A trivalent Apx-fusion protein delivered by E. coli outer membrane vesicles induce protection against Actinobacillus pleuropneumoniae of serotype 1 and 7 challenge in a murine model

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.

Download Full-text

Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00937-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 19

Author(s):

Andreas Bauwens ◽

Lisa Kunsmann ◽

Helge Karch ◽

Alexander Mellmann ◽

Martina Bielaszewska

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Shiga Toxin ◽

Membrane Vesicles ◽

Polymyxin B ◽

Outer Membrane Vesicles ◽

Enterohemorrhagic Escherichia Coli ◽

Content Type ◽

E Coli ◽

Major Virulence Factor

ABSTRACT Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli. Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

Download Full-text

Repression of the Inner Membrane Lipoprotein NlpA by Rns in Enterotoxigenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01714-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1627-1632 ◽

Cited By ~ 23

Author(s):

Maria D. Bodero ◽

M. Carolina Pilonieta ◽

George P. Munson

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Enterotoxigenic Escherichia Coli ◽

Start Codon ◽

Inner Membrane ◽

E Coli ◽

Inner Membrane Protein

ABSTRACT The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions −152 and −195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.

Download Full-text

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

Access Microbiology ◽

10.1099/acmi.ac2020.po0596 ◽

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.

Download Full-text

The Importance of Porins and β-Lactamase in Outer Membrane Vesicles on the Hydrolysis of β-Lactam Antibiotics

International Journal of Molecular Sciences ◽

10.3390/ijms21082822 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2822 ◽

Cited By ~ 2

Author(s):

Si Won Kim ◽

Jung Seok Lee ◽

Seong Bin Park ◽

Ae Rin Lee ◽

Jae Wook Jung ◽

...

Keyword(s):

Outer Membrane ◽

Defense Mechanisms ◽

Antibacterial Properties ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

E Coli ◽

Lactam Antibiotic ◽

Hydrolysis Of ◽

Degradation Ability

Gram-negative bacteria have an outer membrane inhibiting the entry of antibiotics. Porins, found within the outer membrane, are involved in regulating the permeability of β-lactam antibiotics. β-lactamases are enzymes that are able to inactivate the antibacterial properties of β-lactam antibiotics. Interestingly, porins and β-lactamase are found in outer membrane vesicles (OMVs) of β-lactam-resistant Escherichia coli and may be involved in the survival of susceptible strains of E. coli in the presence of antibiotics, through the hydrolysis of the β-lactam antibiotic. In this study, OMVs isolated from β-lactam-resistant E. coli and from mutants, lacking porin or β-lactamase, were evaluated to establish if the porins or β-lactamase in OMVs were involved in the degradation of β-lactam antibiotics. OMVs isolated from E. coli deficient in β-lactamase did not show any degradation ability against β-lactam antibiotics, while OMVs lacking OmpC or OmpF showed significantly lower levels of hydrolyzing activity than OMVs from parent E. coli. These data reveal an important role of OMVs in bacterial defense mechanisms demonstrating that the OmpC and OmpF proteins allow permeation of β-lactam antibiotics into the lumen of OMVs, and antibiotics that enter the OMVs can be degraded by β-lactamase.

Download Full-text

Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

Applied and Environmental Microbiology ◽

10.1128/aem.01011-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 5900-5906 ◽

Cited By ~ 9

Author(s):

Yoshihiro Ojima ◽

Minh Hong Nguyen ◽

Reiki Yajima ◽

Masahito Taya

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Sodium Dodecyl ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Laser Scanning Microscopy ◽

Content Type ◽

E Coli ◽

Enhanced Production ◽

K 12

ABSTRACTMicrobial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation ofEscherichia colicells by overexpression of thebcsBgene was investigated. The flocculation induced by overexpression ofbcsBwas consistent among the variousE. colistrains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeledE. colicells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) inE. coliflocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore,bcsB-inducedE. coliflocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbAand ΔdsbBstrains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production ofE. colicells.

Download Full-text

In-vitro Cytotoxic Activity of E. coli Outer Membrane Vesicles (OMVs) Against Breast Cancer (MCF-7) Cell Line

Medico-Legal Update ◽

10.37506/mlu.v21i1.2355 ◽

2021 ◽

Keyword(s):

Breast Cancer ◽

Cytotoxic Activity ◽

Outer Membrane ◽

Cell Line ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

E Coli ◽

Mcf 7

Download Full-text

Naturally Derived Outer Membrane Vesicles confer Immunity to Salmonella typhimurium in a Murine Model

Open Forum Infectious Diseases ◽

10.1093/ofid/ofw172.1739 ◽

2016 ◽

Vol 3 (suppl_1) ◽

Author(s):

Frances Lee ◽

Christopher Davitt ◽

Jonathan Kurtz ◽

James Mclachlan ◽

Lisa Morici

Keyword(s):

Salmonella Typhimurium ◽

Outer Membrane ◽

Murine Model ◽

Membrane Vesicles ◽

Outer Membrane Vesicles

Download Full-text

Outer membrane vesicles containing OmpA induce mitochondrial fragmentation to promote pathogenesis of Acinetobacter baumannii

Scientific Reports ◽

10.1038/s41598-020-79966-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Varnesh Tiku ◽

Eric M. Kofoed ◽

Donghong Yan ◽

Jing Kang ◽

Min Xu ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Mammalian Cells ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Mouse Lung ◽

Infection Model ◽

Dependent Manner ◽

Mitochondrial Fragmentation ◽

E Coli

AbstractAcinetobacter baumannii is a highly antibiotic resistant Gram-negative bacterium that causes life-threatening infections in humans with a very high mortality rate. A. baumannii is an extracellular pathogen with poorly understood virulence mechanisms. Here we report that A. baumannii employs the release of outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpAAb) to promote bacterial pathogenesis and dissemination. OMVs containing OmpAAb are taken up by mammalian cells where they activate the host GTPase dynamin-related protein 1 (DRP1). OmpAAb mediated activation of DRP1 enhances its accumulation on mitochondria that causes mitochondrial fragmentation, elevation in reactive oxygen species (ROS) production and cell death. Loss of DRP1 rescues these phenotypes. Our data show that OmpAAb is sufficient to induce mitochondrial fragmentation and cytotoxicity since its expression in E. coli transfers its pathogenic properties to E. coli. A. baumannii infection in mice also induces mitochondrial damage in alveolar macrophages in an OmpAAb dependent manner. We finally show that OmpAAb is also required for systemic dissemination in the mouse lung infection model. In this study we uncover the mechanism of OmpAAb as a virulence factor in A. baumannii infections and further establish the host cell factor required for its pathogenic effects.

Download Full-text

Surface Modification of E. coli Outer Membrane Vesicles with Glycosylphosphatidylinositol-Anchored Proteins: Generating Pro/Eukaryote Chimera Constructs

Membranes ◽

10.3390/membranes11060428 ◽

2021 ◽

Vol 11 (6) ◽

pp. 428

Author(s):

Marianne Zaruba ◽

Lena Roschitz ◽

Haider Sami ◽

Manfred Ogris ◽

Wilhelm Gerner ◽

...

Keyword(s):

Surface Modification ◽

Outer Membrane ◽

Extracellular Vesicles ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Functional Surface ◽

Vaccine Strategy ◽

Fluorescent Marker ◽

E Coli ◽

Modular Platform

Extracellular vesicles produced by different types of cells have recently attracted great attention, not only for their role in physiology and pathology, but also because of the emerging applications in gene therapy, vaccine production and diagnostics. Less well known than their eukaryotic counterpart, also bacteria produce extracellular vesicles, in the case of the Gram-negative E. coli the main species is termed outer membrane vesicles (OMVs). In this study, we show for the first time the functional surface modification of E. coli OMVs with glycosylphosphatidylinositol (GPI)-anchored protein, exploiting a process variably described as molecular painting or protein engineering in eukaryotic membranes, whereby the lipid part of the GPI anchor inserts in cell membranes. By transferring the process to bacterial vesicles, we can generate a hybrid of perfectly eukaryotic proteins (in terms of folding and post-translational modifications) on a prokaryotic platform. We could demonstrate that two different GPI proteins can be displayed on the same OMV. In addition to fluorescent marker proteins, cytokines, growth factors and antigens canb be potentially transferred, generating a versatile modular platform for a novel vaccine strategy.

Download Full-text