The first report of bacterial membrane vesicles appeared in the mid-twentieth century
[1]. In this study, the protein exotoxin secreted by
Vibrio cholerae was shown to be resistant to proteases. Transmission electron microscope (TEM) analysis suggested that this exotoxin is located within spherical structures containing components of the bacterial cell envelope. These structures, detected in cell-free supernatants obtained from liquid bacterial cultures in the exponential growth phase
[2], were named membrane vesicles (MVs).
As enveloped structures, MVs have the characteristics of vectors that enable the transport of substances highly sensitive to environmental conditions. They protect proteins enclosed in their lumen against enzymatic decomposition, degradation related to low or high pH and oxidative stress conditions. Therefore, it is not surprising that, in addition to proteins acquiring of nutrients from the environment, pathogenic bacteria also use MVs to transport toxins that directly affect host cells and enzymes promoting bacterial colonization, facilitating the disruption of infected tissues and spreading of infection in the host. We provide examples of the best characterized bacterial virulence factors associated with MVs in Table 1. The enrichment of certain proteins in MVs, at a higher concentration than found in bacteria, suggests a degree of specification for MVs in toxic activity, polymer decomposition, antibiotic inactivation or metal ion sequestration. The small size of MVs (ranging from 20–250 nm in diameter)
[3] permits them to overcome epithelial barriers, such as the gut–blood barrier (GBB), and enter tissues that are not colonized by the bacteria producing them. The presence of surface antigens allows MVs to interact with cells of the host immune system, so that virulence factors they transport can modulate (induce or inhibit) the immune response. MVs can also act as “traps” for antibodies circulating in the inhabited tissue, or for bacteriophages in the natural environment. The great versatility of vesicles is the result of variation in their structure and composition. The secretion of active factors in this form is one of the most complex and diverse mechanisms of bacterial interaction with the environment and other cells
[4].
2. Structure of Membrane Vesicles (MVs) and Mechanisms of Secretion
The production of MVs (both extracellular and intracellular) has been observed in organisms from all three domains of life
[5]. Research on bacterial vesicles has been ongoing for over 60 years, but the mechanisms of their biogenesis are still not fully understood. Several vesicle types have been described in Gram-negative and Gram-positive bacteria. The MVs exhibit the membrane features of the originating bacteria and thus could indicate the nature of their cargos, such as proteins and nucleic acids (
Figure 1).
Figure 1. Mechanisms of bacterial membrane vesicle formation. In gram-negative bacteria, membrane vesicles are produced through membrane blebbing or explosive cell lysis triggered by phage-derived endolysins. Endolysins participate in the formation of cytoplasmic membrane vesicles (CMVs) in Gram-positive bacteria. The cytoplasmic membrane protrudes through holes in the peptidoglycan degraded by phage-derived endolysins. The contents of the membrane vesicles depends on the route of their formation. EMV—explosive membrane vesicle; OIMV—outer-inner membrane vesicle; OMV—outer membrane vesicle; CMV—cytoplasmic membrane vesicle.
OMVs (outer-membrane vesicles) produced by Gram-negative bacteria consist of blebs of bacterial outer membrane containing transmembrane proteins and LPS, with extracellular DNA (eDNA) exposed on the surface of OMVs, with periplasmic content packaged in the lumen of the vesicle. OMVs are produced by many species of pathogenic bacteria, including
Neisseria meningitidis,
Helicobacter pylori,
Escherichia coli (EHEC) and
Salmonella spp.
[6]. Increased secretion of OMVs usually occurs under stressful conditions, and is accompanied with the accumulation of misfolded proteins in the periplasm. According to one MV biogenesis model, the pressure of these defective proteins on the inner surface of the OM is responsible for bulging of the membrane and its detachment from the cell in the form of vesicles
[7].
Outer-inner membrane vesicles (OIMVs) are double-membrane structures and were first observed in cultures of
Pseudomonas aeruginosa [8]. The outer membrane and inner membrane are separated with a thin layer of periplasm with degraded fragments of peptidoglycan. The production of OIMVs are induced in stressful or adverse situations. Cytoplasm present in the lumen of these vesicles contains proteins and also fragments of DNA derived from the chromosome or plasmids and ATP
[8].
Vesicles containing cytoplasm are also produced by Gram-positive bacteria. The release of CMVs (cytoplasmic membrane vesicles) requires local peptidoglycan degradation by internal or external lytic enzymes (digesting both the glycan backbone and peptide bonds in the amino acid chains)
[9]. CMV production has been observed in several model Gram-positive bacteria, including
Bacillus subtilis [10],
Bacillus anthracis and
Staphylococcus aureus [11][12].
The last membrane vesicle type is EMVs (explosive membrane vesicles), which are the most diverse in terms of structure. They arise spontaneously during bacterial cell lysis. Fragments of membrane, together with the outflow of cytoplasm (also periplasm in the case of Gram-negative bacteria), create spherical membrane structures in the environment. Thus, the process of EMV “assembly” is cell-independent and spontaneous, so bacteria are unable to control the content of these vesicles. As a result, each lysing cell produces MVs that differ in size, composition and function. This process has been described in
P. aeruginosa biofilms, where deeply located cells subjected to hypoxia, nutrient deficiency and activation of the SOS system are autolysed through the activity of endolysins, and type R and F pyocins
[13]. EMVs released in this way are an important factor in the virulence of pathogenic
P. aeruginosa strains. As a component of the biofilm matrix, they bind eDNA and polysaccharides, and also bacteria via surface adhesins, which stiffens this structure
[14].
Several recent reviews describe the composition and biogenesis of bacterial membrane vesicles
[6][7][15][16][17]. In this article we present the latest data concerning interactions between MVs and selected human cell types.
3. Conclusions
The molecular understanding of bacterial virulence factors is an important challenge for microbiologists. Modern techniques have enabled the discovery of novel mechanisms that sometimes surprise researchers with their universality. This is the case for membrane vesicles, which play important roles in the interactions of bacteria with cells of other organisms. MVs are not only a new type of secretion system, their great variety of structure and function, action at a distance, and stability in the host system make them an important weapon in the bacterial arsenal. Examples of the best characterized bacterial virulence factors associated with MVs are presented below in
Table 1.
Table 1. Examples of bacteria producing membrane vesicles and active factors discovered inside/outside MVs.
Bacterial Species (Gram-Negative) |
Active Factors |
Reference |
Acholeplasma laidlawii | PG8 |
|
-
adhesins/invasins—enable tight physical contact between bacterium and host cell
-
ABC transporting complexes
-
hydrolases: proteases, nucleases, and glycosylases
-
metallo-β-lactamase
|
|
[18] |
Acinetobacter baumannii |
| |
|
[19] |
Actinobacillus pleuropneumoniae |
| |
|
[20] |
Aggregatibacter actinomycetemcomitans |
| |
|
[21] |
Bartonella henselae |
| |
|
[22] |
Borrelia burgdorferi |
| |
|
[23][24] |
Burkholderia cepacia |
| |
|
[25] |
Campylobacter jejuni |
| |
|
[26] |
Coxiella burnetti |
| |
|
[27] |
Escherichia coli | K1 |
|
-
OmpA—interaction with Ecgp receptor on surface of brain microvascular endothelium leads to cell invasion; may also act in trans to promote cell invasion by other bacterial species
-
K1 antigen—polysaccharide antigen from cell envelope, linear polymer of NeuNac
-
TLR ligands—flagellin, lipoproteins, poly-CpG DNA strands
|
|
[28] |
Escherichia coli | O157: H7
| Shigella dysenteriae |
| |
|
[29] |
enterotoxic | E. coli | (ETEC) |
| |
|
[30] |
enterohemorrhagic | E. coli | (EHEC) |
| |
|
[31] |
extraintestinal pathogenic | E. coli | (ExPEC) |
| |
|
[32] |
Haemophilus influenzae | type B (Hib) |
| |
|
[33] |
Legionella pneumophila |
| |
|
[34] |
Moraxella catarrhalis |
| |
|
[35] |
Neisseria meningitidis | serogroup B |
| |
|
[36] |
Porphyromonas gingivalis |
|
-
gingipains—non-specific proteases degrading elements of host’s tissue and cytokines
-
HmuY—lipoprotein accumulating heme; assists biofilm formation process
-
factors assisting in co-localization with Treponema denticola
|
|
[37] |
Salmonella enterica |
|
-
SopB—protects SCV (Salmonella-containing vacuoles) from degradation by reorganization of actin cytoskeleton
-
SipC—protein assisting in cell invasion process
-
SopA—ubiquitin ligase (E3) disturbing ubiquitin pathway of host cell
-
FljB—flagellin, strong antigen
-
SopE2—guanine nucleotide exchange factor (GEF); by catalysing exchange GDP → GTP disturbs function of Rho-protein family GTPases controlling dynamics of host cell cytoskeleton, which leads to membrane surface deformation and assists invasion process
-
PagK1/2—exact function still unknown; probably assists bacterial proliferation inside SCV
-
SrfN—promotes bacterial survival inside macrophages
|
|
[38] |
Shigella flexneri |
| |
|
[39] |
Treponema denticola |
| |
|
[40] |
Vibrio cholerae |
| |
|
[41] |
Yersinia pestis |
|
-
Ail—surface adhesin; promotes contact with host cells
-
Pla—extracellular protease; activator of plasminogen
-
Caf1—fimbrial antigen F1; main component of OMVs
|
|
[42] |
Bacterial Species (Gram-Positive) |
Active Factors |
Citations |
Bacillus anthracis |
|
-
anthrolysin (ALO)—cholesterol-dependent cytolysin
-
lethal factor (LF)—zinc-protease; hydrolyses several MAPK-kinases (MAPKK), causes disturbance of signalling pathways and cell death
-
edema factor (ED)—calmodulin- and Ca2+-dependent adenylate cyclase; induces uncontrolled increase in cAMP concentration in phagocytic cells thus depleting ATP reserves
|
|
[12][ 12 ] |
Clostridium perfringens |
| |
|
[43][ 43 ] |
Enteroccoccus faeciumEnterokok faecium |
|
-
phospholipids; reduce antibacterial activity of the antibiotic daptomycinfosfolipidy; zmniejszyć działanie przeciwbakteryjne antybiotyku daptomycyny
-
SdrD—collagen-binding proteinSdrD — białko wiążące kolagen
-
PavA—fibronectin-binding proteinPavA — białko wiążące fibronektynę
-
AtlA—autolysin; assists in biofilm formation processAtlA – autolizyna; wspomaga proces powstawania biofilmu
-
Acm—MSCRAMM (microbial surface components recognizing adhesive matrix molecules) group adhesin; binds collagenAcm — MSCRAMM (składniki powierzchni drobnoustrojów rozpoznające cząsteczki matrycy adhezyjnej) z grupy adhezyny; wiąże kolagen
-
Fnm—fibronectin-binding adhesinFnm — adhezyna wiążąca fibronektynę
-
PsaA—lipoprotein; potential component of future vaccinePsaA – lipoproteina; potencjalny składnik przyszłej szczepionki
|
|
[44][ 44 ] |
Mycobacterium tuberculosisPrątek gruźlicy |
|
-
LpqH—lipoprotein; assists in transport processesLpqH — lipoproteina; asystuje w procesach transportowych
-
MPB83—highly immunogenic glycoproteinMPB83 — wysoce immunogenna glikoproteina
-
LprA—lipoprotein; strong TLR2 agonistLprA — lipoproteina; silny agonista TLR2
-
PSTS3—component of ABC transport system connected with phosphorus ion importPSTS3 — element systemu transportowego ABC związany z importem jonów fosforu
-
lipoarabinomannan (LAM)—surface glycolipid with anti-ROS featureslipoarabinomannan (LAM) — glikolipid powierzchniowy o właściwościach anty-ROS
-
mycobactin—surface Fe3+-siderophoremykobaktyna – powierzchnia Fe 3+ -syderofor
|
|
[45][ 45 ] |
Propionibacterium acnes |
| |
|
[46][ 46 ] |
Streptococcus mutans |
|
-
eDNA—important biofilm componenteDNA — ważny składnik biofilmu
-
glucosyltransferases (GtfB/C/D)—produce adhesive extracellular polysaccharides from sucrose substrateglukozylotransferazy (GtfB/C/D) — wytwarzają adhezyjne zewnątrzkomórkowe polisacharydy z substratu sacharozy
-
lipoteichoic acid (LTA)—surface antigen; important in adsorption process in biofilm formationkwas lipotejchojowy (LTA) – antygen powierzchniowy; ważny w procesie adsorpcji w tworzeniu biofilmu
|
|
[47][ 47 ] |
Streptococcus pneumoniae |
|
-
TatD—non-specific DNase enabling degradation of NETs (DNA nets associating with proteins with antimicrobial activities: LL37, myeloperoxidase, neutrophil elastase)TatD — nieswoista DNaza umożliwiająca degradację NET (sieci DNA związane z białkami o działaniu przeciwdrobnoustrojowym: LL37, mieloperoksydaza, elastaza neutrofili)
-
EndA—non-specific DNase located on surface of MVsEndA — nieswoista DNaza zlokalizowana na powierzchni MV
-
PspC—H factor-binding protein; blocks alternative complement pathwayPspC-białko wiążące czynnik H; blokuje alternatywny szlak dopełniacza
-
pneumolysin (Ply)—exotoxin with cytolytic featurespneumolizyna (Ply) – egzotoksyna o właściwościach cytolitycznych
-
PsaA—adhesin; strong surface antigenPsaA – adhezyna; silny antygen powierzchniowy
-
SatA—ABC-type transporter; surface antigenSatA — transporter typu ABC; antygen powierzchniowy
-
AmiA—peptide-binding protein; assists in active transportAmiA — białko wiążące peptydy; asystuje w aktywnym transporcie
-
MalX—maltose and maltodextrin-binding proteinMalX — białko wiążące maltozę i maltodekstrynę
-
PnrA—ABC-type nucleoside transporterPnrA — transporter nukleozydów typu ABC
-
spr1909—penicillin-binding proteinspr1909 — białko wiążące penicylinę
|
|
[48][ 48 ] |