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

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.

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

Science Translational Medicine ◽

10.1126/scitranslmed.aal0033 ◽

2018 ◽

Vol 10 (464) ◽

pp. eaal0033 ◽

Cited By ~ 23

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.

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Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity

Marine Drugs ◽

10.3390/md19110646 ◽

2021 ◽

Vol 19 (11) ◽

pp. 646

Author(s):

Rossella Di Guida ◽

Angela Casillo ◽

Antonietta Stellavato ◽

Celeste Di Meo ◽

Soichiro Kawai ◽

...

Keyword(s):

Chemical Structure ◽

Lipid A ◽

Membrane Integrity ◽

Immunomodulatory Activity ◽

Gram Negative Bacteria ◽

Core Oligosaccharide ◽

Gram Negative ◽

Seriola Quinqueradiata ◽

Specific Polysaccharide

Lipopolysaccharides (LPS) are surface glycoconjugates embedded in the external leaflet of the outer membrane (OM) of the Gram-negative bacteria. They consist of three regions: lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen. Lipid A is the glycolipid endotoxin domain that anchors the LPS molecule to the OM, and therefore, its chemical structure is crucial in the maintenance of membrane integrity in the Gram-negative bacteria. In this paper, we reported the characterization of the lipid A and OS structures from Pseudoalteromonas nigrifaciens Sq02-Rifr, which is a psychrotrophic Gram-negative bacterium isolated from the intestine of Seriola quinqueradiata. The immunomodulatory activity of both LPS and lipid A was also examined.

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Biosynthesis of lipopolysaccharides with different length of the O-specific region as a virulence factor of Gram-negative bacteria

Postępy Higieny i Medycyny Doświadczalnej ◽

10.5604/01.3001.0012.1735 ◽

2018 ◽

Vol 72 ◽

pp. 573-586

Author(s):

Eva Krzyżewska ◽

Jacek Rybka

Keyword(s):

Outer Membrane ◽

Lipid A ◽

Specific Antigen ◽

Gram Negative Bacteria ◽

Bacterial Survival ◽

Core Oligosaccharide ◽

Gram Negative ◽

O Antigen ◽

Length Regulation ◽

External Part

The outer membrane of Gram-negative bacteria is a biological structure with a unique composition that significantly contributes to the survival of bacteria in the unfavorable conditions of the host organism. The lipopolysaccharide constitutes about 70% of the external part of the outer membrane. The LPS molecule is composed of three different parts: lipid A, core oligosaccharide and O antigen. Despite the O-specific antigen being one of the most intensely studied surface structures of bacterial polysaccharides, a number of questions regarding the mechanism of the O antigen biosynthesis and its transport to the cell surface are still unanswered. The paper describes the biosynthesis of the lipopolysaccharide molecule, with particular emphasis on the O-specific chain biosynthesis, the mechanism of lipopolysaccharide length regulation and the influence of the type of synthesized O-specific chains on bacterial survival in adverse host organisms.

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

10.1101/2021.12.16.472937 ◽

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.

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The Chemical Composition of Endotoxin Isolated from Intestinal Strain ofDesulfovibrio desulfuricans

The Scientific World JOURNAL ◽

10.1100/2012/647352 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 6

Author(s):

Jolanta Lodowska ◽

Daniel Wolny ◽

Marzena Jaworska-Kik ◽

Sławomir Kurkiewicz ◽

Zofia Dzierżewicz ◽

...

Keyword(s):

Ulcerative Colitis ◽

Lipid A ◽

Alimentary Tract ◽

Desulfovibrio Desulfuricans ◽

Gram Negative Bacteria ◽

Core Oligosaccharide ◽

Tetradecanoic Acid ◽

Gram Negative ◽

Specific Polysaccharide ◽

Cellular Components

Desulfovibrio desulfuricansanaerobes are constituents of human alimentary tract microflora. There are suggestions that they take part in the pathogenesis of periodontitis and some gastrointestinal inflammatory disorders, such as ulcerative colitis or Crohn’s disease. Endotoxin is one of Gram-negative bacteria cellular components that influence these microorganisms pathogenicity. Endotoxin is a lipid-polisaccharide heteropolymer consisting of three elements: lipid A, core oligosaccharide, and O-specific polysaccharide, also called antigen-O. The biological activity of lipopolysaccharide (LPS) is determined by its structure. In this study, we show that rhamnose, fucose, mannose, glucose, galactose, heptose, and 2-keto-3-deoxyoctulosonic acid (Kdo) are constituents ofD. desulfuricansendotoxin oligosaccharide core and O-antigen. Lipid A of these bacteria LPS is composed of glucosamine disaccharide substituted by 3-acyloxyacyl residues: ester-bound 3-(dodecanoyloxy)tetradecanoic, 3-(hexadecanoyloxy)tetradecanoic acid, and amide-bound 3-(tetradecanoyloxy)tetradecanoic acid.

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

10.1101/2021.12.16.472973 ◽

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.

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Glucosamine Found as a Substituent of Both Phosphate Groups in Bordetella Lipid A Backbones: Role of a BvgAS-Activated ArnT Ortholog

Journal of Bacteriology ◽

10.1128/jb.01875-07 ◽

2008 ◽

Vol 190 (12) ◽

pp. 4281-4290 ◽

Cited By ~ 44

Author(s):

Nico Marr ◽

Alina Tirsoaga ◽

Didier Blanot ◽

Rachel Fernandez ◽

Martine Caroff

Keyword(s):

Lipid A ◽

Biological Activities ◽

Antigenic Determinants ◽

Gram Negative Bacteria ◽

Core Oligosaccharide ◽

Gene Encoding ◽

Gram Negative ◽

Host Immune Responses ◽

Covalently Linked ◽

Phosphate Groups

ABSTRACT Endotoxins are amphipathic lipopolysaccharides (LPSs), major constituents of the outer membrane of gram-negative bacteria. They consist of a lipid region, covalently linked to a core oligosaccharide, to which may be linked a repetitive glycosidic chain carrying antigenic determinants. Most of the biological activities of endotoxins have been associated with the lipid moiety of the molecule: unique to gram-negative bacteria, LPS is a ligand of the mammalian TLR4-MD2-CD14 pathogen recognition receptor complex. Lipid A preparations are often heterogeneous with respect to both the numbers and the lengths of fatty acids and the natures of substituents on the phosphate groups when present. The variants can significantly affect host immune responses. Nine species in the Bordetella genus have been described, and the fine LPS structures of seven of them have been published. In this report, lipids A from Bordetella pertussis Tohama I and B. bronchiseptica strain 4650 were further characterized and revealed to have a glucosamine substituting both lipid A phosphate groups of the diglucosamine backbone. These substitutions have not been previously described for bordetellae. Moreover, a B. pertussis transposon mutation that maps within a gene encoding a Bordetella ArnT (formerly PmrK) glycosyl transferase ortholog does not carry this substitution, thus providing a genetic basis for the modification. Reverse transcriptase PCR of this locus showed that it is Bvg regulated, suggesting that the ability of Bordetella to modify lipid A via this glucosamine modification is a potential virulence trait.

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