Outer Membrane Vesicles Secretion Research Articles

Stress response of Salmonella Newport with various sequence types toward plasma-activated water: Viable but nonculturable state formation and outer membrane vesicle production

This study aims to investigate the response of Salmonella Newport to plasma-activated water (PAW), a novel disinfectant that attracts attention due to its broad-spectrum antimicrobial efficacy and eco-friendliness. In this work, we demonstrated that S. Newport of different sequence types (STs) could be induced into the viable but nonculturable (VBNC) state by PAW treatment. Notably, a remarkable 99.96% of S. Newport ST45 strain entered the VBNC state after a 12-min PAW treatment, which was the fastest observed among the five S. Newport STs (ST31, ST45, ST46, ST166, ST2364). Secretion of outer membrane vesicles was observed in ST45, suggesting a potential strategy against PAW treatment. Genes related to oxidative stress (sodA, katE, trxA), outer membrane proteins (ompA, ompC, ompD, ompF) and virulence (pagC, sipC, sopE2) were upregulated in the PAW-treated S. Newport, especially in ST45. A reduction of 38–65% in intracellular ATP level after PAW treatment was observed, indicating a contributor to the formation of the VBNC state. In addition, a rapid method for detecting the proportion of VBNC cells in food products based on pagC was established. This study contributes to understanding the formation mechanism of the VBNC state in S. Newport under PAW stress and offers insights for controlling microbial risks in the food industry.

Availability of iron ions impacts physicochemical properties and proteome of outer membrane vesicles released by Neisseria gonorrhoeae

Outer membrane vesicles (OMVs) are bilayer structures released by bacteria for various purposes, e.g., response to environmental factors, bacterial communication, and interactions with host cells. One of the environmental variables bacteria need to react is the amount and availability of iron, a crucial element for bacteria biology. We have investigated the impact of the iron amount and availability on OMV secretion by pathogenic Neisseria gonorrhoeae, which, depending on the infection site, challenges different iron availability. N. gonorrhoeae releases OMVs in iron starvation and repletion growth environments. However, OMVs differed in physicochemical features and proteome according to iron amount and availability during the bacteria growth, as was analyzed by Liquid Chromatography-Tandem Mass Spectrometry, Infrared spectroscopy with a Fourier transform infrared spectrometer, and Atomic Force Microscopy. OMVs from iron starvation and repletion conditions had a higher variation in size, different flexibility, and different membrane protein and lipid components than OMVs isolated from control growth conditions. These OMVs also varied qualitatively and quantitatively in their total proteome composition and contained proteins unique for iron starvation and repletion conditions. Thus, the modulation of OMVs' properties seems to be a part of N. gonorrhoeae adaptation to surroundings and indicates a new direction of antigonococcal proceeding.

Sub-MIC antibiotics affect microbial ferrihydrite reduction by extracellular membrane vesicles

Environmental concentrations of antibiotics, usually below MIC, have significant biological effects on bacterial cells. Sub-MIC antibiotics exposure induces bacteria to produce outer membrane vesicles (OMVs). Recently, OMVs is discovered as a novel pathway for dissimilatory iron reducing bacteria (DIRB) to mediate extracellular electron transfer (EET). Whether and how the antibiotic-induced OMVs modulate iron oxides reduction by DIRB have not been studied. This study showed the sub-MIC antibiotics (ampicillin or ciprofloxacin) increased OMVs secretion in Geobacter sulfurreducens, and the antibiotic-induced OMVs contained more redox active cytochromes facilitating iron oxides reduction, especially for the ciprofloxacin-induced OMVs. Deduced from a combination of electron microscopy and proteomic analysis, the influence of ciprofloxacin on SOS response triggered prophage induction and led to the formation of outer-inner membrane vesicles (OIMVs) in, which was a first report in Geobacter species. While ampicillin disrupting cell membrane integrity resulted in more formation of classic OMVs from outer membrane blebbing. The results indicated that the different structure and composition of vesicles were responsible for the antibiotic-dependent regulation on iron oxides reduction. This newly identified regulation on EET-mediated redox reactions by sub-MIC antibiotics expands our knowledge about the impact of antibiotics on microbial processes or “non-target” organisms.

Characterization and Vaccine Potential of Outer Membrane Vesicles from Photobacterium damselae subsp. piscicida

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative fish pathogen with worldwide distribution and broad host specificity that causes heavy economic losses in aquaculture. Although Phdp was first identified more than 50 years ago, its pathogenicity mechanisms are not completely understood. In this work, we report that Phdp secretes large amounts of outer membrane vesicles (OMVs) when cultured in vitro and during in vivo infection. These OMVs were morphologically characterized and the most abundant vesicle-associated proteins were identified. We also demonstrate that Phdp OMVs protect Phdp cells from the bactericidal activity of fish antimicrobial peptides, suggesting that secretion of OMVs is part of the strategy used by Phdp to evade host defense mechanisms. Importantly, the vaccination of sea bass (Dicentrarchus labrax) with adjuvant-free crude OMVs induced the production of anti-Phdp antibodies and resulted in partial protection against Phdp infection. These findings reveal new aspects of Phdp biology and may provide a basis for developing new vaccines against this pathogen.

Intact O-antigen is critical structure for the exceptional tubular shape of outer membrane vesicles in Francisella tularensis

Francisella tularensis is a highly infectious Gram-negative coccobacillus which causes the disease tularemia. The potential for its misuse as a biological weapon has led disease control and prevention centers to classify this bacterium as a category A agent. Bacterial outer membrane vesicles (OMVs) are spherical particles 20–250 nm in size produced by all Gram-negative bacteria and constitute one of the major secretory pathways. Bacteria use them in interacting with both other bacterial cells and eukaryotic (host) cells. OMVs of Francisella contain number of its so far described virulence factors and immunomodulatory proteins. Their role in host–pathogen interactions can therefore be presumed, and the possibility exists also for their potential use in a subunit vaccine. Moreover, Francisella microbes produce both usual spherical and unusual tubular OMVs. Because OMVs emerge from the outermost surface of the bacterial cell, we focused on the secretion of OMVs in several mutant Francisella strains with disrupted surface structures (namely the O-antigen). O-antigen in Francisella is not only the structural component of LPS but also forms another important virulence factor: the O-antigen polysaccharide capsule. Mutant strain phenotypes were evaluated by growth curves, vesiculation rates, their sensitivity to the complement contained in serum, and proliferation inside murine bone marrow macrophages. Morphologies of both OMVs and the bacteria were visualized by electron microscopy. The O-antigen mutant strains were considerably attenuated in serum resistance and intracellular proliferation. All the strains showed lower ability to form the tubular OMVs. Some strains formed tubular protrusions from their outer membrane but their stability was weak. Some hypervesiculating strains were revealed that will serve as source of OMVs for further studies of their protective potential. Our results suggest the presence of LPS and the O-antigen capsule on the surface of Francisella to be critical not only for its virulence but also for the exceptional tubular shape of its OMVs.

High-Yield, Magnetic Harvesting of Extracellular Outer-Membrane Vesicles from Escherichia coli.

Extracellular outer-membrane vesicles (OMVs) are attractive for use as drug nanocarriers, because of their high biocompatibility and ability to enter cells. However, widespread use is hampered by low yields. Here, a high-yield method for magnetic harvesting of OMVs from Escherichia coli is described. To this end, E. coli are grown in the presence of magnetic iron-oxide nanoparticles (MNPs). Uptake of MNPs by E. coli is low and does not increase secretion of OMVs. Uptake of MNPs can be enhanced through PEGylation of MNPs. E. coli growth in the presence of PEGylated MNPs increases bacterial MNP-uptake and OMV-secretion, accompanied by upregulation of genes involved in OMV-secretion. OMVs containing MNPs can be magnetically harvested at 60-fold higher yields than achieved by ultracentrifugation. Functionally, magnetically-harvested OMVs and OMVs harvested by ultracentrifugation are both taken-up in similar numbers by bacteria. Uniquely, in an applied magnetic field, magnetically-harvested OMVs with MNPs accumulate over the entire depth of an infectious biofilm. OMVs harvested by ultracentrifugation without MNPs only accumulate near the biofilm surface. In conclusion, PEGylation of MNPs is essential for their uptake in E. coli and yields magnetic OMVs allowing high-yield magnetic-harvesting. Moreover, magnetic OMVs can be magnetically targeted to a cargo delivery site in the human body.

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria.

The release of bacterial membrane vesicles (BMVs) has become recognized as a key mechanism used by both pathogenic and commensal bacteria to activate innate immune responses in the host and mediate immunity. Outer membrane vesicles (OMVs) produced by Gram-negative bacteria can harbor various immunogenic cargo that includes proteins, nucleic acids and peptidoglycan, and the composition of OMVs strongly influences their ability to activate host innate immune receptors. Although various Gram-negative pathogens can produce OMVs that are enriched in immunogenic cargo compared to their parent bacteria, the ability of OMVs produced by commensal organisms to be enriched with immunostimulatory contents is only recently becoming known. In this study, we investigated the cargo associated with OMVs produced by the intestinal commensal Bacteroides fragilis and determined their ability to activate host innate immune receptors. Analysis of B. fragilis OMVs revealed that they packaged various biological cargo including proteins, DNA, RNA, lipopolysaccharides (LPS) and peptidoglycan, and that this cargo could be enriched in OMVs compared to their parent bacteria. We visualized the entry of B. fragilis OMVs into intestinal epithelial cells, in addition to the ability of B. fragilis OMVs to transport bacterial RNA and peptidoglycan cargo into Caco-2 epithelial cells. Using HEK-Blue reporter cell lines, we identified that B. fragilis OMVs could activate host Toll-like receptors (TLR)-2, TLR4, TLR7 and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), whereas B. fragilis bacteria could only induce the activation of TLR2. Overall, our data demonstrates that B. fragilis OMVs activate a broader range of host innate immune receptors compared to their parent bacteria due to their enrichment of biological cargo and their ability to transport this cargo directly into host epithelial cells. These findings indicate that the secretion of OMVs by B. fragilis may facilitate immune crosstalk with host epithelial cells at the gastrointestinal surface and suggests that OMVs produced by commensal bacteria may preferentially activate host innate immune receptors at the mucosal gastrointestinal tract.

Proteome Analysis of Outer Membrane Vesicles From a Highly Virulent Strain of Haemophilus parasuis.

Haemophilus parasuis has emerged as an important bacterial pathogen in pig husbandry, as H. parasuis can coinfect pigs with a variety of pathogenic microorganisms and further cause an aggravation of the disease. It is crucial to investigate its pathogenetic mechanism. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs), and their potent virulence factors play prominent roles that affect the interaction between bacteria and host. Still, the pathogenesis that is associated with the bacterial OMVs has not been well-elucidated. In this study, we investigated the secretion of OMVs from a clinical H. parasuis isolate strain (H45). In addition, we further analyzed the characterization, the comprehensive proteome, and the virulence potential of OMVs. Our data demonstrated that H. parasuis could secrete OMVs into the extracellular milieu during infection. Using liquid chromatography with tandem mass spectrometry (MS/MS) identification and bio-information analysis, we identified 588 different proteins associated with OMVs. Also, we also analyzed the subcellular location and biological function of those proteins. These proteins are mainly involved in immune and iron metabolism. Moreover, we confirmed the pathogenicity of H. parasuis OMVs by observing a strong inflammatory response in J774A.1 and porcine alveolar macrophages. Taken together, our findings suggested that OMVs from H. parasuis were involved in the pathogenesis of this bacterium during infection.

Inappropriate use of antibiotics exacerbates inflammation through OMV-induced pyroptosis in MDR Klebsiella pneumoniae infection.

The inappropriate use of antibiotics is a severe public health problem worldwide, contributing to the emergence of multidrug-resistant (MDR) bacteria. To explore the possible impacts of the inappropriate use of antibiotics on the immune system, we use Klebsiella pneumoniae (K.pneumoniae) infection as an example and show that imipenem increases the mortality of mice infected by MDR K.pneumoniae. Further studies demonstrate that imipenem enhances the secretion of outer membrane vesicles (OMVs) with significantly elevated presentation of GroEL, which promotes the phagocytosis of OMVs by macrophages that depends on the interaction between GroEL and its receptor, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). OMVs cause the pyroptosis of macrophages and the release of proinflammatory cytokines, which contribute to exacerbated inflammatory responses. We propose that the inappropriate use of antibiotics in the cases of infection by MDR bacteria such as K.pneumoniae might cause damaging inflammatory responses, which underlines the pernicious effects of inappropriate use of antibiotics.

Campylobacter fetus releases S-layered and immunoreactive outer membrane vesicles

The study of outer membrane vesicles (OMVs) became relevant because of their probable important role in the transfer of virulence factors to host cells. Campylobacter fetus is mainly a mammal pathogen whose virulence characterization is still limited. The aim of this study was to evaluate and to characterize the secretion of OMVs in this bacterium. By transmission electron microscopy, we confirmed the production of OMVs in all the strains assayed. Purified OMVs showed a spherical shape and variable size, although comparable to those of other gram-negative bacteria. We also confirmed the presence of the S-layer on the surface of the OMVs of all the strains assayed with the exception of those derived from the NTCC reference strain. In addition, we demonstrated their immunoreactivity by the dot-blot assay. Hence, C. fetus OMVs could contribute to the modulation of the host response and constitute a candidate to be evaluated as an adjuvant of current vaccines used in the veterinary field. This work represents a platform to drive future studies towards the role of these subcellular structures in C. fetus-host interaction.

Outer membrane vesicles as versatile tools for therapeutic approaches.

Budding of the bacterial surface results in the formation and secretion of outer membrane vesicles, which is a conserved phenomenon observed in Gram-negative bacteria. Recent studies highlight that these sphere-shaped facsimiles of the donor bacterium's surface with enclosed periplasmic content may serve multiple purposes for their host bacterium. These include inter- and intraspecies cell-cell communication, effector delivery to target cells and bacterial adaptation strategies. This review provides a concise overview of potential medical applications to exploit outer membrane vesicles for therapeutic approaches. Due to the fact that outer membrane vesicles resemble the surface of their donor cells, they represent interesting nonliving candidates for vaccine development. Furthermore, bacterial donor species can be genetically engineered to display various proteins and glycans of interest on the outer membrane vesicle surface or in their lumen. Outer membrane vesicles also possess valuable bioreactor features as they have the natural capacity to protect, stabilize and enhance the activity of luminal enzymes. Along these features, outer membrane vesicles not only might be suitable for biotechnological applications but may also enable cell-specific delivery of designed therapeutics as they are efficiently internalized by nonprofessional phagocytes. Finally, outer membrane vesicles are potent modulators of our immune system with pro- and anti-inflammatory properties. A deeper understanding of immunoregulatory effects provoked by different outer membrane vesicles is the basis for their possible future applications ranging from inflammation and immune response modulation to anticancer therapy.

Antibiotics Abuse Exacerbates Inflammation Through Outer Membrane Vesicles-Induced Pyroptosis in Multidrug-Resistant Klebsiella Pneumoniae Infection

The abuse of antibiotics is a severe public health problem worldwide, contributing to the emergence of multidrug-resistant (MDR) bacteria. To explore the possible impacts of antibiotic abuse on the immune system, here, using Klebsiella pneumoniae (K. pneumoniae) infection as an example, we showed that imipenem, a carbapenem antibiotic, increased the mortality of mice infected by MDR K. pneumoniae. Further studies demonstrated that imipenem enhanced the secretion of outer membrane vesicles (OMVs) with significantly elevated presentation of GroEL, which promoted the phagocytosis of OMVs by macrophages depending on the interaction between GroEL and its receptor LOX-1. OMVs cause the pyroptosis of macrophages and release of proinflammatory cytokines, which contribute to exacerbated inflammatory responses. We proposed that antibiotic abuse in cases of infection by MDR bacteria might cause severe inflammatory damage, which underlines the perniciousness of antibiotic abuse on the immune system.

Dinoroseobacter shibae Outer Membrane Vesicles Are Enriched for the Chromosome Dimer Resolution Site dif.

Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.

Delivery of Periodontopathogenic Extracellular Vesicles to Brain Monocytes and Microglial IL-6 Promotion by RNA Cargo

Gram-negative bacterial extracellular vesicles (EVs), also known as outer membrane vesicles (OMVs), are secreted from bacterial cells and have attracted research attention due to their role in cell-to-cell communication. During OMV secretion, a variety of cargo such as extracellular RNA (exRNA) is loaded into the OMV. The involvement of exRNAs from a range of bacteria has been identified in several diseases, however, their mechanism of action has not been elucidated. We have recently demonstrated that OMVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans can cross the blood–brain barrier (BBB) and that its exRNA cargo could promote the secretion of proinflammatory cytokines in the brain. However, it was unclear whether the brain immune cells could actually take up bacterial OMVs, which originate outside of the brain, in an appropriate immune response. In the present study, using monocyte-specific live CX3CR1-GFP mice, we visualized OMV-colocalized meningeal macrophages and microglial cells into which bacterial OMVs had been loaded and intravenously injected through tail veins. Our results suggested that meningeal macrophages uptake BBB-crossed OMVs earlier than do cortex microglia. BV2 cells (a murine microglia cell line) and exRNAs were also visualized after OMV treatment and their proinflammatory cytokine levels were observed. Interleukin (IL)-6 and NF-κB of BV2 cells were activated by A. actinomycetemcomitans exRNAs but not by OMV DNA cargo. Altogether, these findings indicate that OMVs can successfully deliver exRNAs into brain monocyte/microglial cells and cause neuroinflammation, implicating a novel pathogenic mechanism in neuroinflammatory diseases.

Non-pathogenic Escherichia coli acquires virulence by mutating a growth-essential LPS transporter.

The molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment.

Francisella tularensis: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation

ABSTRACT Francisella tularensis is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using F. tularensis live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain F. tularensis subsp. tularensis SCHU S4 deleted for the homologous FupA protein exhibited even higher FQ resistance. In addition to an increased FQ minimal inhibitory concentration, LVSΔfupA/B displayed tolerance toward bactericidal compounds including ciprofloxacin and gentamicin. Interestingly, the FupA/B deletion was found to promote increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of vesicles from LVS and LVS∆fupA/B identified 801 proteins, including a subset of 23 proteins exhibiting differential abundance between both strains which may therefore contribute to the reduced antibiotic susceptibility of the FupA/B-deleted strain. We also demonstrated that OMVs are key structural elements of LVSΔfupA/B biofilms providing protection against FQ. These results provide a new basis for understanding and tackling antibiotic resistance and/or persistence of Francisella and other pathogenic members of the Thiotrichales class.

Characterization of outer membrane vesicles of Acetobacter pasteurianus NBRC3283

Acetobacter pasteurianus is characterized as a fermenting bacterium of kurozu, which is a common traditional Japanese black vinegar. Recently, we separated acid-resistant and low Toll-like receptor 4 (TLR4)-stimulatory lipopolysaccharides (LPS) from A.pasteurianus. We also showed that their lipid A parts possessed a novel sugar backbone that is responsible for the low TLR4-stimulatory and acid-resistant properties of the LPS. Outer membrane vesicles (OMVs) are nano-sized spherical structures secreted from many gram-negative bacteria. OMVs contain LPS and act as immunomodulants such as vaccines. In this study, we investigated OMVs secreted from A.pasteurianus. OMV secretion from A.pasteurianus NBRC 3283cells was observed after 2 days in culture by transmission electron microscopy imaging. Thus OMVs were separated from the culture supernatants by ultracentrifugation and then purified by OptiPrep density gradient centrifugation. The OMVs contained several proteins including outer membrane proteins, and several sugars as components of LPS. The OMVs weakly stimulated TLR4 in accordance with the activity of A.pasteurianus LPS. Additionally, the TLR2-stimulating activity of the OMVs was significantly potent, indicating the existence of lipoproteins. Furthermore OMV-like spherical particles were observed in kurozu. Some of these particles are probably derived from A.pasteurianus. These data suggest that A.pasteurianus produce OMVs that contain LPS and probably lipoproteins, and can modulate the innate immune system.

Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport.

Secretion of outer membrane vesicles as a mechanism promoting H. pylori infections

Abstract Helicobacter pylori commonly colonizes the human gastric mucosa. Infections with this microorganism can contribute to serious health consequences, such as peptic ulceration, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma. Chronic persistence of this bacteria in the host organism is probably strongly dependent on the secretion of outer membrane vesicles (OMV). These organelles are small, electron-dense, extracellular structures which are secreted in large amounts during stressful conditions, among others. H. pylori OMV mediate transfer of virulence factors such as toxins and immunomodulatory compounds. They contribute to avoiding a response from the host immune system and inducing chronic gastritis. OMV secretion also affects the formation of cell aggregates, microcolonies and biofilm matrix. Enhanced OMV production is connected to maintenance of direct contact through cell-cell and cell-surface interactions. A key component of OMV, which determines their structural function, is extracellular DNA (eDNA) anchored to the surface of these organelles. eDNA associated with OMV additionally determines the genetic recombination in the process of horizontal gene transfer. H. pylori is naturally competent for genetic transformation and is constantly capable of DNA uptake from the environment. The natural competence state promotes the assimilation of eDNA anchored to the OMV surface. This is probably dependent on ComB and ComEC components, which are involved in the transformation process. For this reason, the OMV secretion mediates intensive exchange of genetic material, promotes adaptation to changing environmental conditions and enables persistent infecting of the gastric mucosa by H. pylori. 1. Introduction. 2. Secretion of outer membrane vesicles by H. pylori. 3. Proteome of H. pylori outer membrane vesicles. 4. Transport of virulence factors through OMV. 4.1. Toxin VacA. 4.2. Oncoprotein CagA. 4.3. Other substances. 5. OMV involvement in biofilm formation. 5.1. Functions of biofilm. 5.2. OMV influence on bacterial biofilm formation. 5.3. OMV influence on biofilm formation by H. pylori. 5.4. Structural function of H. pylori extracellular DNA. 6. Extracellular DNA as an information carrier. 6.1. Influence on virulence. 6.2. Transformation. 6.3. Natural competence of H. pylori. 7. Conclusions

The effect of imipenem and diffusible signaling factors on the secretion of outer membrane vesicles and associated Ax21 proteins in Stenotrophomonas maltophilia.

Outer membrane vesicles (OMVs) are small nanoscale structures that are secreted by bacteria and that can carry nucleic acids, proteins, and small metabolites. They can mediate intracellular communication and play a role in virulence. In this study, we show that treatment with the β-lactam antibiotic imipenem leads to a dramatic increase in the secretion of outer membrane vesicles in the nosocomial pathogen Stenotrophomonas maltophilia. Proteomic analysis of their protein content demonstrated that the OMVs contain the chromosomal encoded L1 metallo-β-lactamase and L2 serine-β-lactamase. Moreover, the secreted OMVs contain large amounts of two Ax21 homologs, i.e., outer membrane proteins known to be involved in virulence and biofilm formation. We show that OMV secretion and the levels of Ax21 in the OMVs are dependent on the quorum sensing diffusible signal system (DSF). More specific, we demonstrate that the S. maltophilia DSF cis-Δ2-11-methyl-dodecenoic acid and, to a lesser extent, the Burkholderia cenocepacia DSF cis-Δ2-dodecenoic acid, stimulate OMV secretion. By a targeted proteomic analysis, we confirmed that DSF-induced OMVs contain large amounts of the Ax21 homologs, but not the β-lactamases. This work illustrates that both quorum sensing and disturbance of the peptidoglycan biosynthesis provoke the release of OMVs and that OMV content is context dependent.