Release Of Outer Membrane Vesicles Research Articles

Helicobacter pylori outer membrane vesicles inhibit human T cell responses via induction of monocyte COX-2 expression.

The modulation of T cell responses by Helicobacter pylori is thought to potentiate both H. pylori persistence and development of gastric pathologies including cancer. Release of outer membrane vesicles (OMV) by H. pylori provides a potential vehicle for modulation of the immune system. Although OMV are thought to have T cell suppressive activity, this has not yet been demonstrated. Their suppressive activity was investigated in this study using the responses of peripheral blood mononuclear cells (PBMC) to T cell stimuli as a readout. We demonstrate that addition of OMV to PBMC significantly inhibits subsequent T cell proliferation in a cyclo-oxygenase-2 (COX-2)-dependent manner. Addition of OMV did not significantly modulate PBMC apoptosis, but induced strong expression of COX-2 by the monocytes present and significantly increased levels of PGE2 and IL-10. These effects were independent of vacuolating cytotoxin expression. Together, these findings demonstrate that OMV can suppress human T cell responses and that the predominant mechanism is not through a direct effect on the T cells but results from the induction of COX-2 expression in monocytes. This increased COX-2 activity may modulate not only H. pylori-directed immune responses but also wider immune responses.

Construction of a System for the Study of Protein‐Induced Membrane Tubules

Communication between cells and the environment is crucial to life, and correspondingly cells have evolved complex signaling pathways. For example, bacteria have a repertoire of cellular structures that mediate adhesion, motility, virulence, etc. Some of the well‐studied systems are pili, flagella, and multiple secretion systems. Recently, more attention has been paid to membranous structures such as secreted vesicles and membrane appendages. These structures have been shown to play roles in adhesion, cell‐to‐cell communication, and pathogenesis. Membrane appendages appear to be very diverse structures with possible distinct assembly mechanisms. For instance, membrane appendages can appear as tubular structures with a size range of 20–70 ηm in width that extend from the bacterial membrane for very long distances. These membrane appendages are sometime referred as nanotubes and in some instances they have been shown to play a role in metabolic exchange. One study showed that Delftia sp. can form nanopods as the result of the release of outer membrane vesicles that fused together1. Other membrane tubules have been shown to contain protein filaments, such as sheathed flagella and unnatural pili.In our laboratory we attempted to construct a heterologous system for bacterial pilus assembly. However, we found that under special conditions we could trick E. coli to elaborate structures that resembled nanotubes. In our initial studies we expressed different proteins from the Pseudomonas aeruginosa Type‐4 pilus system in E. coli Bl21‐AI, E. coli BL21 (DE3), or E. coli NovaBlue (DE3) and induced protein expression with IPTG and/or arabinose when necessary. Using negative staining and electron microscopy, we observed tubular structures (about 20–40 ηm wide, and longer than 1 μm). Remarkably although formation of these extensions was dependent on the major component of the pilus, PilA, it was independent of pre‐pilin cleavage by pre‐pilin peptidase; we found long membrane tubules in the presence of a non‐cleavable pilin. This system promises a means to investigate in detail how bacteria can build nanotubes and how this process is affected by the presence of transmembrane proteins.Support or Funding InformationNIH 5 T32 GM008349

Outer membrane vesicles containing signalling molecules and active hydrolytic enzymes released by a coral pathogen Vibrio shilonii AK1.

Production and release of outer-membrane vesicles (OMVs) is known in many bacteria including human pathogens. To date, OMV release has not been reported in coral-associated bacteria. We discovered that Vibrio shilonii AK1, a well-studied coral pathogen, produces OMVs in culture. Transmission electron microscopy showed that V. shilonii cultures release two types of vesicles, with a single membrane or two membranes, as well as vesicle chain-like morphotype in purified vesicle fraction. No significant difference was observed in the amount of OMVs produced by cultures grown at 20°C or 30°C. OMV proteomic analysis, never before done in a coral isolate, showed that a large number of low abundance proteins were exclusively detected in OMVs released by 20°C cultures. Further, the OMVs purified from AK1 cultures grown at both 20°C and 30°C carry N-acylhomoserine lactone quorum sensing signals, as well as alkaline phosphatase, lipase and chitinase activities. Our results show that V. shilonii OMVs are conduits of signalling molecules, active enzymes and other proteins to its environment. These findings suggest important ecophysiological roles of OMVs in coral reef environment. We discuss the importance of OMV release for V. shilonii fitness and propose several hypotheses as well as a conceptual model.

Pseudomonas aeruginosa Outer Membrane Vesicles Triggered by Human Mucosal Fluid and Lysozyme Can Prime Host Tissue Surfaces for Bacterial Adhesion

Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release outer membrane vesicles (OMVs) in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to phosphate buffered saline (PBS) controls (∼100-fold). Transmission electron microscopy (TEM) and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (∼4-fold, P < 0.01). Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections.

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.

Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. Determining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host.

Activation of Immune and Defense Responses in the Intestinal Mucosa by Outer Membrane Vesicles of Commensal and Probiotic Escherichia coli Strains.

The influence of microbiota in human health is well-known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although, there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well-known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 (EcN) or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i) peripheral blood mononuclear cells (PBMCs) as a model of intestinal barrier disruption, (ii) apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii) colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas, both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier.

Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a’s targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.

Release of outer membrane vesicles in Pseudomonas putida as a response to stress caused by cationic surfactants.

Pseudomonas putida A (ATCC 12633), a degrader of cationic surfactants, releases outer membrane vesicles (OMVs) when grown with tetradecyltrimethylammonium bromide (TTAB) as the sole carbon, nitrogen and energy source. The OMVs exhibit a bilayer structure and were found to be composed of lipopolysaccharides, proteins and phospholipids (PLs) such as cardiolipin, phosphatidylcholine, phosphatidic acid and phosphatidylglycerol (PG). The OMVs showed a marked increase in the PG content, approximately 43 % higher than the amount registered in the parent cells from which the vesicles were derived. After growth of P. putida with TTAB, the amount of lipoprotein covalently cross-linked to the peptidoglycan showed a twofold decrease when compared with values found after growth without the surfactant [16 ± 2 and 28 ± 3 μg (mg cell envelope protein)- 1, respectively]. This decrease in the amount of lipoprotein can be related to areas of loss of contact between the outer membrane and the peptidoglycan and, therefore, to OMV production. In addition, due to its amphiphilic nature, TTAB can contribute to OMV biogenesis, through a physical mechanism, by induction of the curvature of the membrane. Taking into account that OVMs were produced when the cells were grown under external stress, caused by the surfactant, and that TTAB was detected in the vesicles [48 nmol TTAB (nmol PL)- 1], we concluded that this system of TTAB elimination is a mechanism that P. putida A (ATCC 12633) would utilize for alleviating stress caused by cationic surfactants.

The versatile TolC-like Slr1270 in the cyanobacterium Synechocystis sp. PCC 6803.

Here we report on the functional characterization of the hypothetical protein Slr1270, a TolC homologue in Synechocystis sp. PCC 6803. Analysis of a slr1270 insertion deletion mutant and respective wild-type revealed that the mutant presents increased susceptibility to antibiotics. In addition, a detailed study of the exoproteome showed that Slr1270 mediates protein secretion. Among the protein substrates dependent on Slr1270 function, we found the S-layer structural component. Electron microscopy studies of the slr1270 mutant showed that the S-layer is indeed absent. The requirement of functional Slr1270 for protein secretion and drug resistance mechanisms suggests that Slr1270 plays a role similar to that described for TolC in other bacteria. Additional phenotypic traits could also be observed, including slower growth rates at low temperature, impairment in biofilm formation and increased activity of enzymes detoxifying reactive oxygen species. Furthermore, an increased capacity of outer membrane vesicles (OMVs) formation and release was also found in the slr1270 mutant, a feature that has not yet been observed in bacteria lacking TolC. This work highlights the marked physiological fitness that the TolC-like Slr1270 bestows to the photosynthetic model Synechocystis sp. PCC 6803 and presents a valuable model for studying OMVs formation and release.

Intra- and Interspecies Effects of Outer Membrane Vesicles from Stenotrophomonas maltophilia on β-Lactam Resistance.

The treatment ofStenotrophomonas maltophiliainfection with β-lactam antibiotics leads to increased release of outer membrane vesicles (OMVs), which are packed with two chromosomally encoded β-lactamases. Here, we show that these β-lactamase-packed OMVs are capable of establishing extracellular β-lactam degradation. We also show that they dramatically increase the apparent MICs of imipenem and ticarcillin for the cohabituating speciesPseudomonas aeruginosaandBurkholderia cenocepacia.

The relationship between biofilm and outer membrane vesicles: a novel therapy overview.

Microorganisms have the ability of inhabiting nearly every environment through their sophisticated mechanisms of survival such as biofilm formation and release of outer membrane vesicles (OMVs). The biofilm matrix offers microorganism protection and contributes significantly to several clinical challenges, including symptomatic inflammation, antibiotic resistance, recurrence and the spread of infectious emboli. Moreover, bacteria also have another protective mechanism of vesicle production which is used as a means of disseminating toxins to harm their host. A clear understanding of gene expression switch of bacterium from planktonic to biofilm mode offers clinical potentials in treating bacterial infections. In this respect, the treatment of bacterial infections may be achieved through (1) application of RNA interference technology to silence the expression of proteins involved in the process of biofilm formation, (2) utilization of vesicles in delivering antibiotics and (3) use of natural occurred compounds. In this review, we discuss the relationship between biofilm formation and OMV production with respect to tackling biofilm-related clinical challenges. Some prospective considerations in biofilm-associated infections treatment are also discussed.

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.

LytM proteins play a crucial role in cell separation, outer membrane composition, and pathogenesis in nontypeable Haemophilus influenzae.

ABSTRACTLytM proteins belong to a family of bacterial metalloproteases. In Gram-negative bacteria, LytM factors are mainly reported to have a direct effect on cell division by influencing cleavage and remodeling of peptidoglycan. In this study, mining nontypeable Haemophilus influenzae (NTHI) genomes, three highly conserved open reading frames (ORFs) containing a LytM domain were identified, and the proteins encoded by the ORFs were named YebA, EnvC, and NlpD on the basis of their homology with the Escherichia coli proteins. Immunoblotting and confocal analysis showed that while NTHI NlpD is exposed on the bacterial surface, YebA and EnvC reside in the periplasm. NTHI ΔyebA and ΔnlpD deletion mutants revealed an aberrant division phenotype characterized by an altered cell architecture and extensive membrane blebbing. The morphology of the ΔenvC deletion mutant was identical to that of the wild-type strain, but it showed a drastic reduction of periplasmic proteins, including the chaperones HtrA, SurA, and Skp, and an accumulation of β-barrel-containing outer membrane proteins comprising the autotransporters Hap, IgA serine protease, and HMW2A, as observed by proteomic analysis. These data suggest that EnvC may influence the bacterial surface protein repertoire by facilitating the passage of the periplasmic chaperones through the peptidoglycan layer to the close vicinity of the inner face of the outer membrane. This hypothesis was further corroborated by the fact that an NTHI envC defective strain had an impaired capacity to adhere to epithelial cells and to form biofilm. Notably, this strain also showed a reduced serum resistance. These results suggest that LytM factors are not only important components of cell division but they may also influence NTHI physiology and pathogenesis by affecting membrane composition.

Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells.

Production of outer membrane vesicles by the plague pathogen Yersinia pestis.

Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.

Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells.

Vibrio tasmaniensis LGP32, a facultative intracellular pathogen of oyster haemocytes, was shown here to release outer membrane vesicles (OMVs) both in the extracellular milieu and inside haemocytes. Intracellular release of OMVs occurred inside phagosomes of intact haemocytes having phagocytosed few vibrios as well as in damaged haemocytes containing large vacuoles heavily loaded with LGP32. The OMV proteome of LGP32 was shown to be rich in hydrolases (25%) including potential virulence factors such as proteases, lipases, phospholipases, haemolysins and nucleases. One major caseinase/gelatinase named Vsp for vesicular serine protease was found to be specifically secreted through OMVs in which it is enclosed. Vsp was shown to participate in the virulence phenotype of LGP32 in oyster experimental infections. Finally, OMVs were highly protective against antimicrobial peptides, increasing the minimal inhibitory concentration of polymyxin B by 16-fold. Protection was conferred by OMV titration of polymyxin B but did not depend on the activity of Vsp or another OMV-associated protease. Altogether, our results show that OMVs contribute to the pathogenesis of LGP32, being able to deliver virulence factors to host immune cells and conferring protection against antimicrobial peptides.

Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110

Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.

A Structural Approach to Characterizing the Release of Outer Membrane Vesicles in Vibrio vulnificus

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Outer membrane vesicles from pathogenic bacteria initiate an inflammatory response in human endothelial cells

IntroductionGram-negative bacteria release outer membrane vesicles (OMVs) during growth that contain various membrane components involved in eliciting an inflammatory response, including lipopolysaccharide and virulence factors. However, little is known about the role of OMVs in sepsis. The objective of this study was to determine how OMVs, derived from Escherichia (E.) coli, elicit the cellular responses involved in activating the inflammatory cascade, and to determine whether additional virulence factors in pathogenic OMVs augment the inflammatory response. MethodsHuman umbilical endothelial cells were inoculated with OMVs from non-pathogenic E. coli (npOMV) or pathogenic E. coli (pOMV) and analyzed for adhesion protein synthesis, cytokine production, and necrosis factor (NF)-κB translocation. ResultsFlow cytometry demonstrated that human umbilical vein endothelial cells exposed to npOMV or pOMV significantly increased expression of E-selectin and intercellular adhesion molecule, with a large population of cells demonstrating increased expression of both proteins. Interleukin-6 levels were significantly elevated by 4 h after exposure to npOMV and pOMVs. NF-κB translocation to the nucleus was shown to be induced by npOMV and pOMVs. However, the role of additional virulence factors associated with pOMVs remains undefined. ConclusionsBoth npOMVs and pOMVs are capable of initiating the inflammatory cascade in endothelial cells. OMVs trigger NF-κB translocation to the nucleus, resulting in up-regulation of adhesion molecules and cytokines, presumably for the recruitment of leukocytes. By eliciting an inflammatory response, OMVs could facilitate the transition from a localized infection to a systemic response, and ultimately sepsis.

AB0167 Autocitrullinated peptidylarginine deiminase from porphyromonas gingivalis part of an infection model in RA?

BackgroundBacteria release outer membrane vesicles (OMV) which do contain proteins, DNA and enzymes like Peptidylarginine Deiminase from Porphyromonas gingivalis (PPAD) to interact with bacteria and the host immune system. PPAD...