scholarly journals Membrane Distribution of the Pseudomonas Quinolone Signal Modulates Outer Membrane Vesicle Production in Pseudomonas aeruginosa

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Catalina Florez ◽  
Julie E. Raab ◽  
Adam C. Cooke ◽  
Jeffrey W. Schertzer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Small Molecule ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Membrane Curvature ◽  
Outer Membrane Vesicles ◽  
Couple Model ◽  
Clinical Strains ◽  
Bilayer Couple

ABSTRACT The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis. IMPORTANCE Bacterial secretion has been recognized as an essential facet of microbial pathogenesis and human disease. Numerous virulence factors have been found to be transported within outer membrane vesicles (OMVs), and delivery using these biological nanoparticles often results in increased potency. OMV biogenesis is an important but poorly understood process that is ubiquitous among Gram-negative organisms. Our group seeks to understand the biochemical mechanisms behind the formation of OMVs and has developed a model of small-molecule-induced membrane curvature as an important driver of this process. With this work, we demonstrate that PQS, a known small-molecule OMV inducer, must be exported to promote OMV biogenesis in both lab-adapted and clinical strains of Pseudomonas aeruginosa. In supporting and expanding the bilayer-couple model of OMV biogenesis, the current work lays the groundwork for studying environmental and genetic factors that modulate OMV production and, consequently, the packaging and delivery of many bacterial factors. IMPORTANCE Bacterial secretion has been recognized as an essential facet of microbial pathogenesis and human disease. Numerous virulence factors have been found to be transported within outer membrane vesicles (OMVs), and delivery using these biological nanoparticles often results in increased potency. OMV biogenesis is an important but poorly understood process that is ubiquitous among Gram-negative organisms. Our group seeks to understand the biochemical mechanisms behind the formation of OMVs and has developed a model of small-molecule-induced membrane curvature as an important driver of this process. With this work, we demonstrate that PQS, a known small-molecule OMV inducer, must be exported to promote OMV biogenesis in both lab-adapted and clinical strains of Pseudomonas aeruginosa. In supporting and expanding the bilayer-couple model of OMV biogenesis, the current work lays the groundwork for studying environmental and genetic factors that modulate OMV production and, consequently, the packaging and delivery of many bacterial factors.

scholarly journals A Bilayer-Couple Model of Bacterial Outer Membrane Vesicle Biogenesis

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Jeffrey W. Schertzer ◽  
Marvin Whiteley
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Curvature ◽  
Couple Model ◽  
Gram Negative Bacteria ◽  
Outer Membrane Vesicle ◽  
Gram Negative ◽  
Content Type ◽  
Outer Leaflet ◽  
Bilayer Couple

ABSTRACTGram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated. We recently demonstrated that the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) is required for OMV biogenesis inP. aeruginosa. We hypothesized that PQS stimulates OMV formation through direct interaction with the outer leaflet of the outer membrane. To test this hypothesis, we employed a red blood cell (RBC) model that has been used extensively to study small-molecule–membrane interactions. Our results revealed that addition of PQS to RBCs induced membrane curvature, resulting in the formation of membrane spicules (spikes), consistent with small molecules that are inserted stably into the outer leaflet of the membrane. Radiotracer experiments demonstrated that sufficient PQS was inserted into the membrane to account for this curvature and that curvature induction was specific to PQS structure. These data suggest that a low rate of interleaflet flip-flop forces PQS to accumulate in and expand the outer leaflet relative to the inner leaflet, thus inducing membrane curvature. In support of PQS-mediated outer leaflet expansion, the PQS effect was antagonized by chlorpromazine, a molecule known to be preferentially inserted into the inner leaflet. Based on these data, we propose a bilayer-couple model to describeP. aeruginosaOMV biogenesis and suggest that this is a general mechanism for bacterial OMV formation.IMPORTANCEDespite the ubiquity and importance of outer membrane vesicle (OMV) production in Gram-negative bacteria, the molecular details of OMV biogenesis are not fully understood. Early experiments showed that 2-heptyl-3-hydroxy-4-quinolone (PQS) induces OMV formation through physical interaction with the membrane but did not elucidate the mechanism. The present study demonstrates that PQS specifically and reversibly promotes blebbing of model membranes dependent upon the same properties that are required for OMV formation inP. aeruginosa. These results are consistent with a mechanism where expansion of the outer leaflet relative to the inner leaflet induces localized membrane curvature. This “bilayer-couple” model can account for OMV formation under all conditions and is easily generalized to other Gram-negative bacteria. The model therefore raises the possibility of a universal paradigm for vesicle production in prokaryotes with features strikingly different from what is known in eukaryotes.

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 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.

scholarly journals Outer Membrane Machinery and Alginate Synthesis Regulators Control Membrane Vesicle Production in Pseudomonas aeruginosa

2009 ◽  
Vol 191 (24) ◽  
pp. 7509-7519 ◽  
Author(s):  
Yosuke Tashiro ◽  
Ryosuke Sakai ◽  
Masanori Toyofuku ◽  
Isao Sawada ◽  
Toshiaki Nakajima-Kambe ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Serine Proteases ◽  
Membrane Vesicle ◽  
Bacterial Pathogen ◽  
Membrane Vesicles ◽  
Production Mechanism ◽  
Gene Encoding ◽  
Surrounding Environment ◽  
Regulatory Machinery

ABSTRACT The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.

scholarly journals A Critical Threshold of Meningococcal Factor H Binding Protein Expression Is Required for Increased Breadth of Protective Antibodies Elicited by Native Outer Membrane Vesicle Vaccines

2011 ◽  
Vol 18 (5) ◽  
pp. 736-742 ◽  
Author(s):  
Oliver Koeberling ◽  
Isabel Delany ◽  
Dan M. Granoff
Keyword(s):  
Outer Membrane ◽  
Binding Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Factor H ◽  
Critical Threshold ◽  
Recombinant Strains ◽  
Endotoxin Activity ◽  
Group B

ABSTRACTNative outer membrane vesicles (NOMV) (not detergent treated), which are prepared from recombinant strains with attenuated endotoxin activity and overexpressed factor H binding protein (fHbp), elicited broad serum bactericidal antibody responses in mice. The amount of overexpressed fHbp required for optimal immunogenicity is not known. In this study we prepared NOMV vaccines from LpxL1 knockout (ΔLpxL1) mutants with penta-acylated lipooligosaccharide and attenuated endotoxin activity. The recombinant strains had wild-type (1×) fHbp expression or were engineered for 3-fold- or 10-fold-increased fHbp expression (3× or 10× fHbp). Control vaccines included NOMV from ΔLpxL1/ΔfHbp mutants or recombinant fHbp. In mice, only the 10× fHbp NOMV vaccine elicited significantly higher serum IgG anti-fHbp antibody titers than the corresponding 1× fHbp NOMV or recombinant fHbp vaccine. The 10× fHbp NOMV vaccine also elicited higher bactericidal responses (P< 0.05) against five group B strains with heterologous PorA than the recombinant fHbp or 1× fHbp NOMV vaccine. The 3× fHbp NOMV vaccine gave higher bactericidal titers against only one strain. Serum bactericidal titers in mice immunized with the control ΔfHbp NOMV vaccines were <1:10, and bactericidal titers in mice immunized with the 10× fHbp NOMV vaccine were <1:10 after adsorption of anti-fHbp antibodies. Mixing antiserum to NOMV vaccines from fHbp knockout mutants with antiserum to recombinant fHbp did not increase anti-fHbp bactericidal titers. Thus, a critical threshold of increased fHbp expression is required for NOMV vaccines to elicit broad serum bactericidal responses, and the antibodies conferring protection are directed primarily at fHbp.

scholarly journals Cyclodextrins reduce the ability of Pseudomonas aeruginosa outer-membrane vesicles to reduce CFTR Cl− secretion

2019 ◽  
Vol 316 (1) ◽  
pp. L206-L215 ◽  
Author(s):  
Roxanna Barnaby ◽  
Katja Koeppen ◽  
Bruce A. Stanton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Airway Epithelial Cells ◽  
Outer Membrane Vesicles ◽  
Airway Epithelial ◽  
Ussing Chambers ◽  
Planktonic Growth ◽  
Apical Side

Pseudomonas aeruginosa secretes outer-membrane vesicles (OMVs) that fuse with cholesterol-rich lipid rafts in the apical membrane of airway epithelial cells and decrease wt-CFTR Cl− secretion. Herein, we tested the hypothesis that a reduction of the cholesterol content of CF human airway epithelial cells by cyclodextrins reduces the inhibitory effect of OMVs on VX-809 (lumacaftor)-stimulated Phe508del CFTR Cl− secretion. Primary CF bronchial epithelial cells and CFBE cells were treated with vehicle, hydroxypropyl-β-cyclodextrin (HPβCD), or methyl-β-cyclodextrin (MβCD), and the effects of OMVs secreted by P. aeruginosa on VX-809 stimulated Phe508del CFTR Cl− secretion were measured in Ussing chambers. Neither HPβCD nor MβCD were cytotoxic, and neither altered Phe508del CFTR Cl− secretion. Both cyclodextrins reduced OMV inhibition of VX-809-stimulated Phe508del-CFTR Cl− secretion when added to the apical side of CF monolayers. Both cyclodextrins also reduced the ability of P. aeruginosa to form biofilms and suppressed planktonic growth of P. aeruginosa. Our data suggest that HPβCD, which is in clinical trials for Niemann-Pick Type C disease, and MβCD, which has been approved by the U.S. Food and Drug Administration for use in solubilizing lipophilic drugs, may enhance the clinical efficacy of VX-809 in CF patients when added to the apical side of airway epithelial cells, and reduce planktonic growth and biofilm formation by P. aeruginosa. Both effects would be beneficial to CF patients.

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.

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