Items 1 - 75 of 75    (Display the 75 citations in PubMed)
 

ISO 690 Journal of the American Academy of Dermatology, 2017, vol. 76, no 6, p. AB36.

All bacteria lipopolysaccharides are not endotoxins: Structure to activity relationships: vitreoscilla filiformis example. 

SEITE Sophie, BRETON Aude, NOVIKOV Alexey, et al.

 

(http://www.sciencedirect.com/science/article/pii/S0190962217306461)

 

Abstract

Introduction: Endotoxins are the main components of the outer membrane of Gram-negative bacteria. Each bacterium possesses a specific lipopolysaccharides (LPS) structure. Chemically, endotoxins are LPS, but not all LPS are endotoxins. They are composed of two moieties: the polysaccharidic (PS) and the lipid A. The PS moiety is made of the so-called O-antigens linked to core oligosaccharides. They carry the antigenic properties of the molecule and can be used in vaccines as well as in bacterial detection tests. As an example, we studied Vitreoscilla filiformis, a Gram-negative bacterium isolated from spa waters and chosen for its beneficial effects on atopic dermatitis lesions.Methods: First, the detailed structure of the V filiformis lipid A moiety was characterized. Secondly, a comparison of the structure to activity relationships of V filiformis LPS to others LPS, differentiated by the chain-length of their fatty acids, their linkage position on the glucosamines (GlcN), and their impact on the IL-6 and TNF-α inflammatory responses, caspase 3/7 activation and radical oxygen species (ROS) induction, and structurally different ones was performed.Results: Structural differences depend on the bacterial genus, species and the natural heterogeneity of LPS and were associated with differences in biological activities.Conclusions: This study point out the importance to fully characterize the lipid A structures of each bacteria, because LPS could be different in terms of fatty acids (FA) chain-length, FA number, and/or by their linkage position on the glucosamines. These details greatly impact the LPS biological activities. This study also confirms that V filiformis LPS are able to induce immunostimulation of the monocyte system: moderate cytokine release, ROS induction, mitogenicity and caspase activation without cellular death.
 
Microbiology. 2017 Jun;163(6):848-855. doi: 10.1099/mic.0.000476.

Fine structure analysis of lipopolysaccharides in bacteriophage-resistant Pseudomonas aeruginosa PAO1 mutants.

Abstract

Pseudomonas aeruginosa lipopolysaccharides (LPS) serve as primary receptors for many bacteriophages and, consequently, their biosynthesis is frequently affected in phage-resistant mutants. We previously isolated phage-resistant PAO1 mutants using three different phages, and showed that they were affected in the synthesis of LPS. Here we have investigated in detail the effect of mutations in seven genes involved in different steps of the production of core and oligosaccharide chains. The band profile of purified LPS was analysed by PAGE, and we further characterized the O-chains and core structures by MALDI mass spectrometry (MS). Mild LPS extraction conditions and native LPS MS analyses helped unveil lipid A molecular species with three phosphate residues in the close vicinity of the already highly charged inner-core region. No other MS direct analysis has allowed this peculiarity to be demonstrated for native lipid A high-molecular-weight molecular species, in normal growth conditions and without involving separation techniques. The present results shed light on the possible interactions between the phages and the LPS structures in the early phase of infection.

PMID:
 
28682742
 
DOI:
 
10.1099/mic.0.000476

 

Int J Mol Sci. 2017 May 18;18(5). pii: E1080. doi: 10.3390/ijms18051080.

Bordetella holmesii: Lipid A Structures and Corresponding Genomic Sequences Comparison in Three Clinical Isolates and the Reference Strain ATCC 51541.

Abstract

Bordetella holmesii can cause invasive infections but can also be isolated from the respiratory tract of patients with whooping-cough like symptoms. For the first time, we describe the lipid A structure of B. holmesii reference strain ATCC 51541 (alias NCTC12912 or CIP104394) and those of three French B. holmesii clinical isolates originating from blood (Bho1) or from respiratory samples (FR4020 and FR4101). They were investigated using chemical analyses, gas chromatography-mass spectrometry (GC-MS), and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The analyses revealed a common bisphosphorylated β-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. Similar to B. aviumB. hinzii and B. trematum lipids A, the hydroxytetradecanoic acid at the C-2' position are carrying in secondary linkage a 2-hydroxytetradecanoic acid residue resulting of post-traductional biosynthesis modifications. The three clinical isolates displayed characteristic structural traits compared to the ATCC 51541 reference strain: the lipid A phosphate groups are more or less modified with glucosamine in the isolates and reference strain, but the presence of 10:0(3-OH) is only observed in the isolates. This trait was only described in B. pertussis and B. parapertussis strains, as well as in B. petrii isolates by the past. The genetic bases for most of the key structural elements of lipid A were analyzed and supported the structural data.

KEYWORDS: 

Bordetella holmesii; Bordetellae; endotoxin; genomic; lipid A; mass spectrometry; structure

PMID:
 
28524084
 
PMCID:
 
PMC5454989
 
DOI:
 
10.3390/ijms18051080

 

Methods Mol Biol. 2017;1600:167-186. doi: 10.1007/978-1-4939-6958-6_16.

Micromethods for Isolation and Structural Characterization of Lipid A, and Polysaccharide Regions of Bacterial Lipopolysaccharides.

Abstract

Lipopolysaccharides (LPS) are major components of the external membrane of most Gram-negative bacteria, providing them with an effective permeability barrier. They are essentially composed of a hydrophilic polysaccharide region (PS) linked to a hydrophobic one, termed lipid A. The LPS polysaccharide moiety is divided into the core oligosaccharide (OS) and O-chain repetitive elements. Depending on their individual variable fine structures, LPS may be potent immunomodulators. The lipid A structure is a key determinant for LPS activity. However, the presence of the core region, or at least of the highly charged 3-deoxy-d-manno-oct-2-ulosonic acid molecules, is also important for preserving the native lipid A conformation within individual LPS molecules. We describe herein four rapid and practical micromethods for LPS, lipid A, and core OS structural analyses. The first method allows the direct isolation of lipid A from whole bacteria cell mass; the second describes conditions for the sequential release of fatty acids enabling the characterization of their substitution position in the lipid A backbone, to be determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The third one is a microscale procedure for the mass spectra screening of LPS, lipid A, and PS using triethylamine and citric acid. The fourth method is a chromatography procedure for Rough-type LPS on thin-layer-chromatography. These methods were developed to be coupled to mass-spectrometry (e.g., MALDI-MS) but can also be used with other analytical techniques (e.g., chromatography). Examples are given with reference to two major human pathogens: Bordetella pertussis and Pseudomonas aeruginosa; to one porcine pathogen: Actinobacillus pleuropneumoniae; and to commercial samples of Salmonella Minnesota Re595 LPS.

KEYWORDS: 

3-Deoxy-d-manno-oct-2-ulosonic acid; Gas chromatography; Lipooligosaccharide; Lipopolysaccharide; Matrix-assisted laser desorption/ionization mass spectrometry; Polyacrylamide gel electrophoresis; Sodium dodecyl sulfate; Thin-layer chromatography; Triethylamine

PMID:
 
28478567
 
DOI:
 
10.1007/978-1-4939-6958-6_16

 

J Lipid Res. 2017 Mar;58(3):543-552. doi: 10.1194/jlr.M072900. Epub 2017 Jan 25.

Structural and biological characteristics of different forms of V. filiformis lipid A: use of MS to highlight structural discrepancies.

Abstract

Vitreoscilla filiformis is a Gram-negative bacterium isolated from spa waters and described for its beneficial effects on the skin. We characterized the detailed structure of its lipopolysaccharide (LPS) lipid A moiety, an active component of the bacterium that contributes to the observed skin activation properties. Two different batches differing in postculture cell recovery were tested. Chemical analyses and mass spectra, obtained before and after mild-alkali treatments, revealed that these lipids A share the common bisphosphorylated β-(1→6)-linked d-glucosamine disaccharide with hydroxydecanoic acid in an amide linkage. Short-chain FAs, hydroxydecanoic and dodecanoic acid, were found in a 2:1 ratio. The two lipid A structures differed by the relative amount of the hexa-acyl molecular species and phosphoethanolamine substitution of the phosphate groups. The two V. filiformis LPS batches induced variable interleukin-6 and TNF-α secretion by stimulated myelomonocytic THP-1 cells, without any difference in reactive oxygen species production or activation of caspase 3/7. Other different well-known highly purified LPS samples were characterized structurally and used as standards. The structural data obtained in this work explain the low inflammatory response observed for V. filiformis LPS and the previously demonstrated beneficial effects on the skin.

KEYWORDS: 

V. filiformis; cytokines; lipid biochemistry; lipopolysaccharide; mass spectrometry; skin; toll-like receptor

PMID:
 
28122817
 
PMCID:
 
PMC5335584
 [Available on 2018-03-01]
 
DOI:
 
10.1194/jlr.M072900

 

Appl Environ Microbiol. 2016 Oct 14;82(21):6423-6430. Print 2016 Nov 1.

Antimicrobial Peptide Resistance Genes in the Plant Pathogen Dickeya dadantii.

Abstract

Modification of teichoic acid through the incorporation of d-alanine confers resistance in Gram-positive bacteria to antimicrobial peptides (AMPs). This process involves the products of the dltXABCD genes. These genes are widespread in Gram-positive bacteria, and they are also found in a few Gram-negative bacteria. Notably, these genes are present in all soft-rot enterobacteria (Pectobacterium and Dickeya) whose dltDXBAC operons have been sequenced. We studied the function and regulation of these genes in Dickeya dadantii dltB expression was induced in the presence of the AMP polymyxin. It was not regulated by PhoP, which controls the expression of some genes involved in AMP resistance, but was regulated by ArcA, which has been identified as an activator of genes involved in AMP resistance. However, arcA was not the regulator responsible for polymyxin induction of these genes in this bacterium, which underlines the complexity of the mechanisms controlling AMP resistance in D. dadantii Two other genes involved in resistance to AMPs have also been characterized, phoS and phoH dltB, phoS, phoH, and arcA but not dltD mutants were more sensitive to polymyxin than the wild-type strain. Decreased fitness of the dltB, phoS, and phoH mutants in chicory leaves indicates that their products are important for resistance to plant AMPs.

IMPORTANCE: 

Gram-negative bacteria can modify their lipopolysaccharides (LPSs) to resist antimicrobial peptides (AMPs). Soft-rot enterobacteria (Dickeya and Pectobacterium spp.) possess homologues of the dlt genes in their genomes which, in Gram-positive bacteria, are involved in resistance to AMPs. In this study, we show that these genes confer resistance to AMPs, probably by modifying LPSs, and that they are required for the fitness of the bacteria during plant infection. Two other new genes involved in resistance were also analyzed. These results show that bacterial resistance to AMPs can occur in bacteria through many different mechanisms that need to be characterized.

PMID:
 
27565623
 
PMCID:
 
PMC5066359
 
DOI:
 
10.1128/AEM.01757-16
Biochimie. 2015 Jul 8. pii: S0300-9084(15)00214-X. doi: 10.1016/j.biochi.2015.07.006. [Epub ahead of print]

Structure activity characterization of Bordetella petrii lipid A, from environment to human isolates.

Author information:

  • 1Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Bâtiment 409, 91405 Orsay, France.
  • 2Institut Pasteur, Unité de Prévention et Thérapies Moléculaires des Maladies Humaines, URA CNRS 3012, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
  • 3LPS-BioSciences, Bâtiment 409, Université de Paris-Sud, F-91405 Orsay, France.
  • 4Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Bâtiment 409, 91405 Orsay, France; LPS-BioSciences, Bâtiment 409, Université de Paris-Sud, F-91405 Orsay, France. Electronic address: Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser..

 

Abstract

Bordetella petrii, a facultative anaerobic species, is the only known member of the Bordetella genus with environmental origin. However it was also recently isolated from humans. The structures of the B. petrii lipid A moieties of the endotoxins were characterized here for the first time for an environmental strain and compared to that of human isolates. Characterization was achieved using chemical analyses, gas chromatography-mass spectrometry, and Matrix Assisted Laser Desorption Ionisation mass spectrometry. The analyses revealed that the different lipid A structures contain a common bisphosphorylated β-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide as well at the C-3' in ester linkages. Similar to Bordetella pertussis and Bordetella bronchiseptica lipids A, the hydroxytetradecanoic acid at the C-2' position was substituted by tetradecanoic acid. Unlike B. pertussis, the hydroxytetradecanoic acid at the C-2 position was substituted with either 12:0 or 14:0 and/or their 2-OH forms. Depending on the environmental or human origin the structures differed in the length and degree of fatty acid acylation and impacted the IL-6 and TNF-α inflammatory responses tested. In one isolate we showed the presence at the C-3 position of the short-chain 10:0(3-OH), which according to our previous analyses is more characteristic of the human pathogens in the genus like B. pertussis and Bordetella parapertussis.

Copyright © 2015. Published by Elsevier B.V.

PMID: 26164553 [PubMed - as supplied by publisher]
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FEBS Lett. 2015 Jan 2;589(1):165-71. doi: 10.1016/j.febslet.2014.11.042. Epub 2014 Dec 3.

Desulfovibrio desulfuricans isolates from the gut of a single individual: structural and biological lipid A characterization.

Author information:

  • 1Equipe «Endotoxins: Structures and Activities», Institut de Génétique et Microbiologie, Université Paris-Sud, CNRS UMR 8621, Orsay, France.
  • 2Laboratory of Molecular Microbial Ecology and Ecogenomics, Department of Biological Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China.

 

Abstract

The levels of sulfate-reducing bacteria (SRB), including Desulfovibrionaceae, in the gut increase following a fat-enriched diet. Endotoxins from gut microbiota contribute to the inflammation process, leading to metabolic diseases. Thus, we sought to characterize the lipid A structures of Desulfovibrionaceae lipopolysaccharides (LPS) that are associated with the microbiota inflammatory properties. LPS variants were obtained from two SRB isolates from the gut of a single individual. These LPS variants shared similar lipid A moieties with Enterobacterial LPS, but differed from one another with regard to fatty-acid numbers and endotoxic activity. This first complete structural characterization of Desulfovibrio lipid A gives new insights into previously published data on Desulfovibrio lipid A biosynthesis. LPS microdiversity within SRBs illustrates how adaptation can influence pro-inflammatory potential.

Copyright © 2014. Published by Elsevier B.V.

PMID: 25479086 [PubMed - indexed for MEDLINE]
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MBio. 2014 Aug 19;5(4). pii: e01116-14. doi: 10.1128/mBio.01116-14.

Biofilms formed by gram-negative bacteria undergo increased lipid a palmitoylation, enhancing in vivo survival.

Author information:

  • 1Institut Pasteur, Unité de Génétique des Biofilms, Paris, France.
  • 2Equipe Endotoxines, Université de Paris-Sud, CNRS UMR 8621, IGM, Orsay, France.
  • 3Institut Pasteur, Unité de Génétique des Biofilms, Paris, France Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser..

 

Abstract

Bacterial biofilm communities are associated with profound physiological changes that lead to novel properties compared to the properties of individual (planktonic) bacteria. The study of biofilm-associated phenotypes is an essential step toward control of deleterious effects of pathogenic biofilms. Here we investigated lipopolysaccharide (LPS) structural modifications in Escherichia coli biofilm bacteria, and we showed that all tested commensal and pathogenic E. coli biofilm bacteria display LPS modifications corresponding to an increased level of incorporation of palmitate acyl chain (palmitoylation) into lipid A compared to planktonic bacteria. Genetic analysis showed that lipid A palmitoylation in biofilms is mediated by the PagP enzyme, which is regulated by the histone-like protein repressor H-NS and the SlyA regulator. While lipid A palmitoylation does not influence bacterial adhesion, it weakens inflammatory response and enhances resistance to some antimicrobial peptides. Moreover, we showed that lipid A palmitoylation increases in vivo survival of biofilm bacteria in a clinically relevant model of catheter infection, potentially contributing to biofilm tolerance to host immune defenses. The widespread occurrence of increased lipid A palmitoylation in biofilms formed by all tested bacteria suggests that it constitutes a new biofilm-associated phenotype in Gram-negative bacteria.

IMPORTANCE:

Bacterial communities called biofilms display characteristic properties compared to isolated (planktonic) bacteria, suggesting that some molecules could be more particularly produced under biofilm conditions. We investigated biofilm-associated modifications occurring in the lipopolysaccharide (LPS), a major component of all Gram-negative bacterial outer membrane. We showed that all tested commensal and pathogenic biofilm bacteria display high incorporation of a palmitate acyl chain into the lipid A part of LPS. This lipid A palmitoylation is mediated by the PagP enzyme, whose expression in biofilm is controlled by the regulatory proteins H-NS and SlyA. We also showed that lipid A palmitoylation in biofilm bacteria reduces host inflammatory response and enhances their survival in an animal model of biofilm infections. While these results provide new insights into the biofilm lifestyle, they also suggest that the level of lipid A palmitoylation could be used as an indicator to monitor the development of biofilm infections on medical surfaces.

Copyright © 2014 Chalabaev et al.

PMCID: PMC4147861 Free PMC Article
PMID: 25139899 [PubMed - in process]
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Genome Announc. 2013 Oct 24;1(5). pii: e00838-13. doi: 10.1128/genomeA.00838-13.

Draft Genome Sequences of Bordetella hinzii and Bordetella trematum.

Author information:

  • 1Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.

 

Abstract

Bordetella hinzii colonizes the respiratory tracts of poultry but can also infect immunocompromised humans. Bordetella trematum, however, only infects humans, causing ear and wound infections. Here, we present the first draft genome sequences of strains B. hinzii ATCC 51730 and B. trematum CCUG 13902.

PMCID: PMC3813182 Free PMC Article
PMID: 24158552 [PubMed]
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Innate Immun. 2013 Oct 14;20(6):659-672. [Epub ahead of print]

Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications.

Author information:

  • 1Equipe "Endotoxines", I.G.M. Université de Paris-Sud, Orsay, France Present address: Start-up LPS-BioSciences, IGM, Orsay, France.
  • 2Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
  • 3Equipe "Endotoxines", I.G.M. Université de Paris-Sud, Orsay, France.
  • 4Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada Centre for High-Throughput Biology, University of British Columbia, Vancouver, Canada.
  • 5Immunochemistry, N.R.C., Ottawa, Canada.
  • 6Equipe "Endotoxines", I.G.M. Université de Paris-Sud, Orsay, France Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser..

 

Abstract

Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.

© The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

PMID: 24127384 [PubMed - as supplied by publisher]
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Carbohydr Res. 2013 Aug 30;378:56-62. doi: 10.1016/j.carres.2013.05.002. Epub 2013 May 13.

Comparison of lipopolysaccharide structures of Bordetella pertussis clinical isolates from pre- and post-vaccine era.

Author information:

  • 1Endotoxines, Structures et Activités (ESA), Université de Paris-Sud, UMR 8621 CNRS, Institut de Génétique et Microbiologie, F-91405 Orsay, France.

 

Abstract

Endotoxins are lipopolysaccharides (LPS), and major constituents of the outer membrane of Gram-negative bacteria. Bordetella pertussis LPS were the only major antigens, of this agent of whooping-cough, that were not yet analyzed on isolates from the pre- and post-vaccination era. We compared here the LPS structures of four clinical isolates with that of the vaccine strain BP 1414. All physico-chemical analyses, including SDS-PAGE, TLC, and different MALDI mass spectrometry approaches were convergent. They helped demonstrating that, on the contrary to some other B. pertussis major antigens, no modification occurred in the dodecasaccharide core structure, as well as in the whole LPS molecules. These results are rendering these major antigens good potential vaccine components. Molecular modeling of this conserved LPS structure also confirmed the conclusions of previous experiments leading to the production of anti-LPS monoclonal antibodies and defining the main epitopes of these major antigens.

Copyright © 2013 Elsevier Ltd. All rights reserved.

PMID: 23731797 [PubMed - indexed for MEDLINE]
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J Biol Chem. 2013 Apr 26;288(17):11751-60. doi: 10.1074/jbc.M112.434365. Epub 2013 Mar 6.

Minor modifications to the phosphate groups and the C3' acyl chain length of lipid A in two Bordetella pertussis strains, BP338 and 18-323, independently affect Toll-like receptor 4 protein activation.

Author information:

  • 1Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

 

Abstract

Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4'-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3' acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399-BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3' acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.

PMCID: PMC3636864 Free PMC Article
PMID: 23467413 [PubMed - indexed for MEDLINE]
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Rapid Commun Mass Spectrom. 2011 Jul 30;25(14):2043-8. doi: 10.1002/rcm.5084.

A new rapid and micro-scale hydrolysis, using triethylamine citrate, for lipopolysaccharide characterization by mass spectrometry.

Author information:

  • 1Equipe 'ESA: Endotoxines, Structures et Activités', UMR 8621 du CNRS, Institut de Génétique et Microbiologie, Université de Paris-Sud, 91405 Orsay, France.

 

Abstract

Endotoxin (lipopolysaccharide, LPS) is, in general, composed of two moieties: a hydrophilic polysaccharide linked to a hydrophobic lipid A terminal unit and forms a major surface component of gram-negative bacteria. The structural features of LPS moieties play a role in pathogenesis and also involve immunogenicity and diagnostic serology. The major toxic factor of LPS resides in the lipid A moiety, anchored in the outer layer of the bacterium, and its relative biological activity is critically related to fine structural features within the molecule. In establishing relationships between structural features and biological activities of LPS it is of the utmost importance to develop new analytical methods that can be applied to the complete unambiguous characterization of a specific LPS molecule. Herein is presented a practical rapid and sensitive analytical procedure for the mass spectral screening of LPS using triethylamine citrate as an agent for both disaggregation and mild hydrolysis of LPS. It provides improved matrix-assisted laser desorption/ionization (MALDI) mass spectra and, in particular, affords the identification of fragments retaining labile substituents present in the native macromolecular LPS structures. The methods were developed and applied using purified LPS of Escherichia coli and Salmonella enterica, as well as more complex LPS of Actnobacillus pleuropneumoniae.

Copyright © 2011 John Wiley & Sons, Ltd.

PMID: 21698687 [PubMed - indexed for MEDLINE]
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Methods Mol Biol. 2011;739:135-46. doi: 10.1007/978-1-61779-102-4_12.

Micromethods for lipid A isolation and structural characterization.

Author information:

  • 1Equipe "Structure et Activités des Endotoxines", Institut de Génétique et Microbiologie, Université de Paris Sud-XI, Orsay, France.Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

Lipopolysaccharides (LPSs) are major components of the external membrane of Gram-negative bacteria, and act as an effective permeability barrier. They are essentially composed of a hydrophilic polysaccharide region linked to an hydrophobic one, termed lipid A. Depending on their individual variable fine structures, they may be potent immunomodulators. Because of the structural importance and role of lipid A in bacterial pathogenesis, herein we describe two rapid practical micromethods for structural analysis. The first method allows the direct isolation of lipid A from whole bacteria cell mass; the second describes conditions for the sequential release of fatty acids, enabling the determination of their substitution position in the lipid A structure to be determined by matrix-assisted laser desorption/ionization mass spectrometry. Examples are given with reference to two major pathogens: Bordetella pertussis and Pseudomonas aeruginosa.

PMID: 21567324 [PubMed - indexed for MEDLINE]
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Rapid Commun Mass Spectrom. 2011 Apr 30;25(8):1075-81. doi: 10.1002/rcm.4960. Epub 2011 Mar 29.

Structural modifications occurring in lipid A of Bordetella bronchiseptica clinical isolates as demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Author information:

  • 1Endotoxines, Structures et Activités, UMR 8621, GDR 3048, du CNRS, Institut de Génétique et Microbiologie, Université de Paris-Sud, 91405, Orsay, France.

 

Abstract

Bordetella bronchiseptica is a respiratory pathogen in mammal species and its cell surface lipopolysaccharide-endotoxin is a potent virulence factor. In order to better characterize the endotoxin structure to virulence relationships, we studied the lipid A structures of B. bronchiseptica isolates from human and rabbit origins as a function of their virulence phases. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been widely used for the structural characterization of bacterial endotoxins and their lipid A moieties. This method combined with chemical analytical methods proved to be essential for the characterization of small samples and discrete but essential structural modifications. The occurrence of palmitate (C(16)) in the B. bronchiseptica lipid A structures is shown for the first time at two sites. Their presence was also demonstrated for the first time in correlation with the virulence phase of B. bronchiseptica clinical isolates. The recently identified glucosamine modifications of Bordetella lipids A are also reported in these isolates.

Copyright © 2011 John Wiley & Sons, Ltd.

PMID: 21452385 [PubMed - indexed for MEDLINE]
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J Infect Dis. 2010 Dec 15;202(12):1897-906. doi: 10.1086/657409. Epub 2010 Nov 4.

Variability in the lipooligosaccharide structure and endotoxicity among Bordetella pertussis strains.

Author information:

  • 1Department of Microbiology & Immunology, University of British Columbia, Vancouver, British Columbia, Canada.

 

Abstract

Bordetella endotoxins show remarkable structural variability both among each other and in comparison to other gram-negative bacteria. Here we demonstrate that, in contrast to the common Bordetella pertussis laboratory strain and Tohama I derivative BP338, lipooligosaccharide from mouse challenge strain 18-323 is a poor inducer of inflammatory cytokines in human and murine macrophages, is greatly impaired in Toll-like receptor 4-mediated activation of nuclear factor-κB in transfected HEK-293 cells, and functions as a Toll-like receptor 4 antagonist. Comparison of lipid A and lipooligosaccharide structures of B. pertussis strains BP338 and 18-323 revealed that 18-323 (1) lacks the ability to modify its lipid A phosphate groups with glucosamine, (2) is distinct in its acylation at the C3' position of the lipid A diglucosamine backbone, and (3) expresses molecular lipooligosaccharide species that lack a terminal heptose. Our findings have important implications for interpreting previous studies of host defenses to B. pertussis infection in mice and in vitro.

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PMID: 21050116 [PubMed - indexed for MEDLINE]
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Infect Immun. 2010 May;78(5):2060-9. doi: 10.1128/IAI.01346-09. Epub 2010 Feb 22.

Substitution of the Bordetella pertussis lipid A phosphate groups with glucosamine is required for robust NF-kappaB activation and release of proinflammatory cytokines in cells expressing human but not murine Toll-like receptor 4-MD-2-CD14.

Author information:

  • 1Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

 

Abstract

Bordetella pertussis endotoxin is a key modulator of the host immune response, mainly due to the role of its lipid A moiety in Toll-like receptor 4 (TLR4)-mediated signaling. We have previously demonstrated that the lipid A phosphate groups of B. pertussis BP338 can be substituted with glucosamine in a BvgAS-regulated manner. Here we examined the effect of this lipid A modification on the biological activity of B. pertussis endotoxin. We compared purified endotoxin and heat-killed B. pertussis BP338 whole cells that have modified lipid A phosphate groups to an isogenic mutant lacking this modification with respect to their capacities to induce the release of inflammatory cytokines by human and murine macrophages and to participate in the TLR4-mediated activation of NF-kappaB in transfected HEK-293 cells. We found inactivated B. pertussis cells to be stronger inducers of proinflammatory cytokines in THP-1-derived macrophages when lipid A was modified. Most notably, lack of lipid A modification abolished the ability of purified B. pertussis endotoxin to induce the release of inflammatory cytokines by human THP-1-derived macrophages but led to only slightly reduced inflammatory cytokine levels when stimulating murine (RAW 264.7) macrophages. Accordingly, upon stimulation of HEK-293 cells with inactivated bacteria and purified endotoxin, lack of lipid A modification led to impaired NF-kappaB activation only when human, and not when murine, TLR4-MD-2-CD14 was expressed. We speculate that in B. pertussis, lipid A modification has evolved to benefit the bacteria during human infection by modulating immune defenses rather than to evade innate immune recognition.

PMCID: PMC2863497 Free PMC Article
PMID: 20176798 [PubMed - indexed for MEDLINE]
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Appl Environ Microbiol. 2010 Feb;76(3):910-21. doi: 10.1128/AEM.02112-09. Epub 2009 Dec 18.

Association of hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, with cyclic lipopeptide production.

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  • 1Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France. Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C(10)OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.

PMCID: PMC2812987 Free PMC Article
PMID: 20023108 [PubMed - indexed for MEDLINE]
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Innate Immun. 2010 Oct;16(5):288-301. doi: 10.1177/1753425909341807. Epub 2009 Aug 26.

Biofilm-forming Pseudomonas aeruginosa bacteria undergo lipopolysaccharide structural modifications and induce enhanced inflammatory cytokine response in human monocytes.

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  • 1Unité Cytokines & Inflammation, Institut Pasteur, Paris, France. Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

To determine whether growth of bacteria in biofilms triggers a specific immune response, we compared cytokine induction in human monocytes and mouse macrophages by planktonic and biofilm bacteria. We compared Pseudomonas aeruginosa and Staphylococcus aureus, two bacteria often colonizing the airways of cystic fibrosis patients. Planktonic and biofilm S. aureus induced equivalent amounts of cytokine in human monocytes. In contrast, biofilm-forming P. aeruginosa induced a higher production of tumor necrosis factor and interleukin-6 than their planktonic counterpart, both for clinical isolates and laboratory strains. This increased cytokine production was partly dependent on phagocytosis. In contrast, no difference in cytokine induction was observed with mouse macrophages. We investigated the structures of the lipopolysaccharides (LPSs) of these Gram-negative bacteria in biofilm and planktonic cultures of P. aeruginosa. Switch between the two life-styles was shown to cause several reversible LPS structure modifications affecting the lipid A and polysaccharide moieties of both clinical isolates and laboratory strains. In addition, LPS isolated from biofilm-grown bacteria induced slightly more inflammatory cytokines than that extracted from its planktonic counterpart. Our results, therefore, show that P. aeruginosa biofilm LPS undergoes structural modifications that only partially contribute to an increased inflammatory response from human monocytes.

PMID: 19710099 [PubMed - indexed for MEDLINE]
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J Lipid Res. 2009 May;50(5):854-9. doi: 10.1194/jlr.M800454-JLR200. Epub 2008 Nov 17.

Structural characterization of Bordetella parapertussis lipid A.

Author information:

  • 1Equipe Endotoxines, UMR 8619 du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.

 

Abstract

Bordetella parapertussis like B. pertussis, is a causal agent of whooping cough but is not a strictly human pathogen. Because its endotoxin, a major structural component of the Gram-negative outer membrane, is an important virulence factor, we have analyzed the structure of its toxic lipid domain, in one rough and two smooth bacterial strains. Chemical analyses and mass spectra obtained before and after recently developed mild-alkali treatments revealed that the lipids A have the common bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. All three strains have two major molecular species: a tetraacyl and a pentaacyl species. The rough strain is richer in a minor hexaacyl species. Acylation at the C-2, C-3, and C-3' positions was different from that of the B. pertussis lipid A. The C-2 position carries a secondary hexadecanoic acid, the C-3 position is free, and the C-3' position is substituted with hydroxydecanoic acid (not at C-3 as in B. pertussis), and the rough strain hexaacyl species carries a second secondary hexadecanoic acid. Like the lipid A of B. pertussis, the hydroxytetradecanoic acid at the C-2' position was substituted by tetradecanoic acid.

PMCID: PMC2666171 Free PMC Article
PMID: 19017615 [PubMed - indexed for MEDLINE]
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J Bacteriol. 2008 Jun;190(12):4281-90. doi: 10.1128/JB.01875-07. Epub 2008 Apr 18.

Glucosamine found as a substituent of both phosphate groups in Bordetella lipid A backbones: role of a BvgAS-activated ArnT ortholog.

Author information:

  • 1Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.

 

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.

PMCID: PMC2446747 Free PMC Article
PMID: 18424515 [PubMed - indexed for MEDLINE]
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J Lipid Res. 2007 Nov;48(11):2419-27. Epub 2007 Aug 16.

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element.

Author information:

  • 1Equipe Endotoxines, Unité Mixte de Recherche 8619 du Centre National de la Recherche Scientifique, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université de Paris-Sud, Orsay, France.

 

Abstract

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.

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PMID: 17703058 [PubMed - indexed for MEDLINE]
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18. Appl Environ Microbiol. 2007 Mar;73(6):1803-8. Epub 2007 Jan 19.

Simple method for repurification of endotoxins for biological use.

Author information:

  • 1Equipe "Endotoxines," UMR 8619 du CNRS, IBBMC, Université de Paris-Sud, 91405 Orsay, France. Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

A method for obtaining highly purified endotoxin (lipopolysaccharide [LPS]) in a few hours by repurification of commercial or laboratory preparations was devised. It avoids the use of phenol, which is not suitable for phenol-soluble lipopolysaccharides nor for some industrial purposes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry analysis confirmed the integrity of the purified LPSs. The purified products did not activate Toll-like receptor 2 (TLR2), nuclear oligomerization domain 1 (NOD1), or NOD2 but did activate TLR4. Applied to different lipopolysaccharides, the method also improved their mass spectra, thus facilitating their structural analysis.

PMCID: PMC1828799 Free PMC Article
  PMID: 17261511 [PubMed - indexed for MEDLINE]
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19. Eur J Immunol. 2005 Aug;35(8):2459-70.

Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-activating agonists.

Author information:

  • 1Groupe d'Immunité Innée et Signalisation, Institut Pasteur, Paris, France.

 

Abstract

Muropeptides are degradation products of bacterial peptidoglycan (PG) sensed by nucleotide-binding oligomerization domain 1 (NOD1) and NOD2, members of a recently discovered family of pattern recognition molecules (PRM). One of these muropeptides, muramyl dipeptide (MDP) mediates cell signaling by NOD2, exerts adjuvant activity and synergizes with lipopolysaccharide (LPS) to induce pro-inflammatory responses in vitro and in vivo. In contrast, few and contradictory results exist about the stimulatory capacity of NOD1 agonists. Thus, the ability of NOD1 (MurNAc-L-Ala--D-Glu-meso-diaminopimelic acid, MtriDAP) and NOD2 (MurNAc-L-Ala-D-isoGln, MDP; MurNAc-L-Ala--D-Glu-L-Lys, MtriLYS) agonists to activate primary human myeloid cells was examined. We show that both CD14+ monocytes and CD1a+ immature dendritic cells (DC) express NOD1 and NOD2 mRNA. Stimulation of primary human monocytes and DC with highly purified muropeptides (MtriDAP, MDP and MtriLYS) induces release of pro-inflammatory cytokines. We reveal here that NOD1 as well as NOD2 agonists act cooperatively with LPS to stimulate the release of both pro- and anti-inflammatory cytokines in these myeloid cell subsets. Finally, we report that NOD1 as well as NOD2 agonists synergize with sub-active doses of LPS to induce DC maturation, demonstrating that NOD agonists act cooperatively with molecules sensed by Toll-like receptor 4 to instruct the onset of adaptive immune responses.

  PMID: 16021602 [PubMed - indexed for MEDLINE]
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20. Rapid Commun Mass Spectrom. 2005;19(13):1851-7.

The Au(n) cluster probe in secondary ion mass spectrometry: influence of the projectile size and energy on the desorption/ionization rate from biomolecular solids.

Author information:

  • 1Institut de Physique Nucléaire d'Orsay, CNRS-IN2P3, 91406 Orsay, France. Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

A Au-Si liquid metal ion source which produces Au(n) clusters over a large range of sizes was used to study the dependence of both the molecular ion desorption yield and the damage cross-section on the size (n = 1 to 400) and on the kinetic energy (E = 10 to 500 keV) of the clusters used to bombard bioorganic surfaces. Three pure peptides with molecular masses between 750 and 1200 Da were used without matrix. [M+H](+) and [M+cation](+) ion emission yields were enhanced by as much as three orders of magnitude when bombarding with Au(400) (4+) instead of monatomic Au(+), yet very little damage was induced in the samples. A 100-fold increase in the molecular ion yield was observed when the incident energy of Au(9) (+) was varied from 10 to 180 keV. Values of emission yields and damage cross-sections are presented as a function of cluster size and energy. The possibility to adjust both cluster size and energy, depending on the application, makes the analysis of biomolecules by secondary ion mass spectrometry an extremely powerful and flexible technique, particularly when combined with orthogonal time-of-flight mass spectrometry that then allows fast measurements using small primary ion beam currents.

Copyright (c) 2005 John Wiley & Sons, Ltd.

  PMID: 15945024 [PubMed - indexed for MEDLINE]
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21. J Lipid Res. 2005 Aug;46(8):1773-8. Epub 2005 Jun 1.

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization.

Author information:

  • 1Equipe Endotoxines, Unité Mixte de Recherche 8619 du Centre National de la Recherche Scientifique, Institut de Biochimie, Biophysique Moléculaire et Cellulaire, Université de Paris-Sud, 91405 Orsay, France.

 

Abstract

Endotoxins (lipopolysaccharides) are the main components of Gram-negative bacterial outer membranes. A quick and simple way to isolate their lipid region (lipid A) directly from whole bacterial cells was devised. This method using hot ammonium-isobutyrate solvent was applied to small quantities of cells and proved to be indispensable when a rapid characterization of lipid A structure by mass spectrometry was required. Biological activities of endotoxins are directly related to the lipid A structures, which vary greatly with cell growth conditions. This method is suitable for rough- and smooth-type bacteria and very efficient for screening variations in lipid A structures. Data are acquired in a few hours and avoid the use of phenol in extraction.

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  PMID: 15930524 [PubMed - indexed for MEDLINE]
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22. J Proteome Res. 2005 Mar-Apr;4(2):540-5.

Matrix implanted laser desorption ionization (MILDI) combined with ion mobility-mass spectrometry for bio-surface analysis.

Author information:

  • 1Ionwerks, Inc., 2472 Bolsover St., Suite 255, Houston, Texas 77005, USA.

 

Abstract

The implantation of low velocity massive gold cluster ions allows homogeneous incorporation of a metallic matrix into the near-surface region of rat brain tissues. Subsequent analysis by laser desorption ionization mass spectrometry yields spectra exhibiting molecular ion peaks in the mass range up to 35 kDa similar to those observed by matrix-assisted LDI. Matrix-implanted LDI when combined with ion-mobility preseparation promises to be a useful technique for molecular imaging of biotissues with a laser microprobe.

  PMID: 15822932 [PubMed - indexed for MEDLINE]
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23. FEBS Lett. 2005 Jan 3;579(1):18-24.

Structure of the Bordetella trematum LPS O-chain subunit.

Author information:

  • 1Institute for Biological Sciences, National Research Council, 100 Sussex Dr., Ottawa, ON, Canada K1A 0R6.

 

Abstract

Analysis of the O-chain subunit of the lipopolysaccharide (LPS, endotoxin) isolated from Bordetella trematum, a recently identified human pathogen, was undertaken. The polysaccharide (PS) moiety was shown to contain only two O-chain subunits, which differed in the anomeric bond of their first sugar. A trisaccharide fragment resulting from the cleavage of a FucNAc glycosidic bond was isolated after treatment of the PS with anhydrous HF. Nitrous deamination of the LPS led to the release of the following heptasaccharide corresponding to two trisaccharide subunits linked to an anhydromannitol residue. beta-ManNAc3NAmA-(1-4)-beta-ManNAc3NAmA-(1-3)-alpha-FucNAc-(1-4)-beta-ManNAc3NAmA-(1-4)-beta-ManNAc3NAmA-(1-3)-beta-FucNAc-(1-6)-2,5-anhManol.

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  PMID: 15620685 [PubMed - indexed for MEDLINE]
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24. Anal Chem. 2004 Dec 15;76(24):7288-93.

Matrix-implanted laser desorption/ionization mass spectrometry.

Author information:

  • 1Institut de Physique Nucléaire, CNRS-IN2P3, 91406 Orsay, France, Equipe Endotoxines, UMR 8619 du CNRS, IBBMC, Université de Paris-Sud, 91405 Orsay, France.

 

Abstract

The implantation of low-velocity massive gold clusters is shown to be a method of choice for homogeneous incorporation of a metallic matrix into the near-surface region of a solid biopolymer for subsequent laser desorption/ionization (LDI) MS analysis. Matrix implanted (MI)LDI spectra from cluster-implanted pure test peptide or tissue exhibit molecular ion peaks similar to those observed by matrix-assisted LDI. Moreover, the ion emission is very reproducible from any spot on the surface of these test samples. MILDI promises to be a powerful technique for mass spectrometric analysis of native biological samples as demonstrated by the first results on rat brain tissues.

  PMID: 15595871 [PubMed - indexed for MEDLINE]
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25. Rapid Commun Mass Spectrom. 2004;18(4):371-6.

Orthogonal time-of-flight secondary ion mass spectrometric analysis of peptides using large gold clusters as primary ions.

Author information:

  • 1Ionwerks Inc., 2472 Bolsover St, Suite 255, Houston, TX 77030, USA. Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

Secondary ion mass spectrometry (SIMS) for biomolecular analysis is greatly enhanced by the instrumental combination of orthogonal extraction time-of-flight mass spectrometry with massive gold cluster primary ion bombardment. Precursor peptide molecular ion yield enhancements of 1000, and signal-to-noise improvements of up to 20, were measured by comparing SIMS spectra obtained using Au(+) and massive Au(400) (4+) cluster primary ion bombardment of neat films of the neuropeptide fragment dynorphin 1-7. Remarkably low damage cross-sections were also measured from dynorphin 1-7 and gramicidin S during prolonged bombardment with 40 keV Au(400) (4+). For gramicidin S, the molecular ion yield increases slightly as a function of Au(400) (4+) beam fluence up to at least 2 x 10(13) Au(400) (4+)/cm(2). This is in marked contrast to the rapid decrease observed when bombarding with ions such as Au(5) (+) and Au(9) (+). When gramicidin S is impinged with Au(5) (+), the molecular ion yield decreases by a factor of 10 after a fluence of only 8 x 10(12) ions/cm(2). Comparison of these damage cross-sections implies that minimal surface damage occurs during prolonged Au(400) (4+) bombardment. Several practical analytical implications are drawn from these observations.

Copyright 2004 John Wiley & Sons, Ltd.

  PMID: 14966842 [PubMed - indexed for MEDLINE]
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26. Carbohydr Res. 2003 Nov 14;338(23):2431-47.

Structure of bacterial lipopolysaccharides.

Author information:

  • 1Equipe Endotoxines, UMR 8619 du Centre National de la Recherche Scientifique, IBBMC, Université de Paris-Sud, F-91405 Orsay, France.Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

Bacterial lipopolysaccharides are the major components of the outer surface of Gram-negative bacteria They are often of interest in medicine for their immunomodulatory properties. In small amounts they can be beneficial, but in larger amounts they may cause endotoxic shock. Although they share a common architecture, their structural details exert a strong influence on their activity. These molecules comprise: a lipid moiety, called lipid A, which is considered to be the endotoxic component, a glycosidic part consisting of a core of approximately 10 monosaccharides and, in "smooth-type" lipopolysaccharides, a third region, named O-chain, consisting of repetitive subunits of one to eight monosaccharides responsible for much of the immunospecificity of the bacterial cell.

  PMID: 14670707 [PubMed - indexed for MEDLINE]
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27. Mol Microbiol. 2003 May;48(3):725-36.

Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract.

Author information:

  • 1Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

Bordetella bronchiseptica lipopolysaccharide (LPS) expression varies depending on growth conditions, regulated by the Bvg system. A B. bronchiseptica pagP homologue was identified that is required for Bvg-mediated modification of the lipid A core region of LPS that occurs on switching from the Bvg- to the Bvg+ phase. Structural analysis demonstrated that the lipid A of a B. bronchiseptica pagP mutant differed from wild-type lipid A by the absence of a palmitate group in secondary acylation at the C3' position. The putative pagP promoter drove the expression of a green fluorescent protein (GFP) reporter gene in a Bvg-regulated fashion. These data suggest that B. bronchiseptica pagP encodes a Bvg-regulated lipid A palmitoyl transferase that mediates modification of the lipid A as part of the overall Bvg-mediated adaptation of this organism to changing environmental conditions. We also show that pagP is not required for the initial colonization of the mouse respiratory tract by B. bronchiseptica, but is required for persistence of the organism within this organ.

  PMID: 12694617 [PubMed - indexed for MEDLINE]
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28. Nat Immunol. 2003 May;4(5):478-84.

The Drosophila immune system detects bacteria through specific peptidoglycan recognition.

Author information:

  • 1Centre de Génétique Moléculaire du CNRS, F-91198 Gif-sur-Yvette, France.

 

Abstract

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. Here we report that Gram-negative diaminopimelic acid-type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan.

  PMID: 12692550 [PubMed - indexed for MEDLINE]
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29. FEBS Lett. 2003 Jan 30;535(1-3):11-6.

Structural characterization of the O-chain polysaccharide isolated from Bordetella avium ATCC 5086: variation on a theme(1).

Author information:

  • 1Institute for Biological Sciences, NRC, Ottawa, ON, Canada.

 

Abstract

The O-chain polysaccharide (O-PS) of Bordetella avium was isolated from the lipopolysaccharide by mild acid hydrolysis to remove the lipid A, followed by hydrofluorolysis to remove the lipopolysaccharide core oligosaccharide leaving a residual O-PS for structural analysis. High resolution (1)H and (13)C NMR and MALDI studies showed the O-chain to be a polymer composed of 1,4-linked 2-acetamidino-3-[3-hydroxybutanamido]-2,3-dideoxy-beta-D-glucopyranosyluronic acid residues.

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  PMID: 12560070 [PubMed - indexed for MEDLINE]
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30. Microbes Infect. 2002 Jul;4(9):915-26.

Structural and functional analyses of bacterial lipopolysaccharides.

Author information:

  • 1Equipe Endotoxines, UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, Orsay, France.Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

Bacterial lipopolysaccharides (LPSs) are powerful immunomodulators in infected hosts, and may cause endotoxic shock. Most of them share a common architecture but vary considerably in structural motifs from one genus, species, and strain to another. Cells of the innate immune response recognize evolutionarily conserved LPS molecular patterns of endotoxins and structural details thereby greatly influencing their response.

  PMID: 12106784 [PubMed - indexed for MEDLINE]
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31. Anal Chem. 2001 Aug 15;73(16):3804-7.

Direct microextraction and analysis of rough-type lipopolysaccharides by combined thin-layer chromatography and MALDI mass spectrometry.

Author information:

  • 1Equipe Endotoxines, UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, Orsay, France.

 

Abstract

A rapid method for the microscale extraction of lipopolysaccharides (endotoxins, LPSs) from rough-type Gram-negative bacteria was developed using thin-layer chromatography (TLC) combined with improved conditions for LPS analysis by mass spectrometry. TLC of intact bacteria on silica gel plates in an appropriate solvent selectively extracted and separated their LPS components. The bands of molecular species were scraped from the plates after nondestructive visualization, directly mixed with matrix, and analyzed by laser desorption time-of-flight mass spectrometry. Lipids A and Re-type LPSs were analyzed after transfer to a membrane. Adding citric acid to the matrix gave greatly improved mass spectra. The system allows characterization of bacterial LPS at the microscale level and is equally well applicable to heterogeneous LPS and lipid A preparations (Escherichia coli lipid A and Bordetella lipopolysaccharides were used). The technique provides a rapid determination of the heterogeneity of unmodified preparations and the determination of the molecular weight of each separated component.

  PMID: 11534700 [PubMed - indexed for MEDLINE]
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32. J Endotoxin Res. 2001;7(1):63-8.

Structural variability and originality of the Bordetella endotoxins.

Author information:

  • 1Equipe 'Endotoxines', UMR 8619, Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, Orsay, France.Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

Structural studies of Bordetella endotoxins (LPSs) have revealed remarkable differences: (i) between their LPSs and those of other bacterial pathogens; (ii) among the LPSs of the seven identified Bordetella species; and (iii) among the LPSs of some Bordetella strains. The lipid As have the "classical" bisphosphorylated diglucosamine backbone but tend to have fewer and species-specific fatty acid components compared to those of other genera. Nevertheless, three strains of B. bronchiseptica have at least three different fatty acid distributions; however, the recently identified B. hinzii and B. trematum LPSs had identical lipid A structures. The B. pertussis core is a dodecasaccharide multi-branched structure bearing amino and carboxylic groups. Another unusual feature is the presence of free amino sugars in the central core region and a complex distal trisaccharide unit containing five amino groups of which four are acetylated and one is methylated. The B. pertussis LPS does not have O-chains and that of B. trematum had only a single O-unit, unlike the LPSs of all the other species of the smooth-type. The O-chain-free cores of non-B. pertussis LPSs were always built on the B. pertussis core model but most were species-specifically incomplete. The LPS structures of three B. bronchiseptica strains were found to be different from each other. The O-chains of B. bronchiseptica and B. parapertussis were almost identical and had some features in common with B. hinzii O-chain. Serological analyses are consistent with the determined LPS structures.

  PMID: 11521085 [PubMed - indexed for MEDLINE]
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33. FEBS Lett. 2001 Jun 15;499(1-2):1-5.

Helicobacter mustelae lipid A structure differs from that of Helicobacter pylori.

Author information:

  • 1Equipe Endotoxines, UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, Orsay, France.

 

Abstract

The lipid A structure of the Gram-negative bacterium Helicobacter mustelae, a ferret gastric pathogen responsible for the onset of gastric diseases in its host, was investigated. Two variant lipid A structures were found in the same strain. One structure contained a bisphosphorylated beta-(1-->6)-linked D-glucosamine backbone disaccharide with hydroxytetradecanoic acid in amide linkages. Unlike the structure described for the lipid A of the related human Helicobacter pylori gastric pathogen, which contains a C1 phosphate moiety, this lipid A presented phosphate groups at both the C1 and C4' positions, and contained no octadecanoyl fatty acid, which is present in H. pylori. The second lipid A structure had a different fatty acid composition in that 3-OH C(16) replaced most of the amide-linked 3-OH C(14).

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  PMID: 11418100 [PubMed - indexed for MEDLINE]
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34. FEBS Lett. 2000 Nov 17;485(1):40-6.

Chemical and serological characterization of the Bordetella hinzii lipopolysaccharides.

Author information:

  • 1Equipe Endotoxines, UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, Orsay, France.

 

Abstract

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The polysaccharides isolated from its endotoxin (lipopolysaccharide, LPS) were investigated using chemical analyses, NMR, gas-liquid chromatography/mass spectrometry and mass spectrometry by plasma desorption, matrix-assisted laser desorption/ionization and electrospray. The following structure for the O-chain-free LPS was deduced from the experimental results: carbohydrate structure [see text] Mass spectrometry and serology revealed that the O-chains were different from the homopolymer common to Bordetella bronchiseptica and Bordetella parapertussis strains and were composed of a trisaccharide repeating unit. Masses up to 8 kDa were obtained for native LPS molecular species.

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  PMID: 11086162 [PubMed - indexed for MEDLINE]
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35. FEBS Lett. 2000 Jul 14;477(1-2):8-14.

Structure of the Bordetella pertussis 1414 endotoxin.

Author information:

  • 1Equipe Endotoxines, UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris- Sud, F-91405, Orsay, France. Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

The endotoxin (lipopolysaccharide) of Bordetella pertussis, the agent of whooping cough, consists of a lipid A linked to a highly branched dodecasaccharide containing several acid and amino sugars. The elucidation of the polysaccharide structure was accomplished by first analyzing the structures of fragments obtained by hydrolysis and nitrous deamination and then piecing the fragments together. The fine structure of the antigenic distal pentasaccharide, presented here, was determined by chemical analyses as well as by high-resolution nuclear magnetic resonance and mass spectrometry. The complete structure was reconstituted and confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The following structure was derived from the combined experimental data:The detailed structure combined with previously reported serological data now allows the synthesis of its epitopes for potential vaccines.

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  PMID: 10899302 [PubMed - indexed for MEDLINE]
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36. Rapid Commun Mass Spectrom. 2000;14(7):595-9.

Structure of the lipid A of Bordetella hinzii ATCC 51730.

Author information:

  • 1Equipe 'Endotoxines', UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, Orsay, France.

 

Abstract

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The structure of the lipid A of its endotoxin was investigated using chemical analyses, nuclear magnetic resonnance (NMR), gas liquid chromatography/mass spectrometry (GC/MS), plasma desorption mass spectrometry (PDMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The lipid A contains the classical bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid (C14OH) in amide linkages. The lipid A components of B. pertussis, B. bronchiseptica, and B. parapertussis all differ in their acylation pattern but share a residue of tetradecanoyl-3-hydroxytetradecanoic acid in amide linkage at the C-2' position. However, in the B. hinzii species, the tetradecanoic acid (C14) is stoichiometrically replaced by a 2-hydroxytetradecanoic acid (2-C14OH). In the few reported examples of a hydroxylated fatty acid in this position, the substitutions were only partial. The B. hinzii lipid A differs from that of B. pertussis also by replacement of the hydroxydecanoic acid (C10OH) by hydroxydodecanoic acid (C12OH) and by the presence of a hexadecanoic acid (C16) to give a sixth fatty acid. The lipid A was heterogeneous, being composed of three major molecular species: tetra-, penta- and hexaacylated. The fatty acids in ester linkage were localized by PDMS of the native and alkali-treated lipid A. The lipid A components isolated from the O-chain-linked lipopolysaccharides (LPSs) were shown to be more acylated than those from the O-chain-free LPSs.

  PMID: 10775094 [PubMed - indexed for MEDLINE]
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37. FEBS Lett. 2000 Jan 7;465(1):87-92.

Novel variation of lipid A structures in strains of different Yersinia species.

Author information:

  • 1Equipe 'Endotoxines', UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, F-91405, Orsay, France.

 

Abstract

The Yersinia genus includes human and animal pathogens (plague, enterocolitis). The fine structures of the endotoxin lipids A of seven strains of Yersinia enterocolitica, Yersinia ruckeri and Yersinia pestis were determined and compared using mass spectrometry. These lipids differed in secondary acylation at C-2': this was dodecanoic acid (C(12)) for two strains of Y. enterocolitica and Y. ruckeri, tetradecanoic acid (C(14)) in two other Y. enterocolitica and hexadecenoic acid (C(16:1)) in Y. pestis. The enterocolitica lipids having a mass identical to that of Escherichia coli were found to be structurally different. The results supported the idea of a relation between membrane fluidity and environmental adaptability in Yersinia.

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  PMID: 10620712 [PubMed - indexed for MEDLINE]
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38. Rapid Commun Mass Spectrom. 1999;13(22):2252-9.

252Cf-plasma desorption mass spectrometry of unmodified lipid A: fragmentation patterns and localization of fatty acids.

Author information:

  • 1Equipe 'Endotoxines', UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, F-91405 Orsay, France. Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

Abstract

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.

Copyright 1999 John Wiley & Sons, Ltd.

  PMID: 10547633 [PubMed - indexed for MEDLINE]
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39. Nephrol Dial Transplant. 1999 Apr;14(4):853-60.

Molecular aspects of endotoxins relevant to their biological functions.

Author information:

  • 1INSERM U430, Hôpital Broussais, Paris, France.

 

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  PMID: 10328458 [PubMed - indexed for MEDLINE]
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40. Eur J Immunol. 1997 Dec;27(12):3303-9.

Lipopolysaccharide complexed with soluble CD14 binds to normal human monocytes.

Author information:

  • 1INSERM U430, Hôpital Broussais, Paris, France.

 

Abstract

Using flow cytometry we have compared the binding of Neisseria meningitidis lipopolysaccharide labeled with fluorescein isothiocyanate (FITC-LPS) to normal human monocytes in whole blood with the binding to chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO cells). Binding of FITC-LPS to cells was dose dependent, saturable and enhanced in the presence of increasing concentrations of serum. Blockade of membrane CD14 with saturating concentrations of anti-CD14 monoclonal antibody (mAb) My4 inhibited 50% of the binding of FITC-LPS to monocytes and 100% to hCD14-CHO cells. Similarly, removal of membrane CD14 by phosphatidylinositol phospholipase C (PI-PLC) treatment of the cells partially decreased the binding of FITC-LPS to monocytes but totally inhibited the binding to hCD14-CHO-transfected cells. These results suggest that binding of FITC-LPS to monocytes is not only mediated by membrane CD14. Using two-color flow cytometry, we observed that FITC-LPS binds to My4-saturated monocytes in association with soluble (s)CD14 present in serum as revealed by staining with rhodamine-labeled My4 mAb. The binding of FITC-LPS/sCD14 complexes to monocytes treated with saturating amounts of unlabeled My4 prior to addition of the complexes was completely inhibited by anti-CD14 mAb 10G33. When cells were first saturated with a mixture of My4 and 10G33 mAb, washed and further incubated with FITC-LPS/sCD14, inhibition of the binding of LPS was similar to that observed with cells saturated with My4 alone, showing that the binding of FITC-LPS is not mediated by the 10G33 epitope present on mCD14. These results suggest that either the 10G33 epitope on sCD14 is involved in the binding of LPS/sCD14 complexes to the cells, or that 10G33 mAb inhibits the binding of FITC-LPS to sCD14. Taken together, these data indicate that sCD14 which is present in normal serum, in addition to membrane CD14, enables LPS to bind monocytes through an as yet unidentified molecule and that sCD14 does not simply serve as a shuttle for transfer of LPS to membrane CD14.

  PMID: 9464818 [PubMed - indexed for MEDLINE]
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41. Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6712-7.

Alterations of the outer membrane composition in Escherichia coli lacking the histone-like protein HU.

Author information:

  • 1Unité Propre de Recherche 7090, Centre National de la Recherche Scientifique, Laboratoire de Physiologie Bacterienne, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France.

 

Abstract

Escherichia coli cells lacking the histone-like protein HU form filaments and have an abnormal number of anucleate cells. Furthermore, their phenotype resembles that of rfa mutants, the well-characterized deep-rough phenotype, as they show an enhanced permeability that renders them hypersensitive to chloramphenicol, novobiocin, and detergents. We show that, unlike rfa mutants, hupAB mutants do not have a truncated lipopolysaccharide but do have an abnormal abundance of OmpF porin in their outer membrane. While the complete absence of HU does not abolish the osmoregulation of OmpF protein synthesis, the steady-state level of micF RNA, the negative regulator of OmpF, decreases in bacteria lacking HU, increasing the basal level of this membrane protein. These findings demonstrate a novel link between a bacterial chromosomal protein and the outer membrane composition.

PMCID: PMC21223 Free PMC Article
  PMID: 9192630 [PubMed - indexed for MEDLINE]
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42. J Bacteriol. 1997 Jun;179(11):3756-60.

Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution.

Author information:

  • 1Equipe Endotoxines, URA 1116 du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.

 

Abstract

The structures of lipids A isolated from the lipopolysaccharides (LPSs; endotoxins) of three different pathogenic Bordetella bronchiseptica strains were investigated by chemical composition and methylation analysis, gas chromatography-mass spectrometry, nuclear magnetic resonance, and plasma desorption mass spectrometry (PDMS). The analyses revealed that the LPSs contain the classical lipid A bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. Their structures differ from that of the lipid A of Bordetella pertussis endotoxin by the replacement of hydroxydecanoic acid on the C-3 position with hydroxydodecanoic acid or dodecanoic acid and the presence of variable amounts of hexadecanoic acid. The dodecanoic acid is the first nonhydroxylated fatty acid to be found directly linked to a lipid A glucosamine. The lipids A were heterogeneous and composed of one to three major and several minor molecular species. The fatty acids in ester linkage were localized by PDMS of chemically modified lipids A. B. pertussis lipids A are usually hypoacylated with respect to those of enterobacterial lipids A. However, one of the three B. bronchiseptica strains had a major hexaacylated molecular species. C-4 and C-6' hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site of the polysaccharide. The structural variability seen in these three lipids A was unusual for a single species and may have consequences for the pathogenicity of this Bordetella species.

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  PMID: 9171426 [PubMed - indexed for MEDLINE]
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43. Microbiology. 1996 Apr;142 ( Pt 4):971-8.

Epitopes of Bordetella pertussis lipopolysaccharides as potential markers for typing of isolates with monoclonal antibodies.

Author information:

  • 1Equipe Endotoxines, URA-1116 du CNRS, Université de Paris-Sud, Orsay, France.

 

Abstract

Three hybridomas (P1P3, D7 and 60.5) producing monoclonal antibodies (mAbs) against Bordetella pertussis lipopolysaccharide (LPS) were established. All reacted with the LPS from a typical, vaccine strain of B. pertussis (1414), but not with that of a variant strain (A100). Two of these mAbs (P1P3 and 60.5) cross-reacted with a B. bronchiseptica LPS; only one (P1P3) reacted with a B. parapertussis LPS. ELISA reactivities with intact LPSs, and defined partial structures covalently linked to bovine serum albumin, were compared. mAb 60.5 bound to the terminal region of a distal trisaccharide consisting of N-acetylated amino sugars. D7 reacted with a substructure which can be modified in the B. parapertussis and B. bronchiseptica LPSs by addition of a polymeric O-chain. P1P3 bound to a nonacetylated glucosamine substituted with L-glycero-D-manno-heptose, present in the 'core' of the B. pertussis LPS. These mAbs may be useful for rapid typing of Bordetella in clinical isolates.

  PMID: 8936324 [PubMed - indexed for MEDLINE]
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44. Prog Clin Biol Res. 1995;392:103-11.

Use of plasma desorption mass spectrometry in structural analysis of endotoxins: effects on lipid A of different acid treatments.

Author information:

  • 1Equipe "Endotoxines," URA 1116 du Centre National de la Recherche Scientifique, Institut de Biochimie, Université de Paris-Sud, F-91405 Orsay, France.

 

  PMID: 8524917 [PubMed - indexed for MEDLINE]
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45. Rapid Commun Mass Spectrom. 1995;9(8):693-6.

252Cf-plasma desorption mass spectrometry analysis of lipids A obtained by an elimination reaction under mild conditions.

Author information:

  • 1Equipe 'Endotoxines', URA 1116 du Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, Orsay, France.

 

Abstract

Lipids A are the hydrophobic domains of bacterial endotoxic lipopolysaccharides. Since they are responsible for most of the biological activities (both pathogenic and beneficial) of endotoxins, the characterization of their structure is crucial to the understanding of their mode of action. However, the inadequacy of existing methods for preparing certain lipids A has prompted us to devise a new, mild procedure which gives intact products. Use was made of the special features of 252Cf-plasma desorption mass spectrometry for forming molecular ions from these species and giving qualitative and quantitative information from the primary mass spectrum.

  PMID: 7647366 [PubMed - indexed for MEDLINE]
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46. J Biol Chem. 1994 Dec 16;269(50):31881-4.

Distribution of lipid A species between long and short chain lipopolysaccharides isolated from Salmonella, Yersinia, and Escherichia as seen by 252Cf plasma desorption mass spectrometry.

Author information:

  • 1URA 1116 du Centre National de la Recherche Scientifique, Institut de Biochimie, Université de Paris, Orsay, France.

 

Abstract

Smooth type endotoxins of Salmonella, Yersinia, and Escherichia were fractionated into long and short chain lipopolysaccharides by silica gel chromatography. Lipid A was prepared from the fractions and analyzed by plasma desorption mass spectrometry. Both Yersinia and Salmonella endotoxins had a large proportion of aminoarabinose-containing lipopolysaccharide molecular species that were found to be concentrated in the long chain fraction. In the Escherichia endotoxin, hypoacylated lipopolysaccharides (lacking the tetradecanoate and one of the four hydroxytetradecanoates) were found mostly in the short chain fraction. Possible implications of these results for the lipopolysaccharide biosynthetic pathway and for studies on the influence of sugar chain length on the biological effects of endotoxins are discussed.

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47. Microbiology. 1994 Sep;140 ( Pt 9):2459-65.

Specific and cross-reacting monoclonal antibodies to Bordetella parapertussis and Bordetella bronchiseptica lipopolysaccharides.

Author information:

  • 1URA-1116 du CNRS, Université de Paris-Sud, Orsay, France.

 

Abstract

Three groups of monoclonal antibodies (mAbs) were produced that would be useful for immunochemical typing and diagnosis of infections due to Bordetella species, and for the structural analysis of their lipopolysaccharides. PP6, a representative of the first group, recognizes an epitope shared by smooth-type Bordetella parapertussis and Bordetella bronchiseptica lipopolysaccharides (LPS). This epitope is carried by structurally identical polymeric O-chains (POC) present on both LPS molecules. PP8 and PP9 are representatives of the second group of mAbs. The interaction of PP8 and PP9 with B. parapertussis and B. bronchiseptica LPS requires POC, but periodate-sensitive sugar units of the core are also involved in the binding. The mAb BRg1 belongs to the third group, and specifically recognizes B. bronchiseptica LPS. Binding and inhibition studies with various Bordetella LPS molecules, and with their polysaccharide fragments, indicated that BRg1 interacts with a structure located at the hinge between the POC and a core region of the B. bronchiseptica LPS containing periodate-resistant sugars. This suggests that the structures of the hinge regions of the B. parapertussis and B. bronchiseptica LPS are different.

  PMID: 7524956 [PubMed - indexed for MEDLINE]
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48. J Bacteriol. 1994 Aug;176(16):5156-9.

Structural characterization of the lipid A of Bordetella pertussis 1414 endotoxin.

Author information:

  • 1Equipe Endotoxines, URA 1116 du Centre National de la Recherche Scientifique, Institut de Biochimie, Université de Paris-Sud, Orsay, France.

 

Abstract

The structure of Bordetella pertussis 1414 lipid A was investigated by classical methods of chemical analysis as well as plasma desorption mass spectrometry and fast atom bombardment mass spectrometry. Previous analysis showed that it contained a bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkage. The presence of two main molecular species as seen by thin-layer chromatography was confirmed by plasma desorption mass spectrometry, in which the larger signal was attributable to a molecular ion containing two glucosamine, two phosphate, one tetradecanoic acid, one hydroxydecanoic acid, and three hydroxytetradecanoic acid residues. The ion of the smaller signal was lighter by the mass of one hydroxytetradecanoic acid residue (226 Da). The fatty acids in ester linkage were localized by chemical and fast atom bombardment mass spectrometry analysis. C-4 and C-6' hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site for Kdo (3-deoxy-D-manno-octulosonic acid).

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  PMID: 8051033 [PubMed - indexed for MEDLINE]
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49. Carbohydr Res. 1994 Jun 17;259(2):257-75.

Structure of a hexasaccharide proximal to the hydrophobic region of lipopolysaccharides present in Bordetella pertussis endotoxin preparations.

Author information:

  • 1Equipe "Endotoxines" (U.R.A. 1116) du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.

 

Abstract

A branched-chain hexasaccharide containing 3-deoxy-D-manno-oct-2-ulosonic acid was released by detergent-promoted hydrolysis from Bordetella pertussis endotoxin preparations that were first dephosphorylated with aqueous HF and then treated with nitrous acid. Its structure (2) [Formula: See text] was determined by chemical and physical methods. This hexasaccharide is present in all four lipopolysaccharides that make up the B. pertussis strain 1414 (phase 1) endotoxin preparations analysed, and is situated near to the hydrophobic domains. An analogous structure reported previously (ref 7) is erroneous and should be disregarded.

  PMID: 8050099 [PubMed - indexed for MEDLINE]
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50. FEMS Immunol Med Microbiol. 1993 Aug;7(2):119-29.

Structural features involved in the mitogenic activity of Bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice.

Author information:

  • 1Equipe Edotoxines, URA-1116 du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.

 

Abstract

Spleen cells from the C3H/HeJ mouse strain cannot be stimulated by many smooth-type lipopolysaccharides (LPSs), and by the main biologically-active region (lipid A) of these molecules. The genetic origin of this defect (expression of the mutant allele Lpsd at the chromosome 4 locus) was established over 20 years ago, but its biochemical nature has remained undefined. Several investigators have noted, however, that some particular LPSs can bypass this defect, and stimulate the proliferation of C3H/HeJ B lymphocytes. In this study we compare the mitogenic activities of the LPSs isolated from a wild strain (1414) and from a mutant 'rough' strain (A100) of Bordetella pertussis. Both LPS-1414 and LPS-A100 were mitogenic for C3H/HeJ spleen cells, but their lipid A fragments were not. This indicates that a carbohydrate structure proximal to lipid A is involved in the mitogenic activity. However, the isolated polysaccharides were not mitogenic. Four sugars are common to both LPS-1414 and LPS-A100: an heptose, and three sugars bearing free amino groups. After removal of these four sugars from the LPSs by nitrous acid treatment, the recovered lipooligosaccharides were not mitogenic in Lpsd spleen cells. The results suggest that substructures present in lipid A and in this group of four sugars are both required for induction of a mitogenic effect in Lpsd splenocytes, whereas lipid A alone can stimulate Lpsn spleen cells.

  PMID: 8401423 [PubMed - indexed for MEDLINE]
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51. J Biol Chem. 1993 Jun 15;268(17):12321-4.

252Cf plasma desorption mass spectrometry applied to the analysis of underivatized rough-type endotoxin preparations.

Author information:

  • 1URA 1116 du Centre National de la Recherche Scientifique, Institut de Biochimie, Université de Paris-Sud, Orsay.

 

Abstract

Plasma desorption mass spectrometry has recently been used with success to characterize native, underivatized Re- to Rc-type endotoxins in terms of their constituent lipopolysaccharides. The spectra give masses for the major molecular species of lipopolysaccharide present from which their probable compositions could be inferred using the overall composition determined by chemical analyses. Moreover, the relative intensities of the signals are roughly proportional to the abundance of their corresponding molecular species. Native Rc-, Rb-, and Ra-type enterobacterial endotoxins with 5-10 core sugar units have been rendered amenable to plasma-desorption mass spectrometry analysis by improvement in their solubility and the use of cellobiose as an additive. The spectra of four Salmonella and Escherichia endotoxin preparations demonstrated heterogeneity in acylation and phosphorylation. Since these sources of heterogeneity are critical for many biological activities, the spectra underline the need to define the composition of each preparation of endotoxin used in structure-function studies.

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  PMID: 8509370 [PubMed - indexed for MEDLINE]
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52. J Bacteriol. 1993 May;175(10):2988-93.

Comparison of lipids A of several Salmonella and Escherichia strains by 252Cf plasma desorption mass spectrometry.

Author information:

  • 1Université de Paris-Sud, Centre National de la Recherche Scientifique URA 1116, Orsay, France.

 

Abstract

Plasma desorption mass spectrometry has recently been used with success to characterize underivatized lipid A preparations: the major molecular species present give signals indicating their masses, from which probable compositions could be inferred by using the overall composition determined by chemical analyses. In the present study, plasma desorption mass spectrometry was used to compare structures in lipid A preparations isolated from several smooth and rough strains of Escherichia and Salmonella species. Preparations isolated from strains of both genera revealed considerable variation in degree of heterogeneity (number of fatty acids and presence or absence of hexadecanoic acid, phosphorylethanolamine, and aminoarabinose). Molecular species usually associated with Salmonella lipid A were found in preparations from Escherichia sp. In addition, preparations from three different batches of lipid A from one strain of Salmonella minnesota showed significant differences in composition. These results demonstrate that preparations used for biological and structural analyses should be defined in terms of their particular molecular constituents and that no generalizations based on analysis of a single preparation should be made.

PMCID: PMC204617 Free PMC Article
  PMID: 8491718 [PubMed - indexed for MEDLINE]
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53. J Am Geriatr Soc. 1993 May;41(5):550-1.

Dopamine B hydroxylase deficiency responsible for severe dysautonomic orthostatic hypotension in an elderly patient.

Author information:

  • 1Clinique Médicale Harvier, CHU A. Morvan, Brest.

 

Abstract

We report the case of an elderly woman with severe dysautonomic orthostatic hypotension in whom a deficit in dopamine B hydroxylase has been established. In the literature, such a deficit has been described in six young adults with long standing symptoms of postural hypotension. This enzyme catalyses the conversion of dopamine to noradrenaline. In our elderly patient, noradrenaline and adrenaline were undetectable in the plasma, but plasma dopamine was detectable. Treatment with the synthetic amino acid, DL-threo-dihydroxyphenylserine, which is converted to noradrenaline by dopa-decarboxylase, resulted in a significant increase in blood pressure. The mechanism of this acquired deficit is not elucidated.

  PMID: 8486891 [PubMed - indexed for MEDLINE]
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54. FEMS Microbiol Lett. 1992 Oct 15;76(3):275-81.

Characterization of the common antigenic lipopolysaccharide O-chains produced by Bordetella bronchiseptica and Bordetella parapertussis.

Author information:

  • 1Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.

 

Abstract

Representative strains of Bordetella bronchiseptica and B. parapertussis were found to produce smooth lipopolysaccharides (LPS) having identical antigenic O-polysaccharide components composed of linear unbranched polymers of 1,4-linked 2,3-diacetamido-2,3-dideoxy-alpha-L-galacto-pyranosyluronic acid residues. These LPSs differed from the LPS of B. pertussis which produces only rough-type LPS, devoid of O-polysaccharide. While B. bronchiseptica and B. parapertussis had chemically and immunologically identical O-polysaccharide structures, their core oligosaccharide components differed. The core oligosaccharide of B. parapertussis was chemically distinct from the core of B. bronchiseptica which appeared to be structurally and immunologically similar to a core oligosaccharide of B. pertussis.

  PMID: 1427018 [PubMed - indexed for MEDLINE]
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55. J Biol Chem. 1991 Oct 5;266(28):18543-9.

Analysis of unmodified endotoxin preparations by 252Cf plasma desorption mass spectrometry. Determination of molecular masses of the constituent native lipopolysaccharides.

Author information:

  • 1Equipe Endotoxines du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.

 

Abstract

Nine unmodified endotoxin preparations constituted of Re-, Rd-, and Rc-type lipopolysaccharides (2 to 5 glycoses), representing four species of enterobacteria were analyzed by 252Cf plasma desorption mass spectrometry. The constituent lipopolysaccharides were characterized by the ion pair: (M-H)- and its corresponding lipid fragment ion. The lipid fragment ion is produced by cleavage of the glycosidic bond of the 3-deoxy-D-manno-oct-2-ulosonic acid unit that substitutes O-6' of the glucosamin beta 1'-6glucosamine ("lipid A backbone") disaccharide of the lipid A moiety. These lipid fragment ions were identical to the (M-H)- ions seen in the spectra of homologous isolated lipid A preparations that were obtained by hydrolysis (pH 4.5, 100 degrees C) promoted by sodium dodecyl sulfate. Since the molecular components present in the endotoxin preparations analyzed are known, the ion pair (M-H)(-)-lipid fragment ion defines the molecular compositions of each individual lipopolysaccharide. Heterogeneity of the R-type endotoxin preparations analyzed was due almost exclusively to differing lipid A moieties. In three Salmonella minnesota 595 Re endotoxin preparations 10 different lipopolysaccharides were identified, only two of which were common to all three preparations. Of the nine lipopolysaccharides identified in two S. minnesota R7 endotoxin preparations, only two were present in both.

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  PMID: 1917976 [PubMed - indexed for MEDLINE]
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56. J Immunol. 1991 Sep 15;147(6):1899-904.

Binding sites for endotoxins (lipopolysaccharides) on human monocytes.

Author information:

  • 1INSERM U 28, Hôpital Broussais, Paris, France.

 

Abstract

The nature of the binding sites for LPS on human monocytes was investigated using [3H] labeled intact LPS from Neisseria meningitidis and from Salmonella minnesota R7, and the [3H] labeled purified inner core region (PS-OMe) of S.m. R7 LPS. In the presence of serum, intact LPS from enterobacterial and nonenterobacterial strains bound to monocytes in a dose-dependent, saturable, and displaceable fashion. N.m. LPS and LPS from the enterobacterial strain of Escherichia coli 0111-B4 bound to the same sites on monocytes as assessed in competitive binding experiments. Specific binding of intact LPS to monocytes occurred through the CD14 molecule as shown by the ability of mAb and of F(ab')2 fragments of mAb directed against specific epitopes of CD14 to inhibit the binding of [3H]-LPS to cells and by the lack of binding of intact LPS to CD14-deficient cells from patients with paroxysmal nocturnal hemoglobinuria. Specific binding of LPS to monocytes was not mediated by the CD11/CD18 complex because mAb to the alpha and beta chains of the Leu-CAM molecules did not alter the binding of LPS to cells and because LPS did not inhibit the binding of labeled mAb to monocytes. [3H]-PS-OMe also bound in a dose-dependent and displaceable fashion to monocytes involving an unidentified, non-CD14, binding site on the cells. Binding of LPS to monocytes also involved nonsaturable binding sites for hydrophobic structures of LPS as evidenced in binding experiments performed in the absence of serum. These observations indicate that intact LPS may interact with the monocyte membrane in at least three ways including serum-dependent binding to CD14 and to a lectin-like receptor, and serum-independent hydrophobic interactions.

  PMID: 1716284 [PubMed - indexed for MEDLINE]
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57. Kidney Int. 1990 Dec;38(6):1089-94.

Induction of IL-1 during hemodialysis: transmembrane passage of intact endotoxins (LPS).

Author information:

  • 1INSERM U 28, Hôpital Broussais, Paris, France.

 

Abstract

Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

  PMID: 2127434 [PubMed - indexed for MEDLINE]
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58. Cytokine. 1990 Jul;2(4):253-8.

Dissociation between the interleukin 1-inducing capacity and Limulus reactivity of lipopolysaccharides from gram-negative bacteria.

Author information:

  • 1INSERM U28, Hôpital Broussais, Paris, France.

 

Abstract

In this study we compared the interleukin 1 (IL 1)-inducing capacity and the reactivity in the Limulus amoebocyte assay (LAL) of purified lipopolysaccharides (LPSs) from various bacterial strains. LPSs differed greatly in their capacities (on a weight basis) to induce IL 1 release from serum-free cultured human monocytes. LPS species that induced high levels of IL 1 release from human monocytes exhibited a high thiobarbiturate-reactive 2-keto-3-deoxy-octonic acid (KDO) content. No relationship was found between the IL 1-inducing activity and the LAL reactivity of purified LPSs. Filtration experiments in which membranes of decreasing size-exclusion limits were used demonstrated that molecular species of LPS with an apparent Mr below 3,000 may induce IL 1, whereas only species with an apparent Mr above 8,000 are recognized in the LAL assay. The latter observation suggests that the reaction with LAL requires an aggregated form of LPS. These results indicate that biologically active LPS species can cross dialysis membranes in vivo although no LAL reactive material is detected in the blood compartment. The Limulus assay is an insufficient criterion for the absence of LPS in biological fluids.

  PMID: 2129502 [PubMed - indexed for MEDLINE]
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59. Appl Environ Microbiol. 1990 Jun;56(6):1957-9.

Several uses for isobutyric Acid-ammonium hydroxide solvent in endotoxin analysis.

Author information:

  • 1Equipe "Endotoxine," URA 1116 Centre National de la Recherche Scientifique, Université Paris-Sud, 91405 Orsay, France.

 

Abstract

Many steps in the analysis of rough and semirough endotoxins were found to be facilitated by the use of isobutyric acid-ammonium hydroxide solvent.

PMCID: PMC184537 Free PMC Article
  PMID: 16348231 [PubMed]
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60. J Bacteriol. 1990 Feb;172(2):1121-8.

Variations in the carbohydrate regions of Bordetella pertussis lipopolysaccharides: electrophoretic, serological, and structural features.

Author information:

  • 1URA-1116 du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.

 

Abstract

Structural and immunological differences between the two components that are usually present in unequal quantities in Bordetella pertussis endotoxin preparations and are visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been studied by using strains 1414, A100, and 134, all in phase I. According to analyses by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer chromatography, the minor (8%) component of the endotoxin of strain 1414 (endotoxin 1414) appeared to be the predominating component of endotoxins A100 and 134. The masses of the carbohydrate chains isolated from endotoxin A100 and from the major component of endotoxin 1414 were 1,649 and 2,311 atomic mass units, respectively, as determined by 252Cf plasma desorption mass spectrometry. Comparison of the 1H nuclear magnetic resonance spectra of these chains established that four N-acetyl groups, an N-methyl group, and a 6-deoxy function, which characterize the nonreducing, distal trisaccharide of the glycose chain of strain 1414, were absent from that of strain A100. The antigenicity of endotoxin 1414, as measured by enzyme-linked immunosorbent assay, was higher than that of endotoxin A100, but fell below it when the glycose chain of endotoxin 1414 was deprived of seven sugars by treatment with nitrous acid. This observation suggests that at least three (distal, proximal, and intermediate) regions of the glycose chain of endotoxin 1414 carry antigenic determinants. One of these, located in the distal trisaccharide, is absent from both endotoxins A100 and 134.

PMCID: PMC208545 Free PMC Article
  PMID: 2298694 [PubMed - indexed for MEDLINE]
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61. Mol Immunol. 1989 May;26(5):485-94.

Interleukin-1 induction by lipopolysaccharides: structural requirements of the 3-deoxy-D-manno-2-octulosonic acid (KDO).

Author information:

  • 1INSERM U28, Hôpital Broussais, Paris, France.

 

Abstract

We previously showed the importance of the 3-deoxy-D-manno-2-octulosonic acid (KDO) residue in endotoxins (lipopolysaccharides, LPS) for the induction of the synthesis and release of interleukin-1 (IL-1) by human monocytes. We further investigated the effect of some structural variations within the KDO molecule on IL-1 production induced by LPS. Deamination of Bordetella pertussis LPS, followed by mild anhydrous acidic methanolysis released a hexasaccharide (fragment B'), which had a terminal methyl ketoside KDO residue with a methyl-esterified carboxyl group. This fragment was unable to induce IL-1 production by human monocytes. Fragment B' could be converted into an active hexasaccharide by de-esterification (fragment B-OMe), but not by reduction of the methyl ester group. The KDO residues in the LPS of some bacterial species have been shown to be phosphorylated and we observed that these LPS were weak IL-1 inducers. Phosphorylated KDO present in Vibrio cholerae and B. pertussis LPS respond poorly in the thiobarbiturate assay (specific for KDO). However, if these LPS were dephosphorylated with aqueous hydrofluoric acid (HF) their KDO response in this assay was increased 5.4- to 2.6-fold, respectively. In parallel, the HF-treated LPS were more potent IL-1 inducers than untreated endotoxins. These data confirm that the KDO residue(s) present in all endotoxins play(s) a major role in the signal(s) leading to IL-1 production by human monocytes, and show that IL-1 induction by LPS (1) requires a free carboxyl group in the KDO and (2) is correlated with the degree of substitution of the KDO.

  PMID: 2549403 [PubMed - indexed for MEDLINE]
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62. Infect Immun. 1989 Mar;57(3):791-7.

Dissociation of cell-associated interleukin-1 (IL-1) and IL-1 release induced by lipopolysaccharide and lipid A.

Author information:

  • 1Unité d'Immuno-Allergie, Institut Pasteur, Paris, France.

 

Abstract

The capacities of lipopolysaccharide (LPS) and lipid A to trigger mouse BALB/c peritoneal macrophages and to induce the production of cell-associated interleukin-1 (IL-1) and membrane-associated IL-1 and IL-1 release have been compared. Bordetella pertussis lipid A was 1,000 to 10,000 times less efficient than the native LPS to induce IL-1 release by freshly isolated elicited macrophages. When resident macrophages were studied, lipid A, at high concentrations (greater than 2 micrograms/ml), induced significant levels of cell-associated IL-1 but little or no IL-1 release. With synthetic lipid A built up with the Escherichia coli lipid A structure (compound 506), IL-1 activity was present in the supernatants of elicited peritoneal macrophages and to a lesser extent in those of resident macrophages. However, the release of IL-1 induced by synthetic lipid A 506 remained much lower than those induced by rough LPS. Membrane-associated IL-1 could be induced on BALB/c macrophages with LPS and natural or synthetic lipid A, the LPS being the most active. In C3H/HeJ mice, neither natural nor synthetic lipid A could induce detectable cell-associated IL-1, whereas LPS could induce cell-associated and membrane IL-1 activity but no IL-1 release. Our results indicate that fragments of endotoxins may induce the production of IL-1 but the entire structure of the LPS molecule is the most effective to induce intracellular IL-1 production, expression of membrane IL-1, and release of IL-1.

PMCID: PMC313178 Free PMC Article
  PMID: 2537258 [PubMed - indexed for MEDLINE]
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63. Carbohydr Res. 1988 Apr 15;175(2):273-82.

Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin.

Author information:

  • 1Equipe Endotoxines (U.A. 1116) du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.

 

Abstract

Due to the formation of micelles, severance of the hydrophilic (poly- or oligosaccharide) and hydrophobic ("Lipid A") domains of bacterial lipopolysaccharides at pH 3.4 or 4.5 and 100 degrees is slow and sometimes does not proceed at all; partially degraded fragments are usually formed. At pH 3.4 (100 degrees) in aqueous 1% sodium dodecylsulphate (SDS), both lipopolysaccharides of the Bordetella pertussis endotoxin are cleaved within 20-30 min, but 80% of the glycosidically bound phosphate present in the hydrophobic domain is lost. Other endotoxins behave similarly. At pH 4.5 (100 degrees) and in the absence of detergent, hydrolysis of the glycosidic bonds of 3-deoxy-D-manno-2-octulosonic acid residues of the B. pertussis endotoxin is negligible but, in aqueous 1% SDS, severance of the two regions of LPS 1 is complete within 1 h (that of LPS-2 requires 3-4 h), and the glycosidically bound phosphate of the isolated hydrophobic region is preserved. Comparison of the rate of acid-catalysed hydrolysis of the glycosidically bound phosphate present in this "isolated Lipid A" preparation with that of 2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-alpha- and -beta-D-glucopyranose 1-phosphates established that the former 1-phosphate was the alpha anomer.

  PMID: 2900066 [PubMed - indexed for MEDLINE]
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64. Prog Clin Biol Res. 1988;272:89-101.

Characteristics of lipopolysaccharide-induced interleukin-1 production by human monocytes. Clinical relevance in patients undergoing hemodialysis.

Author information:

  • 1INSERM U28, Hôpital Broussais, Paris.

 

  PMID: 3260673 [PubMed - indexed for MEDLINE]
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65. Biochem Biophys Res Commun. 1987 Mar 30;143(3):845-7.

Do endotoxins devoid of 3-deoxy-D-manno-2-octulosonic acid exist?

Abstract

After treatment with aqueous, 50% hydrofluoric acid, a well-known dephosphorylating agent, the presence of 3-deoxy-D-manno-2-octulosonic acid (KDO), an essential and characteristic constituent of endotoxins, can be readily demonstrated in reportedly KDO-deficient bacterial lipopolysaccharides.

  PMID: 3566759 [PubMed - indexed for MEDLINE]
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66. Ann Inst Pasteur Microbiol. 1987 Jan-Feb;138(1):92-8.

The lipopolysaccharides of Brucella abortus and B. melitensis.

  PMID: 3111502 [PubMed - indexed for MEDLINE]
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67. Infect Immun. 1986 Nov;54(2):465-71.

Inability of pyrogenic, purified Bordetella pertussis lipid A to induce interleukin-1 release by human monocytes.

Abstract

Free lipid A of Bordetella pertussis, Neisseria meningitidis, and Escherichia coli lipopolysaccharide (LPS) was prepared by hydrolysis in acetate buffer (pH 4.5); in addition, lipid A from B. pertussis and E. coli was prepared by hydrolysis in mineral acid (HCl). The precipitates obtained were purified by extraction methods in toluene-methanol and are referred to as crude lipid A. Purified lipid A from N. meningitidis and B. pertussis was obtained by extraction in a mixture of chloroform-methanol-water-triethylamine. The different preparations were tested for their pyrogenicity (endogenous pyrogen; EP) and their capacity to trigger the release of interleukin-1 (IL-1; previously known as lymphocyte-activating factor; LAF) by human monocytes. Crude lipid A from E. coli and N. meningitidis were both IL-1 inducers. Crude B. pertussis lipid A (acetate buffer; pH 4.5), which contains a beta-1-6-linked D-glucosamine disaccharide, two phosphoryl groups, and five fatty acids, was pyrogenic and an IL-1 inducer (EP+/LAF+); but crude B. pertussis lipid A (0.25 N HCl), which lacked the glycosidic phosphoryl group, was 1,000-fold less pyrogenic than the diphosphorylated lipid A, yet it retained its IL-1-inducing capacity (EP-/LAF+). Purified N. meningitidis lipid A was not an inducer of IL-1 release and purified B. pertussis lipid A exhibited identical pyrogenicity as the parent LPS but was devoid of any IL-1-release inducing capacity (EP+/LAF-). These results demonstrate that for some endotoxins, purified lipid A is unable to induce IL-1 release by human monocytes; however, it is pyrogenic, supporting the hypothesis that IL-1 and EP are induced by different determinants on the LPS molecule.

PMCID: PMC260184 Free PMC Article
  PMID: 2876960 [PubMed - indexed for MEDLINE]
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68. Eur J Immunol. 1986 Jan;16(1):87-91.

Molecular requirement for interleukin 1 induction by lipopolysaccharide-stimulated human monocytes: involvement of the heptosyl-2-keto-3-deoxyoctulosonate region.

Abstract

Experiments were undertaken to localize in the lipopolysaccharide (LPS) the minimal structural determinants sufficient to initiate the signal leading to interleukin 1 (IL 1) secretion by human monocytes. Our results clearly demonstrated that this signal is triggered by structures present in the so-called inner-core region which chemically consists of 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) and heptose in many LPS of gram-negative bacteria. Thus, the isolated polysaccharide region of Bordetella pertussis endotoxin as well as fragments derived therefrom containing the reducing KDO unit were able to induce similar levels of IL1 induction as the native LPS. Similarly, the trisaccharide alpha-D-manno-heptopyranosyl-(1-3)-alpha-D-manno-heptopyranosyl -(1-5)-3 -deoxy-D-manno-octulosonic acid (hep-hep-KDO), representative for the inner-core region of a large number of enterobacterial LPS, was a very potent IL 1 inducer. Neither KDO monosaccharide, nor the alpha-(2-4)-linked 3-deoxy-D-manno-octulosonic acid disaccharide isolated from Salmonella rough-form LPS promoted the signal indicating that the minimal structure of endotoxin able to induce IL 1 secretion resides in the hep (1-5)-KDO disaccharide.

  PMID: 2419139 [PubMed - indexed for MEDLINE]
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69. Infect Immun. 1984 Nov;46(2):384-8.

Antigenic S-type lipopolysaccharide of Brucella abortus 1119-3.

Abstract

Antigenic phenol-phase soluble lipopolysaccharide isolated from Brucella abortus 1119-3 by hot phenol-water extraction was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, controlled hydrolysis, periodate oxidation, methylation, and 1H and 13C nuclear magnetic resonance studies to be an S-type lipopolysaccharide which could be cleaved to yield a lipid A and an O-chain polysaccharide identified as an unbranched linear homopolymer of 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues. The serological reactivity of bovine antiserum to B. abortus 1119-3 with the lipopolysaccharides of Yersinia enterocolitica serotype O:9 and Vibrio cholerae species has now been related to the occurrence of 1,2-linked N-acylated 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl units in the O-chain polysaccharides of their lipopolysaccharides.

PMCID: PMC261543 Free PMC Article
  PMID: 6437981 [PubMed - indexed for MEDLINE]
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70. Can J Biochem Cell Biol. 1984 Apr;62(4):151-61.

The specific capsular polysaccharide of Streptococcus pneumoniae type 15A (American type 30).

Abstract

The specific capsular polysaccharide of Streptococcus pneumoniae type 15A (American type 30) is composed of D-galactose (three parts), D-glucose (one part), 2-acetamido-2-deoxy-D-glucose (one part), phosphate (one part), and glycerol (one part). Hydrolysis, periodate oxidation, methylation, optical rotation, and nuclear magnetic resonance studies showed that the polysaccharide is a high molecular weight linear polymer of a pentasaccharide repeating unit having the structure: (formula: see text)

  PMID: 6426760 [PubMed - indexed for MEDLINE]
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71. Eur J Biochem. 1984 Feb 15;139(1):195-200.

Structure of the O-chain of the phenol-phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype O:9.

Abstract

The phenol-phase soluble cellular lipopolysaccharide isolated by the phenol/water extraction method from Yersinia enterocolitica serotype O:9 cells was shown by hydrolytic, periodate oxidation, methylation and nuclear magnetic resonance studies to be an S-type lipopolysaccharide with a linear O-antigenic polysaccharide of 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The serological cross-reactivity between Y. enterocolitica serotype O:9 and the lipopolysaccharides of Vibrio cholerae and Brucella species can now be related to the presence of N-acylated 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residues in their respective O-antigenic chains.

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  PMID: 6199199 [PubMed - indexed for MEDLINE]
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72. Carbohydr Res. 1983 Mar 16;114(1):95-102.

Identification of 2-amino-6-O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-D-glucose as a major constituent of the hydrophobic region of the Bordetella pertussis endotoxin.

  PMID: 6303589 [PubMed - indexed for MEDLINE]
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73. Infect Immun. 1980 Mar;27(3):739-45.

Biological activities of fragments derived from Bordetella pertussis endotoxin: isolation of a nontoxic, Shwartzman-negative lipid A possessing high adjuvant properties.

Abstract

Endotoxin from fresly sedimented Bordetella pertussis cells, isolated by the phenol/water procedure when submitted to kinetically controlled, mild acidic hydrolysis released a polysaccharide (polysaccharide 1), a complex lipid (lipid X), and a glycolipid. When treated with somewhat stronger acid, the glycolipid yielded a second polysaccharide (polysaccharide 2) and another complex lipid (lipid A). The intact pertussis endotoxin had all the usual properties of endotoxins extracted from enteric bacteria. Lipid X and the intermediary glycolipid retained all the endotoxic properties of the unfractionated endotoxin. In lipid A, pyrogenicity was reduced to a very low level and toxicity and Shwartzman reactivity were absent; however, this fraction retained most of the endotoxin's antiviral activity, and its adjuvant power was considerably higher than that of the intact endotoxin. Lipid A elicited nonspecific resistance against challenge with certain bacteria, but not against others.

PMCID: PMC550835 Free PMC Article
  PMID: 6247278 [PubMed - indexed for MEDLINE]
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74. Biochem Biophys Res Commun. 1979 Jul 27;89(2):410-3.

Loss of O-methyl groups from methylated 2-amino-2-deoxy-D-glucitol and -D-galactitol derivatives under acidic conditions used during the analysis of glycoproteins.

  PMID: 486171 [PubMed - indexed for MEDLINE]
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75. Eur J Biochem. 1978 Mar 15;84(2):579-89.

A novel type of endotoxin structure present in Bordetella pertussis. Isolation of two different polysaccharides bound to lipid A.

Abstract

The endotoxin of Bordetella pertussis was cleaved by mild acidic hydrolysis to yield a polysaccharide (polysaccharide I, 15%), a glycolipid (63%) and lipid X (2%). Further treatment of the glycolipid with stronger acid released a second polysaccharide (polysaccharide II, 9%) and material similar to lipid A present in enterobacterial endotoxins. Both polysaccharides possess a single molecule of 3-deoxy-2-octulosonic acid as the reducing, terminal sugar. In polysaccharide II the octulosonic acid is phosphorylated in position 5 and presumably substituted in position 4; in polysaccharide I the octulosonic acid is not phosphorylated, but is substituted in position 5. Following treatment of the endotoxin with strong base, a fragment was isolated that contained bound, non-phosphorylated 3-deoxy-2-octulosonic acid, glucosamine phosphate and fatty acids. This indicated that polysaccharide I, like polysaccharide II, was bound to the lipid region of the endotoxin. The endotoxin structure thus defined is different from that proposed for the lipopolysaccharides of enterobacteria.

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  PMID: 205414 [PubMed - indexed for MEDLINE]
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