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

2019 ◽  
Vol 87 (9) ◽  
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.

scholarly journals Horizontal Transfer of the OXA-24 Carbapenemase Gene via Outer Membrane Vesicles: a New Mechanism of Dissemination of Carbapenem Resistance Genes in Acinetobacter baumannii

2011 ◽  
Vol 55 (7) ◽  
pp. 3084-3090 ◽  
Author(s):  
Carlos Rumbo ◽  
Esteban Fernández-Moreira ◽  
María Merino ◽  
Margarita Poza ◽  
Jose Antonio Mendez ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Resistance Genes ◽  
Carbapenem Resistance ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Content Type ◽  
Class D ◽  
Carbapenemase Gene

ABSTRACTThe resistance ofAcinetobacter baumanniistrains to carbapenems is a worrying problem in hospital settings. The main mechanism of carbapenem resistance is the expression of β-lactamases (metalloenzymes or class D enzymes). The mechanisms of the dissemination of these genes amongA. baumanniistrains are not fully understood. In this study we used two carbapenem-resistant clinical strains ofA. baumannii(AbH12O-A2 and AbH12O-CU3) expressing the plasmid-borneblaOXA-24gene (plasmids pMMA2 and pMMCU3, respectively) to demonstrate thatA. baumanniireleases outer membrane vesicles (OMVs) duringin vitrogrowth. The use of hybridization studies enabled us to show that these OMVs harbored theblaOXA-24gene. The incubation of these OMVs with the carbapenem-susceptibleA. baumanniiATCC 17978 host strain yielded full resistance to carbapenems. The presence of the original plasmids harboring theblaOXA-24gene was detected in strain ATCC 17978 after the transformation of OMVs. New OMVs harboringblaOXA-24were released byA. baumanniiATCC 17978 after it was transformed with the original OMV-mediated plasmids, indicating the universality of the process. We present the first experimental evidence that clinical isolates ofA. baumanniimay release OMVs as a mechanism of horizontal gene transfer whereby carbapenem resistance genes are delivered to surroundingA. baumanniibacterial isolates.

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 Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Ryan Finethy ◽  
Sarah Luoma ◽  
Nichole Orench-Rivera ◽  
Eric M. Feeley ◽  
Arun K. Haldar ◽  
...  
Keyword(s):  
Cell Wall ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Binding Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Bacterial Cell Wall ◽  
Cell Wall Component ◽  
Gram Negative

ABSTRACT The Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as “free” LPS aggregates or actively secreted by live bacteria as a component of outer membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli. In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1β (IL-1β) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types. IMPORTANCE The bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of outer membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by bacterial infections. IMPORTANCE The bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of outer membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by bacterial infections.

scholarly journals Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Franz G. Zingl ◽  
Himadri B. Thapa ◽  
Martina Scharf ◽  
Paul Kohl ◽  
Anna M. Müller ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Intestinal Cells ◽  
Gram Negative ◽  
Content Type ◽  
Outer Membrane Porins

ABSTRACT Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

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 ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

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 <i>Escherichia coli</i>, 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 (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) 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.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals Efficacy of Humanized Cefiderocol Exposures over 72 Hours against a Diverse Group of Gram-Negative Isolates in the Neutropenic Murine Thigh Infection Model

2018 ◽  
Vol 63 (2) ◽  
pp. e01040-18 ◽  
Author(s):  
Sean M. Stainton ◽  
Marguerite L. Monogue ◽  
Masakatsu Tsuji ◽  
Yoshinori Yamano ◽  
Roger Echols ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Infection Model ◽  
Diverse Group ◽  
Gram Negative ◽  
Content Type ◽  
Adaptive Resistance

ABSTRACT Herein, we evaluated sustainability of humanized exposures of cefiderocol in vivo over 72 h against pathogens with cefiderocol MICs of 0.5 to 16 μg/ml in the neutropenic murine thigh model. In Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae displaying MICs of 0.5 to 8 μg/ml (n = 11), sustained kill was observed at 72 h among 9 isolates. Postexposure MICs revealed a single 2-dilution increase in one animal compared with controls (1/54 samples, 1.8%) at 72 h. Adaptive resistance during therapy was not observed.

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