Rapid prototyping vaccine approach in mice against multi‐drug resistant Gram‐negative organisms from clinical isolates based on outer membrane vesicles

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from Escherichia coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (kon) and lower dissociation (koff) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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Improved methods for producing outer membrane vesicles in Gram-negative bacteria

Research in Microbiology ◽

10.1016/j.resmic.2004.02.003 ◽

2004 ◽

Author(s):

T Henry

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Improved Methods

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Spheres of Hope, Packets of Doom: the Good and Bad of Outer Membrane Vesicles in Interspecies and Ecological Dynamics

Journal of Bacteriology ◽

10.1128/jb.00012-17 ◽

2017 ◽

Vol 199 (15) ◽

Cited By ~ 24

Author(s):

Jonathan B. Lynch ◽

Rosanna A. Alegado

Keyword(s):

Antimicrobial Activity ◽

Horizontal Gene Transfer ◽

Outer Membrane ◽

Recent Progress ◽

Membrane Vesicles ◽

Stress Relief ◽

Outer Membrane Vesicles ◽

Gram Negative Bacteria ◽

Gram Negative ◽

By Products

ABSTRACT Outer membrane vesicles (OMVs) are proteoliposome nanoparticles ubiquitously produced by Gram-negative bacteria. Typically bearing a composition similar to those of the outer membrane and periplasm of the cells from which they are derived, OMVs package an array of proteins, lipids, and nucleic acids. Once considered inconsequential by-products of bacterial growth, OMVs have since been demonstrated to mediate cellular stress relief, promote horizontal gene transfer and antimicrobial activity, and elicit metazoan inflammation. Recently, OMVs have gained appreciation as critical moderators of interorganismal dynamics. In this review, we focus on recent progress toward understanding the functions of OMVs with regard to symbiosis and ecological contexts, and we propose potential avenues for future OMV studies.

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Gram-Negative Bacterial Outer Membrane Vesicles Inhibit Growth of Multidrug-Resistant Organisms and Induce Wound-Healing Cytokines

Open Forum Infectious Diseases ◽

10.1093/ofid/ofw172.1790 ◽

2016 ◽

Vol 3 (suppl_1) ◽

Cited By ~ 1

Author(s):

Sarah Baker ◽

Christopher Davitt ◽

Lisa Morici

Keyword(s):

Wound Healing ◽

Outer Membrane ◽

Membrane Vesicles ◽

Multidrug Resistant ◽

Outer Membrane Vesicles ◽

Gram Negative ◽

Multidrug Resistant Organisms ◽

Bacterial Outer Membrane

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Outer Membrane Vesicles (OMVs) of Gram-negative Bacteria: A Perspective Update

Frontiers in Microbiology ◽

10.3389/fmicb.2017.01053 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 143

Author(s):

Arif Tasleem Jan

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Gram Negative Bacteria ◽

Gram Negative

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

Applied and Environmental Microbiology ◽

10.1128/aem.03657-12 ◽

2013 ◽

Vol 79 (6) ◽

pp. 1874-1881 ◽

Cited By ~ 102

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).

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Lipopolysaccharide composition determines the preferred route and entry kinetics of bacterial outer membrane vesicles into host cells

10.1101/084848 ◽

2016 ◽

Author(s):

Eloise J O’Donoghue ◽

Douglas F. Browning ◽

Ewa Bielska ◽

Luke Alderwick ◽

Sara Jabbari ◽

...

Keyword(s):

Chemical Composition ◽

Outer Membrane ◽

Real Time ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gram Negative ◽

Immune Priming ◽

Cargo Delivery ◽

Kinetics Of

SUMMARYOuter membrane vesicles are microvesicles shed by Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle - host cell interactions is limited by a lack of methods to study the kinetics of vesicle entry and cargo delivery to host cells in real-time. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded probe targeted to vesicles. We found that route of vesicular uptake, and thus entry kinetics and efficiency of cargo release, are determined by the chemical composition of the bacterial lipopolysaccharide. The presence of O-antigen facilitates receptor-independent entry, which enhances both rate and efficiency of cargo uptake by host cells. Collectively, our findings highlight the chemical composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections.

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Toll-Like Receptors 2 and 4 Modulate Pulmonary Inflammation and Host Factors Mediated by Outer Membrane Vesicles Derived from Acinetobacter baumannii

Infection and Immunity ◽

10.1128/iai.00243-19 ◽

2019 ◽

Vol 87 (9) ◽

Cited By ~ 5

Author(s):

Chad R. Marion ◽

Jaewook Lee ◽

Lokesh Sharma ◽

Kyong-Su Park ◽

Changjin Lee ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Pulmonary Inflammation ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Toll Like Receptors ◽

Gram Negative ◽

Content Type ◽

Intranasal Introduction

ABSTRACT Pneumonia due to Gram-negative bacteria is associated with high mortality. Acinetobacter baumannii is a Gram-negative bacterium that is associated with hospital-acquired and ventilator-associated pneumonia. Bacteria have been described to release outer membrane vesicles (OMVs) that are capable of mediating systemic inflammation. The mechanism by which A. baumannii OMVs mediate inflammation is not fully defined. We sought to investigate the roles that Toll-like receptors (TLRs) play in A. baumannii OMV-mediated pulmonary inflammation. We isolated OMVs from A. baumannii cultures and intranasally introduced the OMVs into mice. Intranasal introduction of A. baumannii OMVs mediated pulmonary inflammation, which is associated with neutrophil recruitment and weight loss. In addition, A. baumannii OMVs increased the release of several chemokines and cytokines in the mouse lungs. The proinflammatory responses were partially inhibited in TLR2- and TLR4-deficient mice compared to those of wild-type mice. This study highlights the important roles of TLRs in A. baumannii OMV-induced pulmonary inflammation in vivo.

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