Outer Membrane Vesicles Proteome Research Articles

Characterization of outer membrane vesicles released by clinical isolates of Neisseria gonorrhoeae.

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1αandIL-1β. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

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.

Quantitative Proteomic Analysis of Outer Membrane Vesicles from Fusobacterium nucleatum Cultivated in the Mimic Cancer Environment.

Fusobacterium nucleatum is a Gram-negative bacterium that has been identified as an important pathogenic gut bacterium associated with colorectal cancer. Compared with the normal intestine, the pH value of the tumor microenvironment is weakly acidic. The metabolic changes of F. nucleatum in the tumor microenvironment, especially the protein composition of its outer membrane vesicles, remain unclear. Here, we systematically analyzed the effect of environmental pH on the proteome of outer membrane vesicles (OMVs) from F. nucleatum by tandem mass tag (TMT) labeling-high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 991 proteins were identified in acidic OMVs (aOMVs) and neutral OMVs (nOMVs), including known virulence proteins and putative virulence proteins. Finally, 306 upregulated proteins and 360 downregulated proteins were detected in aOMVs, and approximately 70% of the expression of OMV proteins was altered under acidic conditions. A total of 29 autotransporters were identified in F. nucleatum OMVs, and 13 autotransporters were upregulated in aOMVs. Interestingly, three upregulated autotransporters (D5REI9, D5RD69, and D5RBW2) show homology to the known virulence factor Fap2, suggesting that they may be involved in various pathogenic pathways such as the pathway for binding with colorectal cancer cells. Moreover, we found that more than 70% of MORN2 domain-containing proteins may have toxic effects on host cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that a number of proteins were significantly enriched in multiple pathways involving fatty acid synthesis and butyrate synthesis. Seven metabolic enzymes involved in fatty acid metabolism pathways were identified in the proteomic data, of which 5 were upregulated and 2 were downregulated in aOMVs, while 14 metabolic enzymes involved in the butyric acid metabolic pathway were downregulated in aOMVs. In conclusion, we found a key difference in virulence proteins and pathways in the outer membrane vesicles of F. nucleatum between the tumor microenvironment pH and normal intestinal pH, which provides new clues for the prevention and treatment of colorectal cancer. IMPORTANCE F. nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. OMVs have been demonstrated to play key roles in pathogenesis by delivering toxins and other virulence factors to host cells. By employing quantitative proteomic analysis, we found that the pH conditions could affect the protein expression of the outer membrane vesicles of F. nucleatum. Under acidic conditions, approximately 70% of the expression of proteins in OMVs was altered. Several virulence factors, such as type 5a secreted autotransporter (T5aSSs) and membrane occupation and recognition nexus (MORN) domain-containing proteins, were upregulated under acidic conditions. A large number of proteins showed significant enrichments in multiple pathways involving fatty acid synthesis and butyrate synthesis. Proteomics analysis of the outer membrane vesicles secreted by pathogenic bacteria in the acidic tumor microenvironment is of great significance for elucidating the pathogenicity mechanism and its application in vaccine and drug delivery vehicles.

Modulation of Klebsiella pneumoniae Outer Membrane Vesicle Protein Cargo under Antibiotic Treatment.

Klebsiella pneumoniae is a nosocomial pathogen and an important propagator of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Like other Gram-negative bacteria, they secrete outer membrane vesicles (OMVs) that distribute virulence and resistance factors. Here, we subjected a K. pneumoniae-XDR to subinhibitory concentrations of meropenem, amikacin, polymyxin B, and a combination of these agents to evaluate changes in the protein cargo of OMVs through liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genome sequencing of the clinical isolate K. pneumoniae strain HCD1 (KpHCD1) revealed the presence of 41 resistance genes and 159 virulence factors. We identified 64 proteins in KpHCD1-OMVs modulated with different antibiotic treatments involved in processing genetic information, environmental information, cell envelope formation, energy metabolism, and drug resistance. The OMV proteome expression profile suggests that OMVs may be associated with pathogenicity, survival, stress response, and resistance dissemination.

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition.

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P.aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.

A PQS-Cleaving Quorum Quenching Enzyme Targets Extracellular Membrane Vesicles of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQDS.b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQDS.b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQDS.b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQDS.b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme.

Analysis of Outer Membrane Vesicles Indicates That Glycerophospholipid Metabolism Contributes to Early Symbiosis Between Sinorhizobium fredii HH103 and Soybean.

Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited. In this work, we isolated and purified OMVs from Sinorhizobium fredii HH103 under free-living conditions that were set as control (C-OMVs) and symbiosis-mimicking conditions that were induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while they inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism, and peptidoglycan biosynthesis. A total of 45 differential lipids were identified through lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched Kyoto Encyclopedia of Genes and Genomes metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by enzyme-linked immunosorbent assay. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically Defined Medium.

Outer membrane vesicles (OMVs) are small vesicles constitutively shed by all Gram-negative bacterium, which have been proposed to play a role in Helicobacter pylori persistence and pathogenesis. The methods currently available for the isolation of H. pylori OMVs are diverse and time-consuming, raising the need for a protocol standardization, which was the main aim of this study. Here, we showed that the chemically defined F12 medium, supplemented with cholesterol, nutritionally supports bacterial growth and maintains H. pylori viability for at least 72 h. Additionally, we developed an abridged protocol for isolation of OMVs from these bacterial cultures, which comprises a low-speed centrifugation, supernatant filtration through a 0.45 μm pore, and two ultracentrifugations for OMVs’ recovery and washing. Using this approach, a good yield of highly pure bona fide OMVs was recovered from cultures of different H. pylori strains and in different periods of bacterial growth, as assessed by nanoparticle tracking analysis, transmission electron microscopy (TEM), and proteomic analyses, confirming the reliability of the protocol. Analysis of the proteome of OMVs isolated from H. pylori F12-cholesterol cultures at different time points of bacterial growth revealed differentially expressed proteins, including the vacuolating cytotoxin VacA. In conclusion, this work proposes a time- and cost-efficient protocol for the isolation of H. pylori OMVs from a chemically defined culture medium that is suitable for implementation in research and in the biopharmaceutical field.

Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells.

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNFα and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response.

Characterization of Outer Membrane Vesicles from Fusobacterium nucleatum

Background/objectives: Fusobacterium nucleatum is an oral pathogen and is associated with the development of colorectal cancer (CRC). This study is to evaluate the ability of outer membrane vesicles (OMV) from F. nucleatum to modulate cellular responses in colonic cells. Methodology: Here we show that infection of colonic epithelial cells with F. nucleatum and its OMV induce pro-inflammatory chemokine and cytokine production and promote an EMT-like pheno- and genotypes in vitro as demonstrated by suppression of E-cadherin and up-regulation of several mesenchymal markers. F. nucleatum and its OMV modulate the barrier function of intestinal monolayers, a process likely related to their demonstrated ability to degrade E-cadherin and suppress its expression. Findings: Analysis of the OMV proteome by mass spectrometry demonstrates that they harbor the known virulence factors that appear to be enriched with proteolytic activity. Novelty/contribution: Taken together, these data indicate that F. nucleatum OMV have the potential to contribute to disease progression in the context of CRC. Keywords: Outer Membrane Vesicles, Fusobacterium nucleatum, Colorectal Cancer, Protease.

Integrative analysis of outer membrane vesicles proteomics and whole-cell transcriptome analysis of eravacycline induced Acinetobacter baumannii strains

BackgroundAcinetobacter baumannii is a multidrug-resistant (MDR) hazardous bacterium with very high antimicrobial resistance profiles. Outer membrane vesicles (OMVs) help directly and/or indirectly towards antibiotic resistance in these organisms. The present study aims to look on the proteomic profile of OMV as well as on the bacterial transcriptome upon exposure and induction with eravacycline, a new synthetic fluorocycline. RNA sequencing analysis of whole-cell and LC-MS/MS proteomic profiling of OMV proteome abundance were done to identify the differential expression among the eravacycline-induced A. baumannii ATCC 19606 and A. baumannii clinical strain JU0126.ResultsThe differentially expressed genes from the RNA sequencing were analysed using R package and bioinformatics software and tools. Genes encoding drug efflux and membrane transport were upregulated among the DEGs from both ATCC 19606 and JU0126 strains. As evident with the induction of eravacycline resistance, ribosomal proteins were upregulated in both the strains in the transcriptome profiles and also resistance pumps, such as MFS, RND, MATE and ABC transporters. High expression of stress and survival proteins were predominant in the OMVs proteome with ribosomal proteins, chaperons, OMPs OmpA, Omp38 upregulated in ATCC 19606 strain and ribosomal proteins, toluene tolerance protein, siderophore receptor and peptidases in the JU0126 strain. The induction of resistance to eravacycline was supported by the presence of upregulation of ribosomal proteins, resistance-conferring factors and stress proteins in both the strains of A. baumannii ATCC 19606 and JU0126, with the whole-cell gene transcriptome towards both resistance and stress genes while the OMVs proteome enriched more with survival proteins.ConclusionThe induction of resistance to eravacycline in the strains were evident with the increased expression of ribosomal and transcription related genes/proteins. Apart from this resistance-conferring efflux pumps, outer membrane proteins and stress-related proteins were also an essential part of the upregulated DEGs. However, the expression profiles of OMVs proteome in the study was independent with respect to the whole-cell RNA expression profiles with low to no correlation. This indicates the possible role of OMVs to be more of back-up additional protection to the existing bacterial cell defence during the antibacterial stress.

Tobramycin reduces key virulence determinants in the proteome of Pseudomonas aeruginosa outer membrane vesicles

Tobramycin is commonly used to treat Pseudomonas aeruginosa lung infections in patients with Cystic Fibrosis (CF). Tobramycin treatment leads to increased lung function and fewer clinical exacerbations in CF patients, and modestly reduces the density of P. aeruginosa in the lungs. P. aeruginosa resides primarily in the mucus overlying lung epithelial cells and secretes outer membrane vesicles (OMVs) that diffuse through the mucus and fuse with airway epithelial cells, thus delivering virulence factors into the cytoplasm that modify the innate immune response. The goal of this study was to test the hypothesis that Tobramycin reduces the abundance of virulence factors in OMVs secreted by P. aeruginosa. Characterization of the proteome of OMVs isolated from control or Tobramycin-exposed P. aeruginosa strain PAO1 revealed that Tobramycin reduced several OMV-associated virulence determinants, including AprA, an alkaline protease that enhances P. aeruginosa survival in the lung, and is predicted to contribute to the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion by primary human bronchial epithelial cells. Deletion of the gene encoding AprA reduced the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion. Moreover, as predicted by our proteomic analysis, OMVs isolated from Tobramycin treated P. aeruginosa had a diminished inhibitory effect on Phe508del-CFTR Cl- secretion compared to OMVs isolated from control P. aeruginosa. Taken together, our proteomic analysis of OMVs and biological validation suggest that Tobramycin may improve lung function in CF patients infected with P. aeruginosa by reducing several key virulence factors in OMVs that reduce CFTR Cl- secretion, which is essential for bacterial clearance from the lungs.

Antibiotics and iron-limiting conditions and their effect on the production and composition of outer membrane vesicles secreted from clinical isolates of extraintestinal pathogenic E. coli.

The focus of this study was to characterize the effect of clinically relevant stress-inducing conditions on the production and composition of proinflammatory outer membrane vesicles (OMVs) produced from ST131 extraintestinal pathogenic Escherichia coli (ExPEC) clinical isolates. A label-free method (relative normalized spectral index quantification, SINQ) was used to identify changes in the respective OMV proteomes following exposure of the ExPEC strains to antibiotics and low iron. Nanoparticle tracking analysis was used to quantify changes in abundance and size of OMVs produced by the gentamicin-resistant (GenR) and gentamicin-sensitive (GenS) ExPEC strains. Up to a 13.1-fold increase in abundance of particles were detected when the gentamicin-sensitive strain was exposed to a range of gentamicin concentrations. In contrast, no increase was observed for the gentamicin-resistant strain. Iron-limiting conditions had minimal effect on OMV production for either strain. Marked changes in the OMV proteome were observed for both strains including increases in Hsp100/Clp proteins, ATP-dependent ClpP protease, and regulatory proteins. These data provide information on changes in the composition of OMV particles derived from ExPEC strains generated in response to clinically relevant conditions. We show that the levels of the proinflammatory OMVs increase for gentamicin-sensitive ExPEC exposed to the antibiotic.

Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors.

Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.

Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.

Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis

Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis.

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.

Porphyromonas gingivalis Outer Membrane Vesicles Exclusively Contain Outer Membrane and Periplasmic Proteins and Carry a Cargo Enriched with Virulence Factors

Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria and have a relevant role in bacteria-host interactions. Using 1D SDS-PAGE and highly sensitive LC-MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain-linked genes and 57 were common to pathogen-derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic-derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 (http://proteomecentral.proteomexchange.org/dataset/PXD000367).

Profiling the Outer Membrane Proteome during Growth and Development of the Social Bacterium Myxococcus xanthus by Selective Biotinylation and Analyses of Outer Membrane Vesicles

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.