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

2020 ◽  
Vol 21 (8) ◽  
pp. 2822 ◽  
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.

scholarly journals Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
E Coli ◽  
Electrophysiological Characterization

Multi-drug-resistance in Gram-negative bacteria is often associated with low permeability of outer membrane. To investigate the role of membrane protein channels in the passage of antibiotics, we extract, purify, reconstitute them into artificial bilayer. Here we demonstrate that using a fusion of native outer membrane vesicles (OMV) facilitates channel reconstitution into bilayer and allows to characterize them in their native environment. Proteins from <i>E. coli</i> (OmpF, OmpC) were overexpressed from the host, and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly only a few channel activities. The asymmetry of the OMV translates after fusion into bilayer with the LPS dominantly present at OMV addition side. Compared to conventional methods, channels fused from OMVs have similar conductance but broader distribution. The further addition of Enrofloxacin yielded higher association but lower dissociation rates attribute to the presence of LPS. We conclude using OMV is a robust approach for functional and structural studies of membrane channels in the native membrane.

scholarly journals Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
E Coli ◽  
Electrophysiological Characterization

Multi-drug-resistance in Gram-negative bacteria is often associated with low permeability of outer membrane. To investigate the role of membrane protein channels in the passage of antibiotics, we extract, purify, reconstitute them into artificial bilayer. Here we demonstrate that using a fusion of native outer membrane vesicles (OMV) facilitates channel reconstitution into bilayer and allows to characterize them in their native environment. Proteins from <i>E. coli</i> (OmpF, OmpC) were overexpressed from the host, and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly only a few channel activities. The asymmetry of the OMV translates after fusion into bilayer with the LPS dominantly present at OMV addition side. Compared to conventional methods, channels fused from OMVs have similar conductance but broader distribution. The further addition of Enrofloxacin yielded higher association but lower dissociation rates attribute to the presence of LPS. We conclude using OMV is a robust approach for functional and structural studies of membrane channels in the native membrane.

scholarly journals Outer membrane vesicles produced by pathogenic strains of Escherichia coli block autophagic flux and exacerbate inflammasome activation

2021 ◽  
Author(s):  
Laure DAVID ◽  
Frederic Taieb ◽  
Marie Penary ◽  
Pierre-Jean Bordignon ◽  
Remy Planes ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Virulence Plasmid ◽  
Autophagic Flux ◽  
Inflammasome Activation ◽  
E Coli ◽  
Intestinal Infections

Escherichia coli (E. coli) strains are responsible for a majority of human extra-intestinal infections, resulting in huge medical, economic and social costs. We had previously shown that HlyF encoded by a large virulence plasmid harbored by pathogenic E. coli is not a hemolysin but a cytoplasmic enzyme leading to the overproduction of outer membrane vesicles (OMVs). Here, we show that these specific OMVs inhibit the autophagic flux by impairing the autophagosome − lysosome fusion, thus preventing the formation of acidic autolysosome and autophagosome clearance. Furthermore, OMVs from E. coli producing HlyF are much more prone to activate the non-canonical inflammasome pathway. Since autophagy and inflammation are crucial in the host′s response to infection especially during sepsis, our findings reveal an unsuspected role of OMVs in the crosstalk between bacteria and their host, highlighting the fact that these extracellular vesicles have exacerbated pathogenic properties compared to OMVs produced by isogenic strains unable to produce a functional HlyF.

scholarly journals Protective role of E. coli outer membrane vesicles against antibiotics

2015 ◽  
Vol 181 ◽  
pp. 1-7 ◽  
Author(s):  
Heramb M. Kulkarni ◽  
R. Nagaraj ◽  
Medicharla V. Jagannadham
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Protective Role ◽  
Outer Membrane Vesicles ◽  
E Coli

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

2017 ◽  
Vol 61 (9) ◽  
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.

scholarly journals The Mutation of Conservative Asp268 Residue in the Peptidoglycan-Associated Domain of the OmpA Protein Affects Multiple Acinetobacter baumannii Virulence Characteristics

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 1972 ◽  
Author(s):  
Jūratė Skerniškytė ◽  
Emilija Karazijaitė ◽  
Julien Deschamps ◽  
Renatas Krasauskas ◽  
Romain Briandet ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Protein A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Clinical Strain ◽  
Infection Model ◽  
Terminal Domain ◽  
Ompa Protein

Acinetobacter baumannii is a nosocomial human pathogen of increasing concern due to its multidrug resistance profile. The outer membrane protein A (OmpA) is an abundant bacterial cell surface component involved in A. baumannii pathogenesis. It has been shown that the C-terminal domain of OmpA is located in the periplasm and non-covalently associates with the peptidoglycan layer via two conserved amino acids, thereby anchoring OmpA to the cell wall. Here, we investigated the role of one of the respective residues, D268 in OmpA of A. baumannii clinical strain Ab169, on its virulence characteristics by complementing the ΔompA mutant with the plasmid-borne ompAD268A allele. We show that while restoring the impaired biofilm formation of the ΔompA strain, the Ab169ompAD268A mutant tended to form bacterial filaments, indicating the abnormalities in cell division. Moreover, the Ab169 OmpA D268-mediated association to peptidoglycan was required for the manifestation of twitching motility, desiccation resistance, serum-induced killing, adhesion to epithelial cells and virulence in a nematode infection model, although it was dispensable for the uptake of β-lactam antibiotics by outer membrane vesicles. Overall, the results of this study demonstrate that the OmpA C-terminal domain-mediated association to peptidoglycan is critical for a number of virulent properties displayed by A. baumannii outside and within the host.

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

2006 ◽  
Vol 189 (5) ◽  
pp. 1627-1632 ◽  
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.

scholarly journals Bacterial glycoengineering as a biosynthetic route to customized glycomolecules

2017 ◽  
Author(s):  
Laura E. Yates ◽  
Dominic C. Mills ◽  
Matthew P. DeLisa
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Recombinant Expression ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Living Systems ◽  
Conjugate Vaccines ◽  
Biosynthetic Machinery ◽  
Opening Up

AbstractBacteria have garnered increased interest in recent years as a platform for the biosynthesis of a variety of glycomolecules such as soluble oligosaccharides, surface-exposed carbohydrates and glycoproteins. The ability to flexibly engineer commonly used laboratory species such asEscherichia colito efficiently synthesize non-native sugar structures by recombinant expression of enzymes from various carbohydrate biosynthesis pathways has allowed for the facile generation of important products such as conjugate vaccines, glycosylated outer membrane vesicles, and a variety of other research reagents for studying and understanding the role of glycans in living systems. This chapter highlights some of the key discoveries and technologies for equipping bacteria with the requisite biosynthetic machinery to generate such products. As the bacterial glyco-toolbox continues to grow, these technologies are expected to expand the range of glycomolecules produced recombinantly in bacterial systems, thereby opening up this platform to an even larger number of applications.

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