scholarly journals Biopearling of Interconnected Outer Membrane Vesicle Chains by a Marine Flavobacterium

2019 ◽  
Vol 85 (19) ◽  
Author(s):  
Tanja Fischer ◽  
Martin Schorb ◽  
Greta Reintjes ◽  
Androniki Kolovou ◽  
Rachel Santarella-Mellwig ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Surface Area ◽  
Algal Blooms ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Glycoside Hydrolases ◽  
Content Type ◽  
Degradative Enzymes

ABSTRACT Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 μm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms. IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.

scholarly journals New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7T: Implications for DNA Content

2013 ◽  
Vol 79 (6) ◽  
pp. 1874-1881 ◽  
Author(s):  
Carla Pérez-Cruz ◽  
Ornella Carrión ◽  
Lidia Delgado ◽  
Gemma Martinez ◽  
Carmen López-Iglesias ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Dna Content ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Freeze Substitution ◽  
Outer Membrane Vesicles ◽  
Negative Bacterium ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type

ABSTRACTOuter membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacteriumShewanella vesiculosaM7Thas revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacteriumShewanella vesiculosaM7Tthat can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).

scholarly journals How the colonic environment influences Enterohaemorrhagic E. coli outer membrane vesicle production, and the interaction between outer membrane vesicles with human host cells

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Daniel Yara ◽  
Regis Stentz ◽  
Tom Wileman ◽  
Stephanie Schuller
Keyword(s):  
Outer Membrane ◽  
Bile Salts ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
T84 Cells ◽  
E Coli

Enterohaemorrhagic E. coli (EHEC) may instigate bloody diarrhoea and haemolytic uraemic syndrome (HUS) due to Shiga toxin (Stx) production. Stx has been detected within outer membrane vesicles (OMVs), which are membrane-derived nanosized proteoliposomes. During colonisation, EHEC encounters many environmental surroundings such as the presence of bile salts and carbon dioxide (CO2). Here, the influence of different intestinal cues on EHEC OMV production was studied. OMV yield was quantified by densitometric analysis of outer membrane proteins F/C and A, following OMV protein separation by SDS-PAGE. Compared to cultures in Luria broth, higher OMV yields were attained following culture in human cell growth medium and simulated colonic environmental medium, with further increases in the presence of bile salts. Interestingly, lower yields were attained in the presence of T84 cells and CO2. The interaction between OMVs and different human cells was also examined by fluorescence microscopy. Here, OMVs incubated with cells showed internalisation by semi confluent but not fully confluent T84 cell monolayers. OMVs were internalised into the lysosomes in confluent Vero and Caco-2 cells, with Stx being transported to the Golgi and then the Endoplasmic reticulum. OMVs were detected within polarised Caco-2 cells, with no impact on the transepithelial electrical resistance by 24 hours. These results suggest that the colonic environmental factors influences OMV production in vivo. Additionally, results highlight the discrepancies which arise when using different cells lines to examine the intestine. Nevertheless, coupled with Stx, OMVs may serve as tools of EHEC which are involved in HUS development.

The search forBrachyspiraouter membrane proteins that interact with the host

2001 ◽  
Vol 2 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Darren J. Trott ◽  
David P. Alt ◽  
Richard L. Zuerner ◽  
Michael J. Wannemuehler ◽  
Thaddeus B. Stanton
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Indirect Evidence ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Surface Proteins ◽  
Brachyspira Pilosicoli ◽  
Variable Surface

AbstractLittle is known about the outer membrane structure ofBrachyspira hyodysenteriae and Brachyspira pilosicolior the role of outer membrane proteins (OMPs) in host colonization and the development of disease. The isolation of outer membrane vesicles fromB. hyodysenteriaehas confirmed that cholesterol is a significant outer membrane constituent and that it may impart unique characteristics to the lipid bilayer structure, including a reduced density. Unique proteins that have been identified in theB. hyodysenteriaeouter membrane include the variable surface proteins (Vsp) and lipoproteins such as SmpA and BmpB. While the function of these proteins remains to be determined, there is indirect evidence to suggest that they may be involved in immune evasion. These data may explain the ability of the organism to initiate chronic infection. OMPs may be responsible for the unique attachment ofB. pilosicolito colonic epithelial cells; however, the onlyB. pilosicoliOMPs that have been identified to date are involved in metabolism. In order to identify furtherB. pilosicoliOMPs we have isolated membrane vesicle fractions from porcine strain 95–1000 by osmotic lysis and isopycnic centrifugation. The fractions were free of contamination by cytoplasm and fla-gella and contained outer membrane. Inner membrane contamination was minimal but could not be completely excluded. An abundant 45-kDa, heat-modifiable protein was shown to have significant homology withB. hyodysenteriaeVsp, and monoclonal antibodies were produced that reacted with fiveB. pilosicoli-specificmembrane protein epitopes. The first of these proteins to be characterized is a unique surface-exposed lipoprotein.

scholarly journals Role of Pseudomonas aeruginosa Peptidoglycan-Associated Outer Membrane Proteins in Vesicle Formation

2012 ◽  
Vol 195 (2) ◽  
pp. 213-219 ◽  
Author(s):  
Aimee K. Wessel ◽  
Jean Liew ◽  
Taejoon Kwon ◽  
Edward M. Marcotte ◽  
Marvin Whiteley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Opportunistic Pathogen ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Content Type

ABSTRACTGram-negative bacteria produce outer membrane vesicles (OMVs) that package and deliver proteins, small molecules, and DNA to prokaryotic and eukaryotic cells. The molecular details of OMV biogenesis have not been fully elucidated, but peptidoglycan-associated outer membrane proteins that tether the outer membrane to the underlying peptidoglycan have been shown to be critical for OMV formation in multipleEnterobacteriaceae. In this study, we demonstrate that the peptidoglycan-associated outer membrane proteins OprF and OprI, but not OprL, impact production of OMVs by the opportunistic pathogenPseudomonas aeruginosa. Interestingly, OprF does not appear to be important for tethering the outer membrane to peptidoglycan but instead impacts OMV formation through modulation of the levels of thePseudomonasquinolone signal (PQS), a quorum signal previously shown by our laboratory to be critical for OMV formation. Thus, the mechanism by which OprF impacts OMV formation is distinct from that for other peptidoglycan-associated outer membrane proteins, including OprI.

scholarly journals Context-Dependent Activation Kinetics Elicited by Soluble versus Outer Membrane Vesicle-Associated Heat-Labile Enterotoxin

2011 ◽  
Vol 79 (9) ◽  
pp. 3760-3769 ◽  
Author(s):  
Halima Chutkan ◽  
Meta J. Kuehn
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Extracellular Vesicle ◽  
Outer Membrane Vesicles ◽  
Cytokine Gene Expression ◽  
Activator Protein 1 ◽  
Content Type ◽  
Creb Phosphorylation ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is the leading cause of traveler's diarrhea and children's diarrhea worldwide. Among its virulence factors, ETEC produces heat-labile enterotoxin (LT). Most secreted LT is associated with outer membrane vesicles that are rich in lipopolysaccharide. The majority of prior studies have focused on soluble LT purified from ETEC periplasm. We investigated the hypothesis that the extracellular vesicle context of toxin presentation might be important in eliciting immune responses. We compared the polarized epithelial cell responses to apically applied soluble LT and LT-containing vesicles (LT+vesicles) as well as controls using a catalytically inactive mutant of LT and vesicles lacking LT. Although vesicle treatments with no or catalytically inactive LT induced a modest amount of interleukin-6 (IL-6), samples containing catalytically active LT elicited higher levels. A combination of soluble LT and LT-deficient vesicles induced significantly higher IL-6 levels than either LT or LT+vesicles alone. The responses to LT+vesicles were found to be independent of the canonical LT pathway, because the inhibition of cyclic AMP response element (CRE)-binding protein (CREB) phosphorylation did not lead to a decrease in cytokine gene expression levels. Furthermore, soluble LT caused earlier phosphorylation of CREB and activation of CRE compared with LT+vesicles. Soluble LT also led to the activation of activator protein 1, whereas LT+vesicle IL-6 responses appeared to be mediated by NF-κB. In summary, the results demonstrate that soluble LT and vesicle-bound LT elicit ultimately similar cytokine responses through distinct different activation pathways.

scholarly journals The Enolase of Borrelia burgdorferi Is a Plasminogen Receptor Released in Outer Membrane Vesicles

2011 ◽  
Vol 80 (1) ◽  
pp. 359-368 ◽  
Author(s):  
A. Toledo ◽  
J. L. Coleman ◽  
C. J. Kuhlow ◽  
J. T. Crowley ◽  
J. L. Benach
Keyword(s):  
Lyme Disease ◽  
Membrane Proteins ◽  
Borrelia Burgdorferi ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Proteinase K ◽  
Dependent Manner ◽  
Content Type

ABSTRACTThe agent of Lyme disease,Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated during its life cycle. In addition to their physiological functions in the organism, these proteins also likely serve different functions in invasiveness and immune evasion. In borreliae, as well as in other bacteria, a number of membrane proteins have been implicated in binding plasminogen. The activation and transformation of plasminogen into its proteolytically active form, plasmin, enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles ofB. burgdorfericontain enolase, a glycolytic-cycle enzyme that catalyzes 2-phosphoglycerate to form phosphoenolpyruvate, which is also a known plasminogen receptor in Gram-positive bacteria. The enolase was cloned, expressed, purified, and used to generate rabbit antienolase serum. The enolase binds plasminogen in a lysine-dependent manner but not through ionic interactions. Although it is present in the outer membrane, microscopy and proteinase K treatment showed that enolase does not appear to be exposed on the surface. However, enolase in the outer membrane vesicles is accessible to proteolytic degradation by proteinase K. Samples from experimentally and tick-infected mice and rabbits as well as from Lyme disease patients exhibit recognition of enolase in serologic assays. Thus, this immunogenic plasminogen receptor released in outer membrane vesicles could be responsible for external proteolysis in the pericellular environment and have roles in nutrition and in enhancing dissemination.

scholarly journals Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages

2011 ◽  
Vol 79 (6) ◽  
pp. 2182-2192 ◽  
Author(s):  
Hyunjin Yoon ◽  
Charles Ansong ◽  
Joshua N. Adkins ◽  
Fred Heffron
Keyword(s):  
Outer Membrane ◽  
Virulence Factors ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Secretion Systems ◽  
Homologous Proteins ◽  
Content Type ◽  
Small Proteins

ABSTRACTSalmonella entericaserovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors.Salmonellavirulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ∼70 amino acids and have high sequence homology to each other (>85% identity).Salmonellalacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all threeSalmonellagene-encoded type III secretion systems (T3SSs)–Salmonellapathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novelSalmonellavirulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.

scholarly journals Pseudomonas Quinolone Signal-Induced Outer Membrane Vesicles Enhance Biofilm Dispersion in Pseudomonas aeruginosa

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Adam C. Cooke ◽  
Catalina Florez ◽  
Elise B. Dunshee ◽  
Avery D. Lieber ◽  
Michelle L. Terry ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Burn Wound ◽  
Content Type ◽  
Enhanced Production ◽  
Biofilm Dispersion ◽  
Biofilm Development

ABSTRACT Bacterial biofilms are major contributors to chronic infections in humans. Because they are recalcitrant to conventional therapy, they present a particularly difficult treatment challenge. Identifying factors involved in biofilm development can help uncover novel targets and guide the development of antibiofilm strategies. Pseudomonas aeruginosa causes surgical site, burn wound, and hospital-acquired infections and is also associated with aggressive biofilm formation in the lungs of cystic fibrosis patients. A potent but poorly understood contributor to P. aeruginosa virulence is the ability to produce outer membrane vesicles (OMVs). OMV trafficking has been associated with cell-cell communication, virulence factor delivery, and transfer of antibiotic resistance genes. Because OMVs have almost exclusively been studied using planktonic cultures, little is known about their biogenesis and function in biofilms. Several groups have shown that Pseudomonas quinolone signal (PQS) induces OMV formation in P. aeruginosa. Our group described a biophysical mechanism for this and recently showed it is operative in biofilms. Here, we demonstrate that PQS-induced OMV production is highly dynamic during biofilm development. Interestingly, PQS and OMV synthesis are significantly elevated during dispersion compared to attachment and maturation stages. PQS biosynthetic and receptor mutant biofilms were significantly impaired in their ability to disperse, but this phenotype was rescued by genetic complementation or exogenous addition of PQS. Finally, we show that purified OMVs can actively degrade extracellular protein, lipid, and DNA. We therefore propose that enhanced production of PQS-induced OMVs during biofilm dispersion facilitates cell escape by coordinating the controlled degradation of biofilm matrix components. IMPORTANCE Treatments that manipulate biofilm dispersion hold the potential to convert chronic drug-tolerant biofilm infections from protected sessile communities into released populations that are orders-of-magnitude more susceptible to antimicrobial treatment. However, dispersed cells often exhibit increased acute virulence and dissemination phenotypes. A thorough understanding of the dispersion process is therefore critical before this promising strategy can be effectively employed. Pseudomonas quinolone signal (PQS) has been implicated in early biofilm development, but we hypothesized that its function as an outer membrane vesicle (OMV) inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs are differentially produced during Pseudomonas aeruginosa biofilm development and provide evidence that effective biofilm dispersion is dependent on the production of PQS-induced OMVs, which likely act as delivery vehicles for matrix-degrading enzymes. These findings lay the groundwork for understanding OMV contributions to biofilm development and suggest a model to explain the controlled matrix degradation that accompanies biofilm dispersion in many species.

scholarly journals Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis tolB Mutant

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Kotaro Takaki ◽  
Yuhei O. Tahara ◽  
Nao Nakamichi ◽  
Yusuke Hasegawa ◽  
Masaki Shintani ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Striking Similarity ◽  
Common Mechanism ◽  
Separation And Purification ◽  
Intercellular Interaction ◽  
Electron Microscopic ◽  
Content Type

ABSTRACT Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of tolB, which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming Buttiauxella agrestis type strain JCM 1090. The ΔtolB mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the ΔtolB mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of ΔtolB mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the B. agrestis ΔtolB mutant to form unique vesicles. IMPORTANCE Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, Buttiauxella agrestis JCM 1090T ΔtolB mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications.

scholarly journals Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Nanomaterials ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1661
Author(s):  
Mei-Hsiu Chen ◽  
Tse-Ying Liu ◽  
Yu-Chiao Chen ◽  
Ming-Hong Chen
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Glioma Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
E Coli ◽  
Tumor Bearing ◽  
Nano Gold ◽  
Gl261 Cell

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.

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