scholarly journals Position-specific secondary acylation determines detection of lipid A by murine TLR4 and caspase-11

2021 ◽  
Author(s):  
Erin M Harberts ◽  
Daniel Grubaugh ◽  
Daniel C. Akuma ◽  
Sunny Shin ◽  
Robert K Ernst ◽  
...  
Keyword(s):  
Lipid A ◽  
Inflammatory Responses ◽  
Chemical Space ◽  
Bacterial Species ◽  
Direct Analysis ◽  
Structural Features ◽  
Gram Negative ◽  
Acyl Chains ◽  
Immune Sensing ◽  
Bacterial Lipid

Immune sensing of the Gram-negative bacterial membrane glycolipid lipopolysaccharide (LPS) is both a critical component of host defense against Gram-negative bacterial infection, and a contributor to hyper-inflammatory response, leading to sepsis and death. Innate immune activation by LPS is due to the lipid A moiety, an acylated di-glucosamine molecule that can activate inflammatory responses via the extracellular sensor TLR4/MD2 or the cytosolic sensor caspase-11 (Casp11). The number and length of acyl chains present on bacterial lipid A structures vary across bacterial species and strains, which affects the magnitude of TLR4 and Casp11 activation. TLR4 and Casp11 are thought to respond similarly to various lipid A structures, as tetra-acylated lipid A structures do not activate either sensor, whereas hexa-acylated structures activate both sensors. However, direct analysis of extracellular and cytosolic responses to the same sources and preparations of LPS/lipid A structures have been limited, and the precise features of lipid A that determine the differential activation of each receptor remain poorly defined. To address this question, we used rationally engineered lipid A isolated from a series of bacterial acyl-transferase mutants that produce novel, structurally defined molecules. Intriguingly, we find that the location of specific secondary acyl chains on lipid A resulted in differential recognition by TLR4- or Casp11, providing new insight into the structural features of lipid A required to activate either TLR4- or Casp11. Our findings indicate that TLR4 and Casp11 sense non-overlapping areas of lipid A chemical space, thereby constraining the ability of Gram-negative pathogens to evade innate immunity.

scholarly journals In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A

2018 ◽  
Vol 10 (464) ◽  
pp. eaal0033 ◽  
Author(s):  
Ahsan R. Akram ◽  
Sunay V. Chankeshwara ◽  
Emma Scholefield ◽  
Tashfeen Aslam ◽  
Neil McDonald ◽  
...  
Keyword(s):  
Lipid A ◽  
Respiratory Infections ◽  
Bacterial Species ◽  
Water Soluble ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Human Lungs ◽  
Fluorescent Peptide ◽  
Bacterial Lipid

Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.

scholarly journals Human TLR4 and noncanonical inflammasome differ in their ability to respond to distinct lipid A variants

2021 ◽  
Author(s):  
Jasmine Alexander-Floyd ◽  
Antonia R. Bass ◽  
Erin M. Harberts ◽  
Daniel Grubaugh ◽  
Joseph D. Buxbaum ◽  
...  
Keyword(s):  
Lipid A ◽  
Inflammatory Responses ◽  
Bacterial Species ◽  
Acyl Chain ◽  
Innate Immune ◽  
Core Oligosaccharide ◽  
Phosphorylation State ◽  
Tlr4 Activation ◽  
Bacterial Lipid ◽  
Immune Detection

Detection of Gram-negative bacterial lipid A by the extracellular sensor, MD-2/TLR4 or the intracellular inflammasome sensors, CASP4 and CASP5, induces robust inflammatory responses. The chemical structure of lipid A, specifically the phosphorylation and acylation state, varies across and within bacterial species, potentially allowing pathogens to evade or suppress host immunity. Currently, it is not clear how distinct alterations in the phosphorylation or acylation state of lipid A affect both human TLR4 and CASP4/5 activation. Using a panel of engineered lipooligosaccharides (LOS) derived from Yersinia pestis with defined lipid A structures that vary in their acylation or phosphorylation state, we identified that differences in phosphorylation state did not affect TLR4 or CASP4/5 activation. However, the acylation state differentially impacted TLR4 and CASP4/5 activation. Specifically, all of the examined tetra-, penta-, and hexa-acylated LOS variants activated CASP4/5-dependent responses, whereas TLR4 responded to penta- and hexa-acylated LOS but did not respond to tetra-acylated LOS or penta-acylated LOS lacking the secondary acyl chain at the 3' position. As expected, lipid A alone was sufficient for TLR4 activation; however, human macrophages required both lipid A and the core oligosaccharide to mount a robust CASP4/5 inflammasome response. Our findings show that human TLR4 and CASP4/5 detect both shared and non-overlapping LOS/lipid A structures, which enables the innate immune system to recognize a wider range of bacterial LOS/lipid A, thereby constraining the ability of pathogens to evade innate immune detection.

scholarly journals Molecular Characterization of Lipopolysaccharide Binding to Humanα-1-Acid Glycoprotein

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Johnny X. Huang ◽  
Mohammad A. K. Azad ◽  
Elizabeth Yuriev ◽  
Mark A. Baker ◽  
Roger L. Nation ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Lipid A ◽  
Inflammatory Responses ◽  
Klebsiella Pneumonia ◽  
Gram Negative Bacteria ◽  
Surface Plasma Resonance ◽  
Core Oligosaccharide ◽  
Gram Negative ◽  
Binding Characteristics ◽  
Bacterial Lipid

The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria:Escherichia coli,Salmonella typhimurium,Klebsiella pneumonia,Pseudomonas aeruginosa, andSerratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide andO-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A,Ra,Rd, andRerough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of theO-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria.

scholarly journals Identification of LpxL, a Late Acyltransferase of Francisella tularensis

2007 ◽  
Vol 75 (11) ◽  
pp. 5518-5531 ◽  
Author(s):  
Molly K. McLendon ◽  
Birgit Schilling ◽  
Jason R. Hunt ◽  
Michael A. Apicella ◽  
Bradford W. Gibson
Keyword(s):  
Francisella Tularensis ◽  
Lipid A ◽  
Structural Features ◽  
Spectrometric Analysis ◽  
Temperature Sensitive ◽  
Gram Negative Bacteria ◽  
Dependent Manner ◽  
Gram Negative ◽  
Acyl Chains

ABSTRACT Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria, and the lipid A region of LPS mediates stimulation of the immune system in a structure-dependent manner. Unlike the LPS of many other gram-negative bacteria, the LPS of Francisella tularensis isolated from in vitro cultures is not proinflammatory. This observed lack of proinflammatory prowess may reflect structural features of the lipid A, such as the number and length of the acyl chains and the single-phosphate group. To better understand this phenotype, we have begun to elucidate LPS biosynthesis in F. tularensis. We present complementation, mutational, and chemical data demonstrating that F. tularensis FTT0232c encodes a functional late acyltransferase enzyme with specificity similar to that of the Escherichia coli LpxL ortholog. Expression of this late acyltransferase complemented the temperature-sensitive and hypoacylated lipid A phenotypes of an E. coli lpxL mutant, expression of FTT0232c is increased during intracellular growth relative to that during in vitro growth, and finally, LPS obtained from a mutant of F. tularensis lacking FTT0232c showed an abundant triacyl lipid A species after mass spectrometric analysis, consistent with the loss of an LpxL late acyltransferase.

scholarly journals The Structure of the Lipid A of Gram-Negative Cold-Adapted Bacteria Isolated from Antarctic Environments

Marine Drugs ◽  
2020 ◽  
Vol 18 (12) ◽  
pp. 592
Author(s):  
Flaviana Di Lorenzo ◽  
Francesca Crisafi ◽  
Violetta La Cono ◽  
Michail M. Yakimov ◽  
Antonio Molinaro ◽  
...  
Keyword(s):  
Lipid A ◽  
Compositional Analysis ◽  
Survival Strategies ◽  
Gram Negative ◽  
Cold Adapted ◽  
Structural Modifications ◽  
Terra Nova Bay ◽  
Drug Synthesis ◽  
Acyl Chains ◽  
Main Component

Gram-negative Antarctic bacteria adopt survival strategies to live and proliferate in an extremely cold environment. Unusual chemical modifications of the lipopolysaccharide (LPS) and the main component of their outer membrane are among the tricks adopted to allow the maintenance of an optimum membrane fluidity even at particularly low temperatures. In particular, the LPS’ glycolipid moiety, the lipid A, typically undergoes several structural modifications comprising desaturation of the acyl chains, reduction in their length and increase in their branching. The investigation of the structure of the lipid A from cold-adapted bacteria is, therefore, crucial to understand the mechanisms underlying the cold adaptation phenomenon. Here we describe the structural elucidation of the highly heterogenous lipid A from three psychrophiles isolated from Terra Nova Bay, Antarctica. All the lipid A structures have been determined by merging data that was attained from the compositional analysis with information from a matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS) and MS2 investigation. As lipid A is also involved in a structure-dependent elicitation of innate immune response in mammals, the structural characterization of lipid A from such extremophile bacteria is also of great interest from the perspective of drug synthesis and development inspired by natural sources.

scholarly journals Global and Targeted Lipid Analysis ofGemmata obscuriglobusReveals the Presence of Lipopolysaccharide, a Signature of the Classical Gram-Negative Outer Membrane

2015 ◽  
Vol 198 (2) ◽  
pp. 221-236 ◽  
Author(s):  
Rajendra Mahat ◽  
Corrine Seebart ◽  
Franco Basile ◽  
Naomi L. Ward
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Direct Evidence ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Gas Chromatography Mass Spectrometry ◽  
Differential Staining ◽  
Gram Negative ◽  
Negative Cell ◽  
Cell Plan

ABSTRACTPlanctomycete bacteria possess many unusual cellular properties, contributing to a cell plan long considered to be unique among the bacteria. However, data from recent studies are more consistent with a modified Gram-negative cell plan. A key feature of the Gram-negative plan is the presence of an outer membrane (OM), for which lipopolysaccharide (LPS) is a signature molecule. Despite genomic evidence for an OM in planctomycetes, no biochemical verification has been reported. We attempted to detect and characterize LPS in the planctomyceteGemmata obscuriglobus. We obtained direct evidence for LPS and lipid A using electrophoresis and differential staining. Gas chromatography-mass spectrometry (GC-MS) compositional analysis of LPS extracts identified eight different 3-hydroxy fatty acids (3-HOFAs), 2-keto 3-deoxy-d-manno-octulosonic acid (Kdo), glucosamine, and hexose and heptose sugars, a chemical profile unique to Gram-negative LPS. Combined with molecular/structural information collected from matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS analysis of putative intact lipid A, these data led us to propose a heterogeneous hexa-acylated lipid A structure (multiple-lipid A species). We also confirmed previous reports ofG. obscuriglobuswhole-cell fatty acid (FA) and sterol compositions and detected a novel polyunsaturated FA (PUFA). Our confirmation of LPS, and by implication an OM, inG. obscuriglobusraises the possibility that other planctomycetes possess an OM. The pursuit of this question, together with studies of the structural connections between planctomycete LPS and peptidoglycans, will shed more light on what appears to be a planctomycete variation on the Gram-negative cell plan.IMPORTANCEBacterial species are classified as Gram positive or negative based on their cell envelope structure. For 25 years, the envelope of planctomycete bacteria has been considered a unique exception, as it lacks peptidoglycan and an outer membrane (OM). However, the very recent detection of peptidoglycan in planctomycete species has provided evidence for a more conventional cell wall and raised questions about other elements of the cell envelope. Here, we report direct evidence of lipopolysaccharide in the planctomyceteG. obscuriglobus, suggesting the presence of an OM and supporting the proposal that the planctomycete cell envelope is an extension of the canonical Gram-negative plan. This interpretation features a convoluted cytoplasmic membrane and expanded periplasmic space, the functions of which provide an intriguing avenue for future investigation.

scholarly journals Aminosugar-based immunomodulator lipid A: synthetic approaches

2018 ◽  
Vol 14 ◽  
pp. 25-53 ◽  
Author(s):  
Alla Zamyatina
Keyword(s):  
Lipid A ◽  
Germ Line ◽  
Bacterial Species ◽  
Complex Mixture ◽  
Structural Heterogeneity ◽  
Structural Features ◽  
Innate Immune ◽  
Outer Leaflet ◽  
Innate Immune Signaling ◽  
Biological Studies

The immediate immune response to infection by Gram-negative bacteria depends on the structure of a lipopolysaccharide (LPS, also known as endotoxin), a complex glycolipid constituting the outer leaflet of the bacterial outer membrane. Recognition of picomolar quantities of pathogenic LPS by the germ-line encoded Toll-like Receptor 4 (TLR4) complex triggers the intracellular pro-inflammatory signaling cascade leading to the expression of cytokines, chemokines, prostaglandins and reactive oxygen species which manifest an acute inflammatory response to infection. The “endotoxic principle” of LPS resides in its amphiphilic membrane-bound fragment glycophospholipid lipid A which directly binds to the TLR4·MD-2 receptor complex. The lipid A content of LPS comprises a complex mixture of structural homologs varying in the acylation pattern, the length of the (R)-3-hydroxyacyl- and (R)-3-acyloxyacyl long-chain residues and in the phosphorylation status of the β(1→6)-linked diglucosamine backbone. The structural heterogeneity of the lipid A isolates obtained from bacterial cultures as well as possible contamination with other pro-inflammatory bacterial components makes it difficult to obtain unambiguous immunobiological data correlating specific structural features of lipid A with its endotoxic activity. Advanced understanding of the therapeutic significance of the TLR4-mediated modulation of the innate immune signaling and the central role of lipid A in the recognition of LPS by the innate immune system has led to a demand for well-defined materials for biological studies. Since effective synthetic chemistry is a prerequisite for the availability of homogeneous structurally distinct lipid A, the development of divergent and reproducible approaches for the synthesis of various types of lipid A has become a subject of considerable importance. This review focuses on recent advances in synthetic methodologies toward LPS substructures comprising lipid A and describes the synthesis and immunobiological properties of representative lipid A variants corresponding to different bacterial species. The main criteria for the choice of orthogonal protecting groups for hydroxyl and amino functions of synthetically assembled β(1→6)-linked diglucosamine backbone of lipid A which allows for a stepwise introduction of multiple functional groups into the molecule are discussed. Thorough consideration is also given to the synthesis of 1,1′-glycosyl phosphodiesters comprising partial structures of 4-amino-4-deoxy-β-L-arabinose modifiedBurkholderialipid A and galactosamine-modifiedFrancisella lipid A. Particular emphasis is put on the stereoselective construction of binary glycosyl phosphodiester fragments connecting the anomeric centers of two aminosugars as well as on the advanced P(III)-phosphorus chemistry behind the assembly of zwitterionic double glycosyl phosphodiesters.

scholarly journals Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products

2020 ◽  
Vol 295 (11) ◽  
pp. 3417-3430 ◽  
Author(s):  
Jitka Petrlova ◽  
Ganna Petruk ◽  
Roland G. Huber ◽  
Eilish W. McBurnie ◽  
Mariena J. A. van der Plas ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Lipid A ◽  
Lipoteichoic Acid ◽  
Structural Features ◽  
Human Neutrophil Elastase ◽  
Gram Negative ◽  
Functional Roles ◽  
Cellular Assays

Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium Escherichia coli. However, the physiological roles of TCP96 in controlling bacterial infections and reducing LPS-induced inflammation are unclear. Here, using various biophysical methods, in silico molecular modeling, microbiological and cellular assays, and animal models, we examined the structural features and functional roles of recombinant TCP96 (rTCP96) in the aggregation of multiple bacteria and the Toll-like receptor (TLR) agonists they produce. We found that rTCP96 aggregates both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa, and their cell-wall components LPS, lipid A, and lipoteichoic acid (LTA). The Gram-negative bacteria E. coli and P. aeruginosa were particularly sensitive to aggregation-induced bacterial permeabilization and killing. As a proof of concept, we show that rTCP96 reduces LPS-induced NF-κB activation in human monocytes, as well as in mouse models of LPS-induced subcutaneous inflammation. Moreover, in a mouse model of subcutaneous inoculation with P. aeruginosa, rTCP96 reduced bacterial levels. Together, these results link TCP-mediated aggregation of endotoxins and bacteria in vitro to attenuation of inflammation and bacterial levels in vivo.

scholarly journals Antimicrobial peptide activity is anticorrelated with lipid a leaflet affinity

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242907
Author(s):  
Nathaniel Nelson ◽  
Belita Opene ◽  
Robert K. Ernst ◽  
Daniel K. Schwartz
Keyword(s):  
Single Molecule ◽  
Lipid A ◽  
Inhibitory Concentration ◽  
Bacterial Species ◽  
Membrane Lipid ◽  
Gram Negative Bacteria ◽  
Supported Bilayers ◽  
Single Molecule Tracking ◽  
Gram Negative ◽  
Outer Membranes

The activity of antimicrobial peptides (AMPs) has significant bacterial species bias, the mechanisms of which are not fully understood. We employed single-molecule tracking to measure the affinity of three different AMPs to hybrid supported bilayers composed of lipid A extracted from four different Gram negative bacteria and observed a strong empirical anticorrelation between the affinity of a particular AMP to a given lipid A layer and the activity of that AMP towards the bacterium from which that lipid A was extracted. This suggested that the species bias of AMP activity is directly related to AMP interactions with bacterial outer membranes, despite the fact that the mechanism of antimicrobial activity occurs at the inner membrane. The trend also suggested that the interactions between AMPs and the outer membrane lipid A (even in the absence of other components, such as lipopolysaccharides) capture effects that are relevant to the minimum inhibitory concentration.

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