hydrolase cleaves the
covalent bonds of peptidoglycans and, when EMN
adhere to bacterial surface, performs their lysis, while
antimicrobial quinolones suppress the growth of other
bacteria. Thus, EMN contributes to the preservation
of competitive advantages by parental bacteria in
polymicrobial communities [27].
The components of EMN have multifunctional
effects, including the interaction between microor
ganisms, the maintenance of biofilm structure, and
the infection of host organisms [62]. EMN possess
such components as different kinds of virulence fac
tors, two LPSs of chemically different structure, spe
ciesspecific communicative signaling molecules
(PQS) and phospholipids, the properties of which are
different from those in the OM. These characteristics
allow EMN (as secreted complexes of insoluble and
soluble components) to perform a lot of different
functions [18, 27]. When bacteria interact with a host
organism and other microbial cells, EMN have both
offensive and defensive capabilities [61, 62]. Some of
these functions of EMN are common for all Gram
negative bacteria, while others are specific for particu
lar bacterial species/strains [33].
It has been shown that EMN are used in quite a
number of processes, including the mechanism of
interspecies communications and microbehost
interactions [9] and intercellular signal transduction
[32, 34, 38]. EMN secrete and transport proteins [63]
and DNA [13, 17, 64], participate in the delivery of
toxins of pathogenic bacteria to eukaryotic host cells
[60], are a tool of bacterial virulence [61]; participate
in the attachment to eukaryotic host cells and inter
nalization [2, 3, 63, 6572]; cause immune activation
and the suppression of host cells [7381] and the lysis
of other bacteria [27, 32, 47, 49, 61]; perform stress
reaction (EMN are produced in response to stress)
[36]; provide bacterial nutrition, the utilization of
unnecessary compounds, and the storage of useful
substances [9]; provide for the survival and protection
of bacteria under different environmental conditions
[8284]; and initiate the formation and maintenance
of the structure of bacterial communities (biofilms)
[13, 14, 85].
Biofilms play an important role, inter alia, in
chronic infections. EMN entering the environment of
normal bacterial growth can supply bacterial DNA,
which is necessary for initial biofilm formation. The
quorum sensing (QS) system plays a certain role in
biofilm formation and in the control of bacterial viru
lence factors [34, 38].
The bacterium P. aeruginosa, which inhabits soils,
ocean, plants, animals and humans, is a model organ
ism for the study of EMN [60]. Analysis of the content

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of extracellular LPS and DNA in the supernatants of

wildtype P. auruginosa and QS mutant cultures has
shown that the amounts of LPS and DNA in EMN
were much higher in the wildtype cultures compared
to the QS mutants. Thus, the action of the QS system
can regulate the release of DNA and LPS present in
EMN [86]. This process can be used in the develop
ment of methods for treating severe infections caused
by P. auruginosa.
Involvement of EMN in pathogenesis. The extracel
lular secretion of different substances is the main mech
anism through which pathogenic bacteria perform the
communication and intoxication of host cells [10]. The
vesicles, which separate from the envelope of growing
bacteria and serve as vehicles for protein and lipid secre
tion by the bacteria, play a considerable role in patho
genesis [60, 65]. The delivery of toxins by EMN is a
powerful mechanism of the virulence of pathogenic
bacteria, modulating the immunity and causing dis
eases (Table) [10, 60]. Thus, the bacterial colonization
of a macroorganism is implemented by the content and
transfer of virulence factors into host cells. Vesicles are
formed in infected tissues under the influence of envi
ronmental factors [19, 36, 3941].
EMN are involved in different aspects of patho
genhost interactions, mediate bacterial attachment
and invasion, demonstrate cytotoxicity, and modulate
the immune response of a host, being bacterial viru
lence factors [66]. The virulence factors revealed in
EMN of different bacteria are presented in the table.
The secretion of virulence factors by pathogenic bac
teria is complicated, because the bacterial envelope
consists of the two lipid bilayers (OM and IM) and the
periplasm. Bacteria release active virulence factors
into the medium and into host organism tissues, using
many strategies that are specific for particular patho
gens [87]. The type II and V secretion systems are two
stage processes in which proteins are transported first
across the inner membrane and then across the outer
membrane. The type I, III and IV secretion systems
are characterized by the transfer of material directly to
the extracellular medium or into another cell. The
type III system is used by pathogenic bacteria for the
transfer of virulence factors. All of these types of secre
tion result in the release of both separate proteins and
their small complexes. Secretion via EMN belongs to
type VI, and its mechanism allows the bacteria to
release large protein and lipid complexes into extracel
lular medium [66].
One of the beststudied functions of EMN in
pathogenesis includes the transfer of virulence factors
and other substances directly into host cells [65]. The
EMN of pathogenic bacteria contain various virulence
factors, including adhesins, toxins, hydrolytic
enzymes, pathogenassociated molecular patterns
(PAMPs), immunomodulators, and other compo
nents that can influence the course of infection, exert
ing a toxigenic effect or activating the innate immune
response [88]. In an infected host, the EMN released

by bacteria can stimulate effective protective mecha

nisms. Thus, EMN, which may be considered anti
gens, can cause both inflammatory and immune
responses in humans and animals [10, 44, 89, 90].
The proteomic analysis of EMN from Campylo
bacter jejuni made it possible to identify more than
150 proteins, including periplasmic and OM proteins,
as well as many factors necessary for bacterial survival
and pathogenesis, including cytolethal distending
toxin (CDT). The periplasmic proteins of EMN from
C. jejuni contained 16 Nbound glycoproteins, which
were immunogenic glycoproteins that were able to
interact with host cells [112].
The bacteria Aeromonas hydrophila and A. salmoni
cida are widespread in the environment and can infect
fish, amphibians, mollusks, and humans [113]. The
infection of humans by bacteria of the genus Aeromo
nas leads to the development of many inflammatory
diseases such as sepsis, meningitis, pneumonia, perito
nitis, conjunctivitis, osteomyelitis, arthroempyesis,
myositis, cholecystitis, cholangitis, urinary tract infec
tions, endocarditis, etc. [114]. The pathogenic charac
teristics of these bacteria are determined by the pro
duction of a great number of virulence factors, includ
ing enzymes such as lipase, chitinase, gelatinase,
hemolysins, cytotoxins, and enterotoxins (membrane
damaging alpha and betahemolysins, cytolytic
enterotoxin, poreforming toxin (aerolysin), thermo
stable and thermolabile enterotoxins, thermostable
and thermolabile metalloproteinases) [115].
Transmission electron microscopy (TEM) has
shown that the bacteria A. hydrophila and A. salmoni
cida in pure cultures release EMN into the medium
[24]. Numerous vesicles appear nearby these bacteria
on the external edges of small colonies. The process of
vesicles budding off the bacterial cell has been shown.
Membrane nanovesicles isolated by differential cen
trifugation and ultrafiltration were visualized by nega
tive contrasting and TEM [24], making it possible to
determine their sizes and ultrastructure. The isolated
EMN population proved to be heterogeneous; the
diameter of its vesicles varied from 10 to 300 nm. On
ultrathin sections of rat intestines, EMN were found
among the aggregates of parietal bacteria of different
species. They often were chains of several bubbles
located inside the glycocalyx between the microvilli of
the apical surface of epithelial cells [24]. Since bacte
rial EMN contain various virulence factors and the
enzymes and proteins characteristic of bacterial OM,
periplasm and cytoplasm, as well as DNA, it may be
supposed that the nanovesicles released by the patho
genic bacteria A. hydrophila and A. salmonicida are
involved in a mechanism of pathogenesis that results in
different diseases of a macroorganism, as well as in the
intercellular interaction both within prokaryotic popu
lations and between pro and eukaryotes [24].
The dynamics of the penetration of EMN released
by bacteria into tissues and cells of a living organism
and their migration inside the cell has been shown in a

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489

Virulence factors in EMN from different bacteria

Bacterial species

Virulence factor

Reference

Actinobacillus leuropnemoniae

Proteases, Apx I

[91]

Actinobacillus actinomycetemcomitans

Leukotoxin

[46 ]

Bacteroides fragilis

Hemagglutinin, hydrolytic enzymes

[92]

Bordetella pertussis

ACHly toxin, Hemagglutinin (FHA), Pertussis toxin (Ptx), adeny [93, 94]
late cyclase toxin

Borrelia burgdorferi

Toxins OspA, OspB, OspD

[67]

Burkholderia cepacia

PLCN, lipase, PSCP, 40kDa protease

[95]

E. coli (ETEC)

Toxins LT

[12]

E. coli (STEC)

Shiga toxin

[16, 96]

E. coli (EHEC)

Cytotoxin ClyA

[97]

Escherichia coli.

Cytotoxic necrotizing factor 1

[98, 99]

A hemolysin
Helicobacter pylori

Toxin VacA

[100, 101]

Legionella pneumophila

Virulence factors: Mip (lpg0791), IcmK/IcmX,

LaiE/LaiF, hydrolytic enzymes

[51]

Moraxella catarrhalis

UspA1/UspA2

[102]

Neisseria meningitides

Virulence factors: PorA, NlpB, NarE

[103]

Pseudomonas aeruginosa

Hemolysin, hydrolytic enzymes, Toxins Cif, PQS

[32 ]

Salmonella typhi

Cytotoxin ClyA

[97]

Shigella flexneri

Toxins IpaB, IpaC, IpaD

[104 ]

Shigella dysenteriae

Shiga toxin

[105]

Treponema denticola

Proteases,dentilysin

[106, 107]

Vibrio anguillarum

Metalloproteinase, hemolysin, phospholipase

[108]

Vibrio cholerae

Toxin RTX

[109 ]

Xanthomonas campestris

Cellulase, glucosidase, xylosidase, avirulent proteins

[54]

Xenorhabdus nematophilus

Chitinase, bacteriocin, adhesin, poreforming toxin

[110 ]

Aeromonas hydrophila,
Aeromonas salmonicida

Lipase, chitinase, gelatinase, hemolysins, cytotoxins and enterotox [111]

ins (membranedamaging alpha and betahemolysins, cytolytic en
terotoxin, poreforming toxin (aerolysin), thermostable and ther
molabile enterotoxins, thermostable and thermolablie metallopro
teinases)

Campylobacter jejuni

The toxin of giant cells (cytolethal distending toxin, CDT)

number of works [2, 3, 63, 65, 6772]. In addition, the

processes of EMN internalization with the surface of
other bacteria, including pathogenic ones, have been
elucidated [104].
Modulation of host immunity. It has been shown that
isolated EMN are triggers of inflammatory processes in
animal and human organisms. For example, EMN iso
lated from Salmonella typhimurium stimulated (to the
same degree as the living bacteria) macrophages and
dendritic cells, in which the expression of MHCII and
CD86 and the production of proinflammatory media
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[112]

tors such as NO, TNF and IL12 substantially

increased [73]. EMN from Salmonella recognized
S. typhimuriumspecific B cells and CD4+ T cells [73].
EMN from P. aeruginosa and Helicobacter pylori caused
the production of interleukins IL8 and IL10 by epi
thelial cells [11, 74, 75]. EMN produced by Porphy
romonas gingivalis caused the disappearance of LPS
receptors CD14 from macrophage surface [76], while
EMN produced by Legionella pneumophila inhibited
the phagosomelysosome fusion [77]. EMN from
group B meningococci stimulated the gene expression
and production of a number of cytokins and inflamma

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tory mediators such as TNF, IL1, MIP1,

interferon in neutrophils [78], while EMN from Neis
seria meningitidis stimulated the proinflammatory
activity of circulating monocytes [79]. EMN from Bor
relia burgdorferi influenced Bcell activity [80], and
EMN from Brucella melitensis stimulated the immuno
protective properties of an organism [81].
Thus, the data considered above show that bacterial
EMN possess the proinflamatory and immunogenic
characteristics of the parent bacteria and can stimulate
the development of innate and adaptive immunity of a
host organism. They contribute to the maturation of
antigenpresenting cells and stimulate the expression
of proinflammatory cytokins, interleukins, and other
mediators. The presence of the above properties sug
gests that EMN can be used as an additional tool to
stimulate specific immunity against pathogens, in par
ticular, when creating vaccines against infectious dis
eases.
EMN applications in biotechnology and medicine.
The possibility of using EMN isolated from different
bacteria as cellfree vaccines has been extensively inves
tigated in recent years [10]. EMN constitutively
secreted by bacteria into the medium contain many
bacterial antigens. EMN have immunogenic properties
and, in the presence of natural adjuvants and the ability
to easily penetrate mammalian cells, which can be
intensified by the methods of genetic engineering, are
promising for vaccine development. The immunization
of mice with EMN isolated from a culture of Salmonella
typhimurium was accompanied by the generation of Sal
monellaspecific antibodies. The immunized mice were
not infected with living Salmonella [73].
Preparations of EMN are used for vaccination
against meningitis [116, 117]. Thus, EMN vaccines
obtained from the wildtype culture of Neisseria men
ingitidis are the only effective preparations against
serogroup B meningococcal infection [118]. The first
EMNbased vaccines were used during outbreaks of
meningitis (serogroup B meningococci) in Cuba,
Norway, Brazil, and New Zealand; however, up to
now, there has been no general vaccine against the
meningococci of this serogroup [119].
Proteomic analysis makes it possible to assess the
composition and content of complex EMN vaccines.
The identification of their proteins shows that, in con
trast to ordinary vaccines, EMN vaccines contain peri
plasmic, membraneassociated, and cytoplasmic pro
teins [103]. They were shown to contain 74 antigens,
including outer membrane proteins: PorA, PorB,
RmpM, and OpcA. The peculiar features of EMN vac
cines are the natural orientation of superficial mem
brane antigens and stability [118]. An experimental
vaccine on the basis of EMN obtained from three sero
group B N. meningitidis strains was used to immunize
rabbits, and its efficiency was assessed by the induction
of antibodies in blood serum and the bactericidal activ
ity against other meningococcal strains. The results
showed that the vaccine could induce an effective

response of rabbit bactericidal antibodies against

N. meningitidis strains of serogroups A, C, E, W135,
and X. Analysis of their specificity showed that the
antiLPS antibodies PorA and NadA induced by the
EMN vaccine were responsible for the majority of bac
tericidal activity against N. meningitides strains from
other serogroups [120].
The EMN from the cholera vibrio and Bordetella
pertusis were successfully used as vaccines against
cholera [121124]. EMNbased vaccines against
whooping cough [121] and pneumonia [125] have
been developed and applied.
The efficiency, acceptability, and safety of EMN
vaccines used in practical medicine have been proved.
The currently developed EMN vaccines against vari
ous infections with additional crossprotective poten
tial will include a number of crossprotective univer
sal antigens. On this basis, it is possible to construct a
vaccine that will defend against all respective bacterial
infections [118].
***
The data presented in this review show that EMN
secretion is an evolutionarily fixed process inherent in
all prokaryotic organisms. EMN secretion allows the
bacteria to secrete a complex of soluble and insoluble
compounds. EMN provide the effects of bacterial
enzymes outside the cell and contribute to bacterial
survival, performing protective, offensive and adaptive
functions. Their production is a bacterial stress
response, which includes protein secretion, adhesion
and internalization into other cells, the activation and
suppression of host immunity. EMN are necessary for
bacterial nutrition, biofilm formation, virulence, and
pathogenesis. The major role of EMN secreted by
Gramnegative and Grampositive bacteria and
archaea consists in providing their communication
with other prokaryotic and eukaryotic cells. In recent
years, EMN have been actively used in medical prac
tice as vaccine carriers. The interest in EMN as vac
cines increases as more and more molecular features
of vesiculation in bacteria and possibilities of using
EMN to control infectious diseases are revealed [126].
It has been shown that genetically modified EMN can
perform the overexpression of antigens. The works on
bioengineering make it possible to create EMN with
modified properties [127132].
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