scholarly journals Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans

2021 ◽  
Vol 118 (30) ◽  
pp. e2026246118
Author(s):  
Tzu-Han Lo ◽  
Hung-Lin Chen ◽  
Cheng-I Yao ◽  
I-Chun Weng ◽  
Chi-Shan Li ◽  
...  
Keyword(s):  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Therapeutic Interventions ◽  
Inflammasome Activation ◽  
Promoting Effect ◽  
Galectin 3 ◽  
Self Association ◽  
Intracellular Binding ◽  
Association Property

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, β-galactoside–binding proteins, bind to various gram-negative bacterial LPS, which display β-galactoside–containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1β production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis—a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

scholarly journals Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis

2020 ◽  
Vol 11 ◽  
Author(s):  
Maia L. Elizagaray ◽  
Marco Túlio R. Gomes ◽  
Erika S. Guimaraes ◽  
Martín Rumbo ◽  
Daniela F. Hozbor ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Inflammasome Activation

Heterologous Expression of 3-O-Deacylase in Acinetobacter baumannii Modulates the Endotoxicity of Lipopolysaccharide

2015 ◽  
Vol 25 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Farzad Badmasti ◽  
Fereshteh Shahcheraghi ◽  
Seyed Davar Siadat ◽  
Saeid Bouzari ◽  
Soheila Ajdary ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Recombinant Dna ◽  
Inflammatory Responses ◽  
Acyl Chain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Negative Ion ◽  
Recombinant Dna Technology ◽  
Whole Cells

The lipopolysaccharide (LPS) of <i>Acinetobacter baumannii</i> is a potent stimulator of proinflammatory cytokines, such as interleukin-6 (IL-6). The 3-O-deacylase (PagL)-modifying enzyme that removes the 3-O-linked acyl chain from the disaccharide backbone of lipid A provides the opportunity to develop a new therapeutic compound that could reduce detrimental inflammatory responses. The plasmid pMMB66EH-PagL obtained by recombinant DNA technology was electroporated into <i>A. baumannii</i> ATCC 19606. Compared with wild-type LPS, outer membrane vesicles and inactivated whole cells of engineered bacteria had a statistically significant decreased ability to produce IL-6. Structural analysis of lipid A by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the profile of lipid A fractions under PagL expression was changed. Taken together, our data showed that recombinant penta-acylated lipid A had less immunoreactivity and that the tetra-acylated version of lipid A with TLR4 antagonist activity decreased the induction of IL-6 production in the murine macrophage cell line J774 A.1.

scholarly journals LPS Remodeling Triggers Formation of Outer Membrane Vesicles inSalmonella

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Wael Elhenawy ◽  
Michael Bording-Jorgensen ◽  
Ezequiel Valguarnera ◽  
M. Florencia Haurat ◽  
Eytan Wine ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mass Spectrometry Analysis ◽  
Membrane Remodeling ◽  
Macrophage Infection ◽  
Spectrometry Analysis

ABSTRACTOuter membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employingSalmonella entericaserovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in apagLmutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane.IMPORTANCEThe role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis inS. Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. AnS. Typhimurium ΔpagLstrain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are produced by bacteria is important to advance our understanding of the host-pathogen interactions.

scholarly journals Lipopolysaccharide Recognition in the Crossroads of TLR4 and Caspase-4/11 Mediated Inflammatory Pathways

2020 ◽  
Vol 11 ◽  
Author(s):  
Alla Zamyatina ◽  
Holger Heine
Keyword(s):  
Nlrp3 Inflammasome ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Structural Features ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Structural Basis ◽  
Intracellular Bacteria ◽  
Inflammatory Caspases

The innate immune response to lipopolysaccharide is essential for host defense against Gram-negative bacteria. In response to bacterial infection, the TLR4/MD-2 complex that is expressed on the surface of macrophages, monocytes, dendritic, and epithelial cells senses picomolar concentrations of endotoxic LPS and triggers the production of various pro-inflammatory mediators. In addition, LPS from extracellular bacteria which is either endocytosed or transfected into the cytosol of host cells or cytosolic LPS produced by intracellular bacteria is recognized by cytosolic proteases caspase-4/11 and hosts guanylate binding proteins that are involved in the assembly and activation of the NLRP3 inflammasome. All these events result in the initiation of pro-inflammatory signaling cascades directed at bacterial eradication. However, TLR4-mediated signaling and caspase-4/11-induced pyroptosis are largely involved in the pathogenesis of chronic and acute inflammation. Both extra- and intracellular LPS receptors—TLR4/MD-2 complex and caspase-4/11, respectively—are able to directly bind the lipid A motif of LPS. Whereas the structural basis of lipid A recognition by the TLR4 complex is profoundly studied and well understood, the atomic mechanism of LPS/lipid A interaction with caspase-4/11 is largely unknown. Here we describe the LPS-induced TLR4 and caspase-4/11 mediated signaling pathways and their cross-talk and scrutinize specific structural features of the lipid A motif of diverse LPS variants that have been reported to activate caspase-4/11 or to induce caspase-4/11 mediated activation of NLRP3 inflammasome (either upon transfection of LPS in vitro or upon infection of cell cultures with intracellular bacteria or by LPS as a component of the outer membrane vesicles). Generally, inflammatory caspases show rather similar structural requirements as the TLR4/MD-2 complex, so that a “basic” hexaacylated bisphosphorylated lipid A architecture is sufficient for activation. However, caspase-4/11 can sense and respond to much broader variety of lipid A variants compared to the very “narrow” specificity of TLR4/MD-2 complex as far as the number and the length of lipid chains attached at the diglucosamine backbone of lipid A is concerned. Besides, modification of the lipid A phosphate groups with positively charged appendages such as phosphoethanolamine or aminoarabinose could be essential for the interaction of lipid A/LPS with inflammatory caspases and related proteins.

scholarly journals Hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing

2018 ◽  
Author(s):  
Shouwen Chen ◽  
Dahai Yang ◽  
Ying Wen ◽  
Zhiwei Jiang ◽  
Lingzhi Zhang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bacterial Colonization ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Bacterial Toxin ◽  
Critical Event ◽  
Inflammasome Activation ◽  
Immune Recognition ◽  
Bacterial Outer Membrane

AbstractInflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains uncovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cells (IECs). Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion, gut inflammation, and enterocyte pyroptosis in orally-infected mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.SignificanceSensing of lipopolysaccharide (LPS) in the cytosol triggers non-canonical inflammasome-mediated innate responses. Recent work revealed that bacterial outer membrane vesicles (OMVs) enables LPS to access the cytosol for extracellular bacteria. However, since intracellular OMVs are generally constrained in endosomes, how OMV-derived LPS gain access to the cytosol remains unknown. Here, we reported that hemolysin largely bound with OMVs and entered cells through dynamin-dependent endocytosis. Intracellular hemolysin significantly impaired OMVs-constrained vacuole integrity and increased OMV-derived LPS exposure to the cytosolic sensor, which promoted non-canonical inflammasome activation and restricted bacterial gut infections. This work reveals the role of hemolysin in promoting non-canonical inflammasome activation and alerting host immune recognition, which provides insights into the more sophisticated biological functions of hemolysin upon infection.

scholarly journals LOS oligosaccharide modification enhances dendritic cell responses to meningococcal native outer membrane vesicles expressing a non‐toxic lipid A

2013 ◽  
Vol 16 (4) ◽  
pp. 519-534 ◽  
Author(s):  
Hannah E. Jones ◽  
Alastair Copland ◽  
Hendrik Jan Hamstra ◽  
Jonathan Cohen ◽  
Jeremy Brown ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Outer Membrane ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Cell Responses

Outer membrane vesicles obtained from Bordetella pertussis Tohama expressing the lipid A deacylase PagL as a novel acellular vaccine candidate

Vaccine ◽  
2011 ◽  
Vol 29 (8) ◽  
pp. 1649-1656 ◽  
Author(s):  
Cristian J.A. Asensio ◽  
María Emilia Gaillard ◽  
Griselda Moreno ◽  
Daniela Bottero ◽  
Eugenia Zurita ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Vaccine Candidate ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Acellular Vaccine

scholarly journals The Attenuated Protective Effect of Outer Membrane Vesicles Produced by a mcr-1 Positive Strain on Colistin Sensitive Escherichia coli

2021 ◽  
Vol 11 ◽  
Author(s):  
Xue Li ◽  
Lang Sun ◽  
Congran Li ◽  
Xinyi Yang ◽  
Xiukun Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protective Effect ◽  
Outer Membrane ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Bacterial Cells ◽  
Colistin Resistance ◽  
E Coli ◽  
Positive Strain

Resistance to colistin, especially mobilized colistin resistance (mcr), is a serious threat to public health since it may catalyze a return of the “pre-antibiotic era”. Outer membrane vesicles (OMVs) play a role in antibiotic resistance in various ways. Currently, how OMVs participate in mcr-1-mediated colistin resistance has not been established. In this study, we showed that both OMVs from the mcr-1 negative and positive Escherichia coli (E. coli) strains conferred dose-dependent protection from colistin. However, OMVs from the mcr-1 positive strain conferred attenuated protection when compared to the OMVs of a mcr-1 negative strain at the same concentration. The attenuated protective effect of OMVs was related to the reduced ability to absorb colistin from the environment, thus promoting the killing of colistin sensitive E. coli strains. Lipid A modified with phosphoethanolamine was presented in the OMVs of the mcr-1 positive E. coli strain and resulted in decreased affinity to colistin and less protection. Meanwhile, E. coli strain carrying the mcr-1 gene packed more unmodified lipid A in OMVs and kept more phosphoethanolamine modified lipid A in the bacterial cells. Our study provides a first glimpse of the role of OMVs in mcr-1 -mediated colistin resistance.

scholarly journals Outer membrane vesicles harboring modified lipid A moiety augment the efficacy of an influenza vaccine exhibiting reduced endotoxicity in a mouse model

Vaccine ◽  
2017 ◽  
Vol 35 (4) ◽  
pp. 586-595 ◽  
Author(s):  
Tae-Young Lee ◽  
Chang-Ung Kim ◽  
Eun-Hye Bae ◽  
Sang-Hwan Seo ◽  
Dae Gwin Jeong ◽  
...  
Keyword(s):  
Mouse Model ◽  
Influenza Vaccine ◽  
Outer Membrane ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles

scholarly journals LptO (PG0027) Is Required for Lipid A 1-Phosphatase Activity in Porphyromonas gingivalis W50

2017 ◽  
Vol 199 (11) ◽  
Author(s):  
Minnie Rangarajan ◽  
Joseph Aduse-Opoku ◽  
Ahmed Hashim ◽  
Graham McPhail ◽  
Zofia Luklinska ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Phosphatase Activity ◽  
Porphyromonas Gingivalis ◽  
Virulence Factors ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Content Type ◽  
Maldi Tof ◽  
Mutant Strains

ABSTRACT Porphyromonas gingivalis produces outer membrane vesicles (OMVs) rich in virulence factors, including cysteine proteases and A-LPS, one of the two lipopolysaccharides (LPSs) produced by this organism. Previous studies had suggested that A-LPS and PG0027, an outer membrane (OM) protein, may be involved in OMV formation. Their roles in this process were examined by using W50 parent and the ΔPG0027 mutant strains. Inactivation of PG0027 caused a reduction in the yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and the ΔPG0027 mutant strains were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Lipid A from W50 cells contained bis-P-pentaacyl, mono-P-pentaacyl, mono-P-tetraacyl, non-P-pentaacyl, and non-P-tetraacyl species, whereas lipid A from ΔPG0027 mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl (m/z 1,688) and mono-P-tetraacyl (m/z 1,448) lipid A from ΔPG0027 showed that both contained lipid A 1-phosphate, suggesting that the ΔPG0027 mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the ΔPG0027 mutant strains were similar, whereas the phosphatase activity in the periplasm of the ΔPG0027 mutant was lower than that in W50, supporting a role for PG0027 in lipid A dephosphorylation. W50 OMVs were enriched in A-LPS, and its lipid A did not contain nonphosphorylated species, whereas lipid A from the ΔPG0027 mutant (OMVs and cells) contained similar species. Thus, OMVs in P. gingivalis are apparently formed in regions of the OM enriched in A-LPS devoid of nonphosphorylated lipid A. Conversely, dephosphorylation of lipid A through a PG0027-dependent process is required for optimal formation of OMVs. Hence, the relative proportions of nonphosphorylated and phosphorylated lipid A appear to be crucial for OMV formation in this organism. IMPORTANCE Gram-negative bacteria produce outer membrane vesicles (OMVs) by “blebbing” of the outer membrane (OM). OMVs can be used offensively as delivery systems for virulence factors and defensively to aid in the colonization of a host and in the survival of the bacterium in hostile environments. Earlier studies using the oral anaerobe Porphyromonas gingivalis as a model organism to study the mechanism of OMV formation suggested that the OM protein PG0027 and one of the two lipopolysaccharides (LPSs) synthesized by this organism, namely, A-LPS, played important roles in OMV formation. We suggest a novel mechanism of OMV formation in P. gingivalis involving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs.

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