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Find video protocols related to scientific articles indexed in Pubmed.
Serological characterization of the enterobacterial common antigen substitution of the lipopolysaccharide of Yersinia enterocolitica O:3.
Microbiology (Reading, Engl.)
PUBLISHED: 11-20-2014
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Enterobacterial common antigen (ECA) is a polysaccharide present in all members of Enterobacteriaceae anchored either via phosphatidylglycerol (PG) or lipopolysaccharide (LPS) to the outer leaflet of outer membrane (ECAPG and ECALPS, respectively). Only the latter form is ECA-immunogenic. We earlier demonstrated that Yersinia enterocolitica O:3 and its rough (O-specific polysaccharide-negative) mutants were ECA-immunogenic implicating that they contained ECALPS however, it was not known which part of the LPS core region was involved in ECA binding. To address this, we used a set of three deep-rough LPS mutants for rabbit immunizations. The obtained polyvalent antisera were: (i) analyzed for the presence of anti-LPS and anti-ECA antibodies; (ii) treated with caprylic acid (CA) to precipitate IgM antibodies and protein aggregates; and (iii) adsorbed with live ECA-negative bacteria to obtain specific anti-ECA antisera. We could demonstrate the presence of antibodies specific for both ECAPG and ECALPS in all antisera obtained. Both CA treatment and adsorption with ECA-negative bacteria efficiently removed anti-LPS antibodies resulting in specific anti-ECA sera. The LPS of the ECALPS positive deepest-rough mutant contained only lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues of the inner core suggesting that ECALPS was linked to the Kdo-region of LPS in Y. enterocolitica O:3.
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Occurrence of an unusual hopanoid-containing lipid A among lipopolysaccharides from Bradyrhizobium species.
J. Biol. Chem.
PUBLISHED: 11-06-2014
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The chemical structures of the unusual hopanoid-containing lipids A of the lipopolysaccharides (LPS) from three strains of Bradyrhizobium (slow-growing rhizobia) have been established. They differed considerably from other Gram-negative bacteria in regards to the backbone structure, the number of ester-linked long chain hydroxylated fatty acids as well as the presence of a tertiary residue that consisted of at least one molecule of carboxyl-bacteriohopanediol or its 2-methyl derivative. The structural details of this type of lipid A were established using 1D and 2D NMR spectroscopy, chemical composition analyses and mass spectrometry techniques (ESI FT-ICR-MS and MALDI-TOF-MS). In these lipids A the glucosamine disaccharide characteristic for enterobacterial lipid A was replaced by a 2,3-dideoxy-2,3-diamino-D-glucopyranosyl- (GlcpN3N) disaccharide, deprived of phosphate residues, and substituted by an ?-D-Manp-(1?6)-?-D-Manp disaccharide substituting C-4' of the non-reducing (distal) GlcpN3N, and one residue of galacturonic acid (D-GalpA) ?-(1?1)-linked to the reducing (proximal) amino sugar residue. Amide-linked 12:0(3-OH) and 14:0(3-OH) were identified. Some hydroxy groups of these fatty acids were further esterified by long (?-1)-hydroxylated fatty acids (VLCFAs) comprising 26-34 carbon atoms. As confirmed by mass spectrometry techniques, these long chain fatty acids could form two or three acyloxyacyl residues. The triterpenoid derivatives were identified as 34-carboxyl-bacteriohopane-32,33-diol and 34-carboxyl-2?-methyl-bacteriohopane-32,33-diol and were covalently linked to the (?-1)-hydroxy group of VLCFA in bradyrhizobial lipid A. Bradyrhizobium japonicum possessed lipid A species with two hopanoid residues.
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Chemistry of Lipid?A: At the Heart of Innate Immunity.
Chemistry
PUBLISHED: 11-05-2014
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Invited for the cover of this issue is an international collaboration of groups from Italy, Germany, Ireland, Switzerland, Austria, Spain, and Slovenia. The image depicts the chemical aspects behind (and responsible for) the bioactivity of lipid A. Read the full text of the article at 10.1002/chem.201403923.
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Chemistry of Lipid?A: At the Heart of Innate Immunity.
Chemistry
PUBLISHED: 10-30-2014
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In many Gram-negative bacteria, lipopolysaccharide (LPS) and its lipid?A moiety are pivotal for bacterial survival. Depending on its structure, lipid?A carries the toxic properties of the LPS and acts as a potent elicitor of the host innate immune system via the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) receptor complex. It often causes a wide variety of biological effects ranging from a remarkable enhancement of the resistance to the infection to an uncontrolled and massive immune response resulting in sepsis and septic shock. Since the bioactivity of lipid?A is strongly influenced by its primary structure, a broad range of chemical syntheses of lipid?A derivatives have made an enormous contribution to the characterization of lipid?A bioactivity, providing novel pharmacological targets for the development of new biomedical therapies. Here, we describe and discuss the chemical aspects regarding lipid?A and its role in innate immunity, from the (bio)synthesis, isolation and characterization to the molecular recognition at the atomic level.
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Pyranoside-into-Furanoside Rearrangement: New Reaction in Carbohydrate Chemistry and Its Application in Oligosaccharide Synthesis.
Chemistry
PUBLISHED: 09-01-2014
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Great interest in natural furanoside-containing compounds has challenged the development of preparative methods for their synthesis. Herein a novel reaction in carbohydrate chemistry, namely a pyranoside-into-furanoside (PIF) rearrangement permitting the transformation of selectively O-substituted pyranosides into the corresponding furanosides is reported. The discovered process includes acid-promoted sulfation accompanied by rearrangement of the pyranoside ring into a furanoside ring followed by solvolytic O-desulfation. This process, which has no analogy in organic chemistry, was shown to be a very useful tool for the synthesis of furanoside-containing complex oligosaccharides, which was demonstrated by synthesizing disaccharide derivatives ?-D-Galp-(1?3)-?-D-Galf-OPr, 3-O-s-lactyl-?-D-Galf-(1?3)-?-D-Glcp-OPr, and ?-L-Fucf-(1?4)-?-D-GlcpA-OPr related to polysaccharides from the bacteria Klebsiella pneumoniae and Enterococcus faecalis and the brown seaweed Chordaria flagelliformis.
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Location-specific expression of chemokines, TNF-? and S100 proteins in a teat explant model.
Innate Immun
PUBLISHED: 08-17-2014
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The distal compartments of the udder are the first to interact with invading pathogens. The regulatory and effector functions of two major teat regions [Fürstenberg's rosette (FR); teat cistern (TC)] are largely unknown. The objective of this study was to establish an in vitro model with explants of the FR and the TC to analyse their response towards Escherichia coli LPS and Staphylococcus aureus lipoteichoic acid (LTA). Quantitative stereological analysis confirmed differences in the cellular composition of FR and TC explants. Chemokine (CXCL8, CCL5, CCL20) and TNF-? mRNA were expressed at low levels in both locations. Explant stimulation with LPS increased the mRNA abundance of all tested chemokines and TNF-?. Stimulation with LTA only induced CCL20 and CXCL8. LPS- and LTA-stimulated explant supernatants contained CXCL8 and CXCL3. Supernatants significantly attracted neutrophils in vitro. Compared with TC, the FR showed high constitutive mRNA expression of S100 proteins (A8, A9, A12). In the TC, both LPS and LTA significantly induced S100A8, whereas S100A9 and S100A12 expression was only induced by LPS. The novel model system underpins the role of the teat for recognising pathogens and shaping a pathogen- and location-specific immune response.
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The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections.
Front Cell Infect Microbiol
PUBLISHED: 08-14-2014
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Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn's disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative ATP binding cassette-transporter, located on the MAP-specific LSP14. In the present study, we generated an mptD knockout strain (MAP?mptD) by specialized transduction. In order to investigate the potential role of mptD in the host, we performed infection experiments with macrophages. By this, we observed a significantly reduced cell number of MAP?mptD early after infection, indicating that the mutant was hampered with respect to adaptation to the early macrophage environment. This important role of mptD was supported in mouse infection experiments where MAP?mptD was significantly attenuated after peritoneal challenge. Metabolic profiling was performed to determine the cause for the reduced virulence and identified profound metabolic disorders especially in the lipid metabolism of MAP?mptD. Overall our data revealed the mptD gene to be an important factor for the metabolic adaptation of MAP required for persistence in the host.
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Structure of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas sobria strain Pt312.
Carbohydr. Res.
PUBLISHED: 04-29-2014
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The O-specific polysaccharide (OPS) obtained by mild-acid degradation of the lipopolysaccharide from Aeromonas sobria strain Pt312 was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, NOESY, H-detected (1)H,(13)C HSQC, and HMBC experiments. The sequence of the sugar residues was determined using (1)H,(1)H NOESY and (1)H,(13)C HMBC experiments. It was found that the OPS was built up of disaccharide repeating units composed of GlcpNAc and non-stoichiometrically O-acetylated Rhap residues, and had the structure.
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Biosynthesis of the unique wall teichoic acid of Staphylococcus aureus lineage ST395.
MBio
PUBLISHED: 04-10-2014
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ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an ?-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage ?187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to ?187 and enabled ?187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.
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Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes.
Nat Commun
PUBLISHED: 03-17-2014
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Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic Bradyrhizobium strain, symbiont of Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A. Biophysical analyses of reconstituted liposomes indicate that this hopanoid-lipid A structure reinforces the stability and rigidity of the outer membrane. In addition, the bacterium produces other hopanoid molecules not linked to LPS. A hopanoid-deficient strain, lacking a squalene hopene cyclase, displays increased sensitivity to stressful conditions and reduced ability to survive intracellularly in the host plant. This unusual combination of hopanoid and LPS molecules may represent an adaptation to optimize bacterial survival in both free-living and symbiotic states.
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Structural studies of the lipopolysaccharide from the fish pathogen Aeromonas veronii strain Bs19, serotype O16.
Mar Drugs
PUBLISHED: 01-27-2014
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Chemical analyses, mass spectrometry, and NMR spectroscopy were applied to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas veronii strain Bs19, serotype O16. ESI-MS revealed that the most abundant LPS glycoforms have tetra-acylated or hexa-acylated lipid A species, consisting of a bisphosphorylated GlcN disaccharide with an AraN residue as a non-stoichiometric substituent, and a core oligosaccharide composed of Hep?Hex?HexN?Kdo?P?. Sugar and methylation analysis together with 1D and 2D ¹H and ¹³C NMR spectroscopy were the main methods used, and revealed that the O-specific polysaccharide (OPS) of A. veronii Bs19 was built up of tetrasaccharide repeating units with the structure: ?4)-?-D-Quip3NAc-(1?3)-?-L-Rhap-(1?4)-?-D-Galp-(1?3)-?-D-GalpNAc-(1?. This composition was confirmed by mass spectrometry. The charge-deconvoluted ESI FT-ICR MS recorded for the LPS preparations identified mass peaks of SR- and R-form LPS species, that differed by ?m = 698.27 u, a value corresponding to the calculated molecular mass of one OPS repeating unit (6dHexNAc6dHexHexHexNAc-H?O). Moreover, unspecific fragmentation spectra confirmed the sequence of the sugar residues in the OPS and allowed to assume that the elucidated structure also represented the biological repeating unit.
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Identification of novel immune phenotypes for allergic and nonallergic childhood asthma.
J. Allergy Clin. Immunol.
PUBLISHED: 01-16-2014
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Childhood asthma is classified into allergic asthma (AA) and nonallergic asthma (NA), yet both are treated identically, with only partial success.
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Bioavailability and allergoprotective capacity of milk-associated conjugated linoleic acid in a murine model of allergic airway inflammation.
Int. Arch. Allergy Immunol.
PUBLISHED: 01-10-2014
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Cross-sectional epidemiological studies have demonstrated that farm milk from traditional farm settings possesses allergoprotective properties. Up to now, it has not been clarified which milk ingredient is responsible for protection against allergic diseases. As farm milk is rich in conjugated linoleic acids (CLA), it is hypothesized that this n-3 polyunsaturated fatty acid family contributes to the allergoprotective capacity of farm milk. We aim to prove this hypothesis in a murine model of allergic airway inflammation.
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Neutrophil elastase-mediated increase in airway temperature during inflammation.
J. Cyst. Fibros.
PUBLISHED: 01-03-2014
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How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection.
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Structural investigation of rhamnose-rich polysaccharides from Streptococcus dysgalactiae bovine mastitis isolate.
Carbohydr. Res.
PUBLISHED: 01-03-2014
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Different rhamnose-rich polysaccharides (RRP) were identified in the cell envelope of the Gram-positive bovine mastitis isolate Streptococcus dysgalactiae 2023. Structural investigations of the 1D and 2D nuclear magnetic resonance experiments as well as chemical analyses identified as main components l-Rha and d-GalNAc. Two main RRP were characterized, namely 1 being composed of the repeating unit {?3)-?-l-Rhap-(1?2)-[?-d-GalpNAc-(1?3)-?-d-GalpNAc-(1?3)-]?-l-Rhap-(1} and 2 possessing the repeat [?2)-?-l-Rhap-(1?3)-?-l-Rhap-(1?].
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Structure and genetic basis of Yersinia similis serotype O:9 O-specific polysaccharide.
Innate Immun
PUBLISHED: 12-24-2013
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The O-polysaccharide (OPS, O-Ag) cap of LPS is a major virulence factor of Yersinia species and also serves as a receptor for the binding of lytic bacteriophage ?R1-37. Currently, the OPS-based serotyping scheme for the Yersinia pseudotuberculosis complex includes 21 known O-serotypes that follow three distinct lineages: Y. pseudotuberculosis sensu stricto, Y. similis and the Korean group of strains. Elucidation of the Y. pseudotuberculosis complex OPS structures and characterization of the OPS genetics (altogether 18 O-serotypes studied thus far) allows a better understanding of the relationships among the various O serotypes and will facilitate the analysis of the evolutionary processes giving rise to new serotypes. Here we present the characterization of the OPS structure and gene cluster of Y. similis O:9. Bacteriophage ?R1-37, which uses the Y. similis O:9 OPS as a receptor, also infects a number of Y. enterocolitica serotypes, including O:3, O:5,27, O:9 and O:50. The Y. similis O:9 OPS structure resembled none of the receptor structures of the Y. enterocolitica strains, suggesting that ?R1-37 can recognize several surface receptors, thus promoting broad host specificity.
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The WbaK acetyltransferase of Salmonella enterica group E gives insights into O antigen evolution.
Microbiology (Reading, Engl.)
PUBLISHED: 09-06-2013
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O antigens are polysaccharides consisting of repeat units of three to eight sugars, generally assembled by genes in a discrete O antigen gene cluster. Salmonella enterica produces 46 forms of O antigen, and most of the variation is determined by genes in the gene cluster. However in some cases the structures are modified by enzymes encoded outside of the gene cluster, and several such modifications have been reported for Salmonella enterica group E, some with the genes on bacteriophages and one gene at a distant chromosomal site. We identified the enzyme, WbaK, that is responsible for O-acetylating the subgroup E1 O antigen, and found that the gene is located just downstream of the gene cluster as currently known. The wbaK gene appears to have been imported by a recombination event that also replaced the last 37 bp of the wbaP gene, indicating that homologous recombination was involved. Some of the group E strains we studied must have the original gene cluster, as they lack wbaK and the sequence downstream of wbaP is very similar to that in several other S. enterica O antigen gene clusters. In effect the gene cluster was extended by one gene in subgroup E1. It appears that a function that is usually encoded by a gene outside of the gene cluster has been added to the gene cluster, in this case giving an example of how such gene clusters can evolve.
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Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O : 3.
Microbiology (Reading, Engl.)
PUBLISHED: 06-19-2013
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Yersinia enterocolitica serotype O?:?3 produces two types of lipopolysaccharide (LPS) molecules to its surface. In both types the lipid A (LA) structure is substituted by inner core (IC) octasaccharide to which either outer core (OC) hexasaccharide or homopolymeric O-polysaccharide (OPS) is linked. In addition, enterobacterial common antigen (ECA) can be covalently linked to LPS, however, via an unknown linkage. To elucidate the relationship between ECA and LPS in Y. enterocolitica O?:?3 and the effect of temperature on their expression, LPS was isolated from bacteria grown at 22 °C and 37 °C by consequent hot phenol/water and phenol-chloroform-light petroleum extractions to obtain LPS preparations free of ECA linked to glycerophospholipid. In immunoblotting, monoclonal antibodies TomA6 and 898, specific for OPS and ECA, respectively, reacted both with ladder-like bands and with a slower-migrating smear suggesting that the ECA and OPS epitopes coexist on the same molecules. These results were supported by immunoblotting with a monovalent Y. enterocolitica O?:?3 ECA-specific rabbit antiserum. Also, two or three 898-positive (and monovalent-positive) TomA6-negative bands migrated at the level of the LA-IC band in LPS samples from certain OC mutants, most likely representing LA-IC molecules carrying 1-3 ECA repeat units but no OPS. These bands were also present in Y. enterocolitica O?:?9 OC mutants; however, coexistence of ECA and OPS in the same molecules could not be detected. Finally, the LA-IC-ECA bands were missing from LPS of bacteria grown at 37 °C and also the general reduction in wild-type bacteria of ECA-specific monovalent-reactive material at 37 °C suggested that temperature regulates the expression of ECA. Indeed, RNA-sequencing analysis showed significant downregulation of the ECA biosynthetic gene cluster at 37 °C.
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Structure and immunogenicity of the rough-type lipopolysaccharide from the periodontal pathogen Tannerella forsythia.
Clin. Vaccine Immunol.
PUBLISHED: 04-24-2013
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Tannerella forsythia is a Gram-negative anaerobic organism that inhabits subgingival plaque biofilms and is covered with a so far unique surface layer composed of two glycoproteins. It belongs to the so-called "red complex" of bacteria comprising species that are associated with periodontal disease. While the surface layer glycoprotein glycan structure had been elucidated recently and found to be a virulence factor, no structural data on the lipopolysaccharide (LPS) of this organism were available. In this study, the T. forsythia LPS structure was partially elucidated by a combined mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) approach and initial experiments to characterize its immunostimulatory potential were performed. The T. forsythia LPS is a complex, rough-type LPS with a core region composed of one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue, three mannose residues, and two glucosamine residues. MS analyses of O-deacylated LPS proved that, in addition, one phosphoethanolamine residue and most likely one galactose-phosphate residue were present, however, their positions could not be identified. Stimulation of human macrophages with T. forsythia LPS resulted in the production of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha in a dose-dependent manner. The response to T. forsythia LPS was observed only upon stimulation in the presence of fetal calf serum (FCS), whereas no cytokine production was observed in the absence of FCS. This finding suggests that the presence of certain additional cofactors is crucial for the immune response induced by T. forsythia LPS.
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Chemical structures of the secondary cell wall polymers (SCWPs) isolated from bovine mastitis Streptococcus uberis.
Carbohydr. Res.
PUBLISHED: 04-23-2013
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The cell envelope of Gram-positive bacteria is decorated with a variety of polysaccharides. In this study wall teichoic acid (WTA) and neutral polysaccharides were isolated from the cell envelope of bovine mastitis Streptococcus uberis. The polysaccharides were released by lysozyme treatment, and purified by hydrophobic interaction chromatography. Further separation was achieved utilizing anion-exchange chromatography which yielded two products, that is, a neutral polysaccharide with a high content of Rha and less Glc (rhamnan) and an anionic phosphate-rich one containing glycerol and Glc (WTA). The structures of these molecules were elucidated applying 1D and 2D nuclear magnetic resonance experiments as well as chemical analyses. In the rhamnan sample two independent molecules were identified, that is, a glucorhamnan with the structure ?2)-?-L-Rhap-(1?3)-[?-D-Glcp-(1?2)-]?-L-Rhap-(1?, and a homopolymeric rhamnan ?2)-?-L-Rhap-(1?3)-?-L-Rhap-(1?. The WTA comprised a polyphosphoglycerol chain substituted nonstoichiometrically with ?-Glcp.
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Endotoxicity of Lipopolysaccharide as a Determinant of T-cell Mediated Colitis Induction in Mice.
Gastroenterology
PUBLISHED: 03-22-2013
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The intestinal microbiota is an important determinant of the mucosal response. In patients with inflammatory bowel diseases (IBD), the mucosal immune system has inappropriate interactions with the intestinal microbiota. We investigated how the composition of the intestinal microbiota affects its endotoxicity and development of colitis in mice.
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Structural and immunochemical studies of the lipopolysaccharide from the fish pathogen, Aeromonas bestiarum strain K296, serotype O18.
Mar Drugs
PUBLISHED: 02-25-2013
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Chemical analyses and mass spectrometry were used to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas bestiarum strain K296, serotype O18. ESI-MS revealed that the most abundant A. bestiarum LPS glycoforms have a hexa-acylated or tetra-acylated lipid A with conserved architecture of the backbone, consisting of a 1,4-bisphosphorylated ?-(1?6)-linked D-GlcN disaccharide with an AraN residue as a non-stoichiometric substituent and a core oligosaccharide composed of Kdo1Hep6Hex1HexN1P1. 1D and 2D NMR spectroscopy revealed that the O-specific polysaccharide (OPS) of A. bestiarum K296 consists of a branched tetrasaccharide repeating unit containing two 6-deoxy-l-talose (6dTalp), one Manp and one GalpNAc residues; thus, it is similar to that of the OPS of A. hydrophila AH-3 (serotype O34) in both the sugar composition and the glycosylation pattern. Moreover, 3-substituted 6dTalp was 2-O-acetylated and additional O-acetyl groups were identified at O-2 and O-4 (or O-3) positions of the terminal 6dTalp. Western blots with polyclonal rabbit sera showed that serotypes O18 and O34 share some epitopes in the LPS. The very weak reaction of the anti-O34 serum with the O-deacylated LPS of A. bestiarum K296 might have been due to the different O-acetylation pattern of the terminal 6dTalp. The latter suggestion was further confirmed by NMR.
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Wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens.
Nat Commun
PUBLISHED: 01-28-2013
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Mobile genetic elements (MGEs) encoding virulence and resistance genes are widespread in bacterial pathogens, but it has remained unclear how they occasionally jump to new host species. Staphylococcus aureus clones exchange MGEs such as S. aureus pathogenicity islands (SaPIs) with high frequency via helper phages. Here we report that the S. aureus ST395 lineage is refractory to horizontal gene transfer (HGT) with typical S. aureus but exchanges SaPIs with other species and genera including Staphylococcus epidermidis and Listeria monocytogenes. ST395 produces an unusual wall teichoic acid (WTA) resembling that of its HGT partner species. Notably, distantly related bacterial species and genera undergo efficient HGT with typical S. aureus upon ectopic expression of S. aureus WTA. Combined with genomic analyses, these results indicate that a glycocode of WTA structures and WTA-binding helper phages permits HGT even across long phylogenetic distances thereby shaping the evolution of Gram-positive pathogens.
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Structural studies of the O-antigenic polysaccharide of the bovine mastitis isolate Escherichia coli serotype O174.
Carbohydr. Res.
PUBLISHED: 01-18-2013
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Escherichia coli is one of the major causative agents of bovine mastitis, a disease affecting dairy herds. The lipopolysaccharide (LPS) of E. coli was plays a prominent role during infection. Here, we report on the O-antigen chemical structure of the LPS from Escherichia coli strain 2188 (serotype O174:H28) isolated from a mastitis-diseased cow. The structure of the OPS was analyzed by 1 and 2D NMR spectroscopy, and methylation analysis, which identified the branched repeating tetrasaccharide biological unit. [structure: see text].
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Testosterone increases susceptibility to amebic liver abscess in mice and mediates inhibition of IFN? secretion in natural killer T cells.
PLoS ONE
PUBLISHED: 01-02-2013
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Amebic liver abscess (ALA), a parasitic disease due to infection with the protozoan Entamoeba histolytica, occurs age and gender dependent with strong preferences for adult males. Using a mouse model for ALA with a similar male bias for the disease, we have investigated the role of female and male sexual hormones and provide evidence for a strong contribution of testosterone. Removal of testosterone by orchiectomy significantly reduced sizes of abscesses in male mice, while substitution of testosterone increased development of ALA in female mice. Activation of natural killer T (NKT) cells, which are known to be important for the control of ALA, is influenced by testosterone. Specifically activated NKT cells isolated from female mice produce more IFN? compared to NKT cells derived from male mice. This high level production of IFN? in female derived NKT cells was inhibited by testosterone substitution, while the IFN? production in male derived NKT cells was increased by orchiectomy. Gender dependent differences were not a result of differences in the total number of NKT cells, but a result of a higher activation potential for the CD4(-) NKT cell subpopulation in female mice. Taken together, we conclude that the hormone status of the host, in particular the testosterone level, determines susceptibility to ALA at least in a mouse model of the disease.
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Chemical structure of wall teichoic acid isolated from Enterococcus faecium strain U0317.
Carbohydr. Res.
PUBLISHED: 08-11-2011
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Wall teichoic acid (WTA) was isolated from Enterococcus faecium strain U0317 and structurally characterized using (1)H, (13)C, and (31)P NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, HMQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods and HF treatment followed by GLC and GLC-MS analyses. The repeating unit of WTA consisted of two residues of 2-acetamido-2-deoxy-D-galactose, glycerol (Gro), and phosphate, and has the structure shown below: [See formula in text].
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Identification of the lipopolysaccharide core of Yersinia pestis and Yersinia pseudotuberculosis as the receptor for bacteriophage ?A1122.
J. Bacteriol.
PUBLISHED: 07-15-2011
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?A1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. ?A1122 infects Y. pestis grown both at 20 °C and at 37 °C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37 °C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30 °C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also ?A1122 sensitive when grown at 22 °C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous ?A1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (L-glycero-D-manno-heptose/D-glucopyranose)-Kdo/Ko (3-deoxy-D-manno-oct-2-ulopyranosonic acid/D-glycero-D-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully ?A1122 resistant. Our data thus conclusively demonstrated that the ?A1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.
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Cytokine-inducing lipoteichoic acids of the allergy-protective bacterium Lactococcus lactis G121 do not activate via Toll-like receptor 2.
Glycobiology
PUBLISHED: 06-11-2011
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It was established in a mouse model that the cowshed Gram-positive bacterium Lactococcus lactis G121 modulates the immune system resulting in allergy protection. However, the molecules and mechanisms involved in this process have not been elucidated yet. Lipoteichoic acids (LTAs) represent one major cell envelope component of Gram-positive bacteria that is considered a pathogen-associated molecular pattern. In the investigations presented here, the isolation as well as the structural and functional analyses of the LTA of L. lactis G121 were performed. Extraction with butan-1-ol and purification by hydrophobic interaction chromatography yielded pure LTA. Structural investigations included chemical analytical methods, nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization Fourier-transformed ion cyclotron mass spectrometry. LTA comprised a heterogeneous mixture of molecules composed of a 1,3-linked poly(glycerol phosphate) backbone which was randomly substituted at C-2 by D-alanine and ?-D-galactopyranose. The lipid anchor constituents were kojibiose linked to a heterogeneous diglyceride comprising in total six different fatty acid compositions. This LTA preparation possesses Toll-like receptor 2- (TLR2) and TLR4-independent cytokine-inducing activities in human mononuclear cells.
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Deletion of the glycosyltransferase bgsB of Enterococcus faecalis leads to a complete loss of glycolipids from the cell membrane and to impaired biofilm formation.
BMC Microbiol.
PUBLISHED: 04-06-2011
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Deletion of the glycosyltransferase bgsA in Enterococcus faecalis leads to loss of diglucosyldiacylglycerol from the cell membrane and accumulation of its precursor monoglucosyldiacylglycerol, associated with impaired biofilm formation and reduced virulence in vivo. Here we analyzed the function of a putative glucosyltransferase EF2890 designated biofilm-associated glycolipid synthesis B (bgsB) immediately downstream of bgsA.
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The lipopolysaccharide of the mastitis isolate Escherichia coli strain 1303 comprises a novel O-antigen and the rare K-12 core type.
Microbiology (Reading, Engl.)
PUBLISHED: 03-03-2011
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Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[?4)-?-D-Quip3NAc-(1?3)-?-L-Fucp2OAc-(1?4)-?-D-Galp-(1?3)-?-D-GalpNAc-(1?]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli 1303 was also determined. This cluster, located between the gnd and galF genes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal LD-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core - namely terminal LD-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolate E. coli 1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.
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Serodiversity of opsonic antibodies against Enterococcus faecalis--glycans of the cell wall revisited.
PLoS ONE
PUBLISHED: 02-15-2011
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In a typing system based on opsonic antibodies against carbohydrate antigens of the cell envelope, 60% of Enterococcus faecalis strains can be assigned to one of four serotypes (CPS-A to CPS-D). The structural basis for enterococcal serotypes, however, is still incompletely understood. Here we demonstrate that antibodies raised against lipoteichoic acid (LTA) from a CPS-A strain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies also bind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-C and CPS-D strains resistant to opsonization by anti-LTA, we purified a novel diheteroglycan with a repeating unit of ?6)-?-Galf-(1?3)- ?-D-Glcp-(1? with O-acetylation in position 5 and lactic acid substitution at position 3 of the Galf residue. The purified diheteroglycan, but not LTA absorbed opsonic antibodies from whole cell antiserum against E. faecalis type 2 (a CPS-C strain) and type 5 (CPS-D). Rabbit antiserum raised against purified diheteroglycan opsonized CPS-C and CPS-D strains and passive protection with diheteroglycan-specific antiserum reduced bacterial counts by 1.4-3.4 logs in mice infected with E. faecalis strains of the CPS-C and CPS-D serotype. Diheteroglycan-specific opsonic antibodies were absorbed by whole bacterial cells of E. faecalis FA2-2 (CPS-C) but not by its isogenic acapsular cpsI-mutant and on native PAGE purified diheteroglycan co-migrated with the gene product of the cps-locus, suggesting that it is synthesized by this locus. In summary, two polysaccharide antigens, LTA and a novel diheteroglycan, are targets of opsonic antibodies against typeable E. faecalis strains. These cell-wall associated polymers are promising candidates for active and passive vaccination and add to our armamentarium to fight this important nosocomial pathogen.
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Unusual outer membrane lipid composition of the gram-negative, lipopolysaccharide-lacking myxobacterium Sorangium cellulosum So ce56.
J. Biol. Chem.
PUBLISHED: 02-14-2011
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The gram-negative myxobacterium Sorangium cellulosum So ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. Careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (LPS) are missing in this microbe. Biochemical analysis gave no evidence for the presence of LPS in the membranes of So ce56. By analyzing the lipid composition of its outer membrane sphingolipids were identified as the major lipid class, together with ornithine-containing lipids (OL) and ether lipids. A detailed analysis of these lipids resulted in the identification of more than 50 structural variants within these three classes, which possessed several interesting properties regarding to LPS replacement, mediators in myxobacterial differentiation, as well as potential bioactive properties. The sphingolipids with the basic structure C9-methyl-C(20)-sphingosine possessed as an unusual trait C9-methylation, which is common to fungi but highly uncommon to bacteria. Such sphingolipids have not been found in bacteria before, and they may have a function in myxobacterial development. The OL, also identified in myxobacteria for the first time, contained acyloxyacyl groups, which are also characteristic for LPS and might replace those in certain functions. Finally, the ether lipids may serve as biomarkers in myxobacterial development.
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The genetics and structure of the O-specific polysaccharide of Yersinia pseudotuberculosis serotype O:10 and its relationship with Escherichia coli O111 and Salmonella enterica O35.
Glycobiology
PUBLISHED: 02-14-2011
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The O-specific polysaccharide (OPS) is a variable constituent of the lipopolysaccharide of Gram-negative bacteria. The polymorphic nature of OPSs within a species is usually first defined serologically, and the current serotyping scheme for Yersinia pseudotuberculosis consists of 21 O serotypes of which 15 have been characterized genetically and structurally. Here, we present the structure and DNA sequence of Y. pseudotuberculosis O:10 OPS. The O unit consists of one residue each of d-galactopyranose, N-acetyl-d-galactosamine (2-amino-2-deoxy-d-galactopyranose) and d-glucopyranose in the backbone, with two colitose (3,6-dideoxy-l-xylo-hexopyranose) side-branch residues. This structure is very similar to that shared by Escherichia coli O111 and Salmonella enterica O35. The gene cluster sequences of these serotypes, however, have only low levels of similarity to that of Y. pseudotuberculosis O:10, although there is significant conservation of gene order. Within Y. pseudotuberculosis, the O10 structure is most closely related to the O:6 and O:7 structures.
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Structural characterization of the O-specific polysaccharide from the lipopolysaccharide of the fish pathogen Aeromonas bestiarum strain P1S.
Carbohydr. Res.
PUBLISHED: 01-31-2011
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The O-specific polysaccharide obtained by mild-acid degradation of lipopolysaccharide of Aeromonas bestiarum P1S was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy. The sequence of the sugar residues was determined using (1)H,(1)H NOESY and (1)H,(13)C HMBC experiments. The O-specific polysaccharide was found to be a high-molecular-mass polysaccharide composed of tetrasaccharide repeating units of the structure [formula in text]. Since small amounts of a terminal Quip3N residue were identified in methylation analysis, it was assumed that the elucidated structure also represented the biological repeating unit of the O-specific polysaccharide.
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Identification and structural determination of the capsular polysaccharides from two Acinetobacter baumannii clinical isolates, MG1 and SMAL.
Carbohydr. Res.
PUBLISHED: 01-12-2011
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The structures of the capsular polysaccharides (CPSs) of the two clinical isolates Acinetobacter baumannii SMAL and MG1 were elucidated. Hot phenol/water extractions of the dry biomasses, followed by enzymatic digestions and repeated ultracentrifugations led to the isolation of polysaccharides that were negative in Western blot analysis utilizing an anti-lipid A antibody, thus proving that they were not the LPS O-antigens but CPSs. Their structures were established on the basis of NMR spectroscopy and GC-MS analyses. The A. baumannii MG1 CPS consisted of a linear aminopolysaccharide with acyl substitution heterogeneity at the N-4 amino group of QuipN4N: 4)-?-d-GlcpNAc-(1?4)-?-l-GalpNAcA-(1?3)-?-d-QuipNAc4NR-(1? R=3-hydroxybutyrryl or acetyl. The repeating unit of the CPS produced by strain SMAL is a pentasaccharide, already reported for the O-antigen moiety from A. baumannii strain ATCC 17961:
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Microbe-associated molecular patterns in innate immunity: Extraction and chemical analysis of gram-negative bacterial lipopolysaccharides.
Meth. Enzymol.
PUBLISHED: 09-07-2010
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Bacterial lipopolysaccharides (LPSs) are the major component of the outer membrane of Gram-negative bacteria. They have a structural role since they contribute to the cellular rigidity by increasing the strength of cell wall and mediating contacts with the external environment that can induce structural changes to allow life in different conditions. Furthermore, the low permeability of the outer membrane acts as a barrier to protect bacteria from host-derived antimicrobial compounds. They also have a very important role in the elicitation of the animal and plant host innate immunity since they are microbe-associated molecular patterns, namely, they are glycoconjugates produced only by Gram-negative bacteria and are recognized as a molecular hallmark of invading microbes. LPSs are amphiphilic macromolecules generally comprising three defined regions distinguished by their genetics, structures, and function: the lipid A, the core oligosaccharide and a polysaccharide portion, the O-chain. In some Gram-negative bacteria, LPS can terminate with the core portion to form rough-type LPS (R-LPS, LOS). In this chapter, we will describe the isolation of both kinds of LPSs and their full chemical analysis, pivotal operations in the complete description of the primary structure of such important glycoconjugates.
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Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core.
J. Biol. Chem.
PUBLISHED: 07-01-2010
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Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.
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Genetic characterisation and structural analysis of the O-specific polysaccharide of Yersinia pseudotuberculosis serotype O:1c.
Innate Immun
PUBLISHED: 04-23-2010
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Many, but not all, of the current 21 serotypes of Yersinia pseudotuberculosis have been investigated with regard to the chemical structures of their O-specific polysaccharide (OPS) and the genetic basis of their biosynthesis. Completion of the genetics and structures of the remaining serotypes will enhance our understanding of the emerging relationship between genetics and structures within this species. Here, we present a structural and genetic analysis of the Y. pseudotuberculosis serotype O:1c OPS. Our results showed that this OPS has the same backbone as Y. pseudotuberculosis O:2b, but with a 3,6-dideoxy-D-ribo-hexofuranose (paratofuranose, Parf) side-branch instead of a 3,6-dideoxy-D-xylo-hexopyranose (abequopyranose, Abep). The 3-end of the gene cluster is the same as for O:2b and has the genes for synthesis of the backbone and for processing the completed repeat unit. The 5-end of the cluster consists of the same genes as O:1b for synthesis of Parf and a related gene for its transfer to the repeating unit backbone.
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Temperature-induced changes in the lipopolysaccharide of Yersinia pestis affect plasminogen activation by the pla surface protease.
Infect. Immun.
PUBLISHED: 04-05-2010
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The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane beta-barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37 degrees C than in cells grown at 20 degrees C; the difference in enzymatic activity by far exceeded the increase in the cellular content of the Pla protein. Y. pestis modifies its LPS structure in response to growth temperature. We purified His(6)-Pla under denaturing conditions and compared various LPS types for their capacity to enhance plasmin formation by His(6)-Pla solubilized in detergent. Reactivation of His(6)-Pla was higher with Y. pestis LPSs isolated from bacteria grown at 37 degrees C than with LPSs from cells grown at 25 degrees C. Lack of O antigens and the presence of the outer core region as well as a lowered level of acylation in LPS were found to enhance the Pla-LPS interaction. Genetic substitution of arginine 138, which is part of a three-dimensional protein motif for binding to lipid A phosphates, decreased both the enzymatic activity of His(6)-Pla and the amount of Pla in Y. pestis cells, suggesting the importance of the Pla-lipid A phosphate interaction. The temperature-induced changes in LPS are known to help Y. pestis to avoid innate immune responses, and our results strongly suggest that they also potentiate Pla-mediated proteolysis.
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Low endotoxin release from Escherichia coli and Bacteroides fragilis during exposure to moxifloxacin.
Chemotherapy
PUBLISHED: 03-29-2010
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Bacterial endotoxin is known to act as a potent trigger of disseminated coagulation and septic shock. During clinical antibiotic treatment, endotoxin may be released from Gram-negative bacteria. It is known that antibiotic classes differ in their ability to induce endotoxin release.
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Glycosylation of wall teichoic acid in Staphylococcus aureus by TarM.
J. Biol. Chem.
PUBLISHED: 02-25-2010
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Wall teichoic acid (WTA) glycopolymers are major constituents of cell envelopes in Staphylococcus aureus and related gram-positive bacteria with important roles in cell wall maintenance, susceptibility to antimicrobial molecules, biofilm formation, and host interaction. Most S. aureus strains express polyribitol phosphate WTA substituted with D-alanine and N-acetylglucosamine (GlcNAc). WTA sugar modifications are highly variable and have been implicated in bacteriophage susceptibility and immunogenicity, but the pathway and enzymes of staphylococcal WTA glycosylation have remained unknown. Revisiting the structure of S. aureus RN4220 WTA by NMR analysis revealed the presence of canonical polyribitol phosphate WTA bearing only alpha-linked GlcNAc substituents. A RN4220 transposon mutant resistant to WTA-dependent phages was identified and shown to produce altered WTA, which exhibited faster electrophoretic migration and lacked completely the WTA alpha-GlcNAc residues. Disruption of a gene of unknown function, renamed tarM, was responsible for this phenotype. Recombinant TarM was capable of glycosylating WTA in vitro in a UDP-GlcNAc-dependent manner, thereby confirming its WTA GlcNAc-transferase activity. Deletion of the last seven amino acids from the C terminus abolished the activity of TarM. tarM-related genes were found in the genomes of several WTA-producing bacteria, suggesting that TarM-mediated WTA glycosylation is a general pathway in gram-positive bacteria. Our study represents a basis for dissecting the biosynthesis and function of glycosylated WTA in S. aureus and other bacteria.
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Immunochemical studies of trehalose-containing major glycolipid from Tsukamurella pulmonis.
Carbohydr. Res.
PUBLISHED: 02-18-2010
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The chemical structure of the major glycolipid present in the chloroform-methanol extract of bacterial biomass of Tsukamurella pulmonis is reported. This compound was purified by TLC and HPLC. The sugar analysis revealed only glucose. Detailed chemical analyses, NMR, and MALDI FT-ICR-mass spectrometric studies identified 2,3-di-O-acyl-alpha-d-glucopyranosyl-(1-->1)-alpha-d-glucopyranose as the final structure. Short branched fatty acids (4:0 or 5:0) were linked to C-3 and saturated, mono, and diunsaturated 18:0, 18:1, 18:2, 20:1, 20:2, and 20:0 to C-2 of the same glucose residue. ELISA tests revealed the weak cross-reactivity of the glycolipid with rabbit antisera against cells of T. paurometabola, Rhodococcus wratislaviensis, and Nocardiopsis dassonvillei.
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The structure of a novel neutral lipid A from the lipopolysaccharide of Bradyrhizobium elkanii containing three mannose units in the backbone.
Chemistry
PUBLISHED: 01-21-2010
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The chemical structure of the lipid A of the lipopolysaccharide (LPS) from Bradyrhizobium elkanii USDA 76 (a member of the group of slow-growing rhizobia) has been established. It differed considerably from lipids A of other Gram-negative bacteria, in that it completely lacks negatively charged groups (phosphate or uronic acid residues); the glucosamine (GlcpN) disaccharide backbone is replaced by one consisting of 2,3-dideoxy-2,3-diamino-D-glucopyranose (GlcpN3N) and it contains two long-chain fatty acids, which is unusual among rhizobia. The GlcpN3N disaccharide was further substituted by three D-mannopyranose (D-Manp) residues, together forming a pentasaccharide. To establish the structural details of this molecule, 1D and 2D NMR spectroscopy, chemical composition analyses and high-resolution mass spectrometry methods (electrospray ionisation Fourier-transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) and tandem mass spectrometry (MS/MS)) were applied. By using 1D and 2D NMR spectroscopy experiments, it was confirmed that one D-Manp was linked to C-1 of the reducing GlcpN3N and an alpha-(1-->6)-linked D-Manp disaccharide was located at C-4 of the non-reducing GlcpN3N (alpha-linkage). Fatty acid analysis identified 12:0(3-OH) and 14:0(3-OH), which were amide-linked to GlcpN3N. Other lipid A constituents were long (omega-1)-hydroxylated fatty acids with 26-33 carbon atoms, as well as their oxo forms (28:0(27-oxo) and 30:0(29-oxo)). The 28:0(27-OH) was the most abundant acyl residue. As confirmed by high-resolution mass spectrometry techniques, these long-chain fatty acids created two acyloxyacyl residues with the 3-hydroxy fatty acids. Thus, lipid A from B. elkanii comprised six acyl residues. It was also shown that one of the acyloxyacyl residues could be further acylated by 3-hydroxybutyric acid (linked to the (omega-1)-hydroxy group).
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Structural characterization of the major glycolipids from Arthrobacter globiformis and Arthrobacter scleromae.
Carbohydr. Res.
PUBLISHED: 01-16-2010
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Arthrobacter is a genus of Gram-positive bacteria widely distributed in soil. The ability to catabolize a variety of xenobiotics has shown their potential as a detoxifying agent. Recently, Arthrobacter has been also recognized as an opportunistic pathogen. Glycolipids from A. scleromae, a clinical isolate, and A. globiformis, from soil, were isolated by chloroform-methanol extraction and subsequently purified using column chromatography and high-performance liquid chromatography. Structural studies were carried out utilizing specific chemical degradation, matrix-assisted laser-desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT ICR-MS), and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The major glycolipids in A. scleromae and A. globiformis were found to be a diglycosylglycerol with the structure alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Gro (Man A-Man B-Gro; G1), and a monoglycosylglycerol with the structure beta-Galp-(1-->3)-Gro (G2). Glycolipids were acylated at positions 1 of Gro and 6 of Man B in the case of G1 and at positions 1 and 2 of Gro in the case of G2. The distribution of the fatty acids was different in both species. A. scleromae glycolipids contained heptadecanoic acid while in the A. globiformis glycolipids mainly pentadecanoic acid could be detected. The substitution by hexadecanoic acid was proportionally similar in both species. The taxonomical value of major glycolipids from Arthrobacter is also presented.
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Arabinogalactan isolated from cowshed dust extract protects mice from allergic airway inflammation and sensitization.
J. Allergy Clin. Immunol.
PUBLISHED: 01-07-2010
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Extract from cowshed dust (CDE) is a source of immunomodulating substances. We have previously shown that such substances protect from experimental allergic disorders in a mouse model of asthma.
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The structure of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas bestiarum strain 207.
Carbohydr. Res.
PUBLISHED: 01-04-2010
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The O-specific polysaccharide obtained by mild-acid degradation of Aeromonas bestiarum 207 lipopolysaccharide was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy. The sequence of the sugar residues was determined by ROESY and HMBC experiments. It is concluded that the O-polysaccharide is composed of branched pentasaccharide repeating units of the following structure: [structure: see the text]
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Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter lwoffii F78.
J. Exp. Med.
PUBLISHED: 12-07-2009
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The pre- and postnatal environment may represent a window of opportunity for allergy and asthma prevention, and the hygiene hypothesis implies that microbial agents may play an important role in this regard. Using the cowshed-derived bacterium Acinetobacter lwoffii F78 together with a mouse model of experimental allergic airway inflammation, this study investigated the hygiene hypothesis, maternal (prenatal) microbial exposure, and the involvement of Toll-like receptor (TLR) signaling in prenatal protection from asthma. Maternal intranasal exposure to A. lwoffii F78 protected against the development of experimental asthma in the progeny. Maternally, A. lwoffii F78 exposure resulted in a transient increase in lung and serum proinflammatory cytokine production and up-regulation of lung TLR messenger RNA. Conversely, suppression of TLRs was observed in placental tissue. To investigate further, the functional relevance of maternal TLR signaling was tested in TLR2/3/4/7/9(-/-) knockout mice. The asthma-preventive effect was completely abolished in heterozygous offspring from A. lwoffii F78-treated TLR2/3/4/7/9(-/-) homozygous mother mice. Furthermore, the mild local and systemic inflammatory response was also absent in these A. lwoffii F78-exposed mothers. These data establish a direct relationship between maternal bacterial exposures, functional maternal TLR signaling, and asthma protection in the progeny.
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Structural characterization of teichoic acids from Lactobacillus brevis.
Carbohydr. Res.
PUBLISHED: 11-12-2009
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Teichoic acids are a major constituent of the cell wall of Gram-positive bacteria. Structural characterization of lipoteichoic and teichoic acids isolated from Lactobacillus brevis was undertaken using 1D and 2D NMR experiments as well as chemical methodology. Compositional analysis indicated the presence of high amounts of glycerol, glucose, and alanine. In the case of LTA octadecenoic acid was also detected. The basic LTA/WTA structure was identified as 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at C-2 of the glycerol residues with D-Ala or alpha-D-Glc. In the case of LTA a higher amount of Ala could be detected and partial alanylation at position C-6 of the Glc could also be observed.
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The periplasmic chaperone Skp facilitates targeting, insertion, and folding of OmpA into lipid membranes with a negative membrane surface potential.
Biochemistry
PUBLISHED: 09-29-2009
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The basic biochemical and biophysical principles by which chaperone-bound membrane proteins are targeted to the outer membrane of Gram-negative bacteria for insertion and folding are unknown. Here we compare spontaneous folding of outer membrane protein A (OmpA) of Escherichia coli from its urea-unfolded form and from the complex with its periplasmic chaperone Skp into lipid bilayers. Skp facilitated folding of OmpA into negatively charged membranes containing dioleoylphosphatidylglycerol (DOPG). In contrast, Skp strongly inhibited folding of OmpA when bilayers were composed of dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine (DOPC). These results indicate that the positively charged Skp targets OmpA to a negatively charged membrane, which facilitates the release of OmpA from its complex with Skp for subsequent folding and membrane insertion. The dual functionality of Skp as a chaperone and as a targeting protein is ideal to mediate the transport of OmpA and other outer membrane proteins across the periplasm in a folding-competent form to the outer membrane, which is negatively charged on its periplasmic side. OmpA (pI 5.5) folded most efficiently above its isoelectric point. In the absence of Skp and in contrast to folding into DOPC bilayers, insertion and folding of OmpA were retarded for membranes containing DOPG at neutral or basic pH because of electrostatic repulsion. When folding of OmpA was performed near its isoelectric point, urea dilution led to a more compact aqueous form of OmpA previously characterized by fluorescence, which folded at a much slower rate. Under conditions where two different aqueous conformations of OmpA coexisted, e.g., in the titration region of OmpA, the last step of OmpA folding could be well described by two parallel pseudo-first-order kinetic phases. In this kinetic model, the contribution of the faster folding process, but not the changes in the rate constants, determined the folding yields obtained at different pH. The faster phase dominated when the experimental conditions favored the less compact form of aqueous OmpA.
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Characterization of the specific O-polysaccharide structure and biosynthetic gene cluster of Yersinia pseudotuberculosis serotype O:15.
Innate Immun
PUBLISHED: 09-01-2009
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Yersinia pseudotuberculosis serotyping scheme contains 21 serotypes based on the distribution of about 30 different O-factors within the species. The chemical structures of LPSs and the genetic basis of their biosynthesis has been determined for a number of Y. pseudotuberculosis strains representing different serotypes; thus, an overall picture of the relationship between genetics and structures is emerging. In this work, we have performed a structural and genetic analysis of the Y. pseudotuberculosis serotype O:15 O-specific polysaccharide. Our results showed that the set-up of the Y. pseudotuberculosis O:15 gene cluster is a hybrid between those of Y. pseudotuberculosis serotypes O:1b and O:5a, possibly due to a single recombination event. The hybrid nature could also be seen in the structure of the O-specific polysaccharide repeating unit pentasaccharide. It contains a tetrameric backbone identical to that of O:5a while the branching paratofuranose residue is identical to that of O:1b.
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Identification and role of a 6-deoxy-4-keto-hexosamine in the lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3.
Chemistry
PUBLISHED: 08-22-2009
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The outer core (OC) region of Yersinia enterocolitica serotype O:3 lipopolysaccharide is a hexasaccharide essential for the integrity of the outer membrane. It is involved in resistance against cationic antimicrobial peptides and plays a role in virulence during early phases of infection. We show here that the proximal residue of the OC hexasaccharide is a rarely encountered 4-keto-hexosamine, 2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (Sugp) and that WbcP is a UDP-GlcNAc-4,6-dehydratase enzyme responsible for the biosynthesis of the nucleotide-activated form of this rare sugar converting UDP-2-acetamido-2-deoxy-D-glucopyranose (UDP-D-GlcpNAc) to UDP-2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (UDP- Sugp). In an aqueous environment, the 4-keto group of this sugar was present in the 4-dihydroxy form, due to hydration. Furthermore, evidence is provided that the axial 4-hydroxy group of this dihydroxy function was crucial for the biological role of the OC, that is, in the bacteriophage and enterocoliticin receptor structure and in the epitope of a monoclonal antibody.
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WaaA of the hyperthermophilic bacterium Aquifex aeolicus is a monofunctional 3-deoxy-D-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis.
J. Biol. Chem.
PUBLISHED: 06-22-2009
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The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli DeltawaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate.
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Novel interactions of glycosaminoglycans and bacterial glycolipids mediate binding of enterococci to human cells.
J. Biol. Chem.
PUBLISHED: 04-23-2009
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Enterococcus faecalis is among the most important nosocomial pathogens. The intestinal mucosa is considered to be the main site used by these bacteria for entrance and dissemination. A better understanding of the mechanisms involved in colonization and invasion of enterococci may help to devise methods to prevent infections in hospitalized patients. Glycosaminoglycans, which are present on the surface of all eukaryotic cells, were investigated with regard to their role as host receptors for adhesion of E. faecalis. Competitive binding assays, enzymatic digestion, and reduction of the sulfation of the glycosaminoglycan chains indicated that heparin and heparan sulfate, but not chondroitin sulfate B, played important roles in adhesion of E. faecalis 12030 to Caco2 cells. By using proteinases and carbohydrate oxidation by sodium meta-periodate to modify the bacterial surface, it could be demonstrated that a sugar-containing molecule rather than a protein is the bacterial ligand mediating adhesion to eukaryotic cells. Preincubation of Caco2 cells with the enterococcal glycolipid diglucosyldiacylglycerol but not other carbohydrate cell wall components inhibited bacterial binding. These results may indicate that heparin and/or heparan sulfate on host epithelial cells and diglucosyldiacylglycerol, either itself or as a partial structure of lipoteichoic acid, are involved in enterococcal adhesion to colonic epithelia, the first step in translocation from the intestinal tract.
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Binding regions of outer membrane protein A in complexes with the periplasmic chaperone Skp. A site-directed fluorescence study.
Biochemistry
PUBLISHED: 04-23-2009
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Periplasmic Skp facilitates folding and membrane insertion of many outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. We have examined the binding sites of outer membrane protein A (OmpA) from Escherichia coli in its complexes with the membrane protein chaperone Skp and with Skp and lipopolysaccharide (LPS) by site-directed fluorescence spectroscopy. Single-Trp OmpA mutants, W(n)-OmpA, with tryptophan at position n in the polypeptide chain were isolated in the unfolded form in 8 M urea. In five beta(x)W(n)-OmpA mutants, the tryptophan was located in beta-strand x, in four l(y)W(n)-OmpA mutants, in outer loop y, and in three t(z)W(n)-OmpA mutants in turn z of the beta-barrel transmembrane domain (TMD) of OmpA. PDW(286)-OmpA contained tryptophan in the periplasmic domain (PD). After dilution of the denaturant urea in aqueous solution, spectra indicated a more hydrophobic environment of the tryptophans in beta(x)W(n) mutants in comparison to l(y)W(n)-OmpA and t(z)W(n)-OmpA, indicating that the loops and turns form the surface of hydrophobically collapsed OmpA, while the strand regions are less exposed to water. Addition of Skp increased the fluorescence of all OmpA mutants except PDW(286)-OmpA, demonstrating binding of Skp to the entire beta-barrel domain but not to the PD of OmpA. Skp bound the TMD of OmpA asymmetrically, displaying much stronger interactions with strands beta(1) to beta(3) in the N-terminus than with strands beta(5) to beta(7) in the C-terminus. This asymmetry was not observed for the outer loops and the periplasmic turns of the TMD of OmpA. The fluorescence results demonstrated that all turns and loops l(1), l(2), and l(4) were as strongly bound to Skp as the N-terminal beta-strands. Addition of five negatively charged LPS per one preformed Skp.W(n)-OmpA complex released the C-terminal loops l(2), l(3), and l(4) of the TMD of OmpA from the complex, while its periplasmic turn regions remained bound to Skp. Our results demonstrate that interactions of Skp.OmpA complexes with LPS change the conformation of OmpA in the Skp complex for facilitated insertion and folding into membranes.
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Natural killer T cells activated by a lipopeptidophosphoglycan from Entamoeba histolytica are critically important to control amebic liver abscess.
PLoS Pathog.
PUBLISHED: 04-16-2009
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The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jalpha18(-/-) mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using alpha-galactosylceramide (alpha-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d(-/-) mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-gamma but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28:0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-gamma production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to alpha-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess.
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The O-specific polysaccharide structure and biosynthetic gene cluster of Yersinia pseudotuberculosis serotype O:11.
Carbohydr. Res.
PUBLISHED: 03-06-2009
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In the Yersinia pseudotuberculosis serotyping scheme, 21 serotypes are present originating from about 30 different O-factors distributed within the species. With regard to the chemical structures of lipopolysaccharides (LPSs) and the genetic basis of their biosynthesis, a number, but not all, of Y. pseudotuberculosis strains representing different serotypes have been investigated. In order to present an overall picture of the relationship between genetics and structures, we have been working on the genetics and structures of various Y. pseudotuberculosis O-specific polysaccharides (OPSs). Here, we present a structural and genetic analysis of the Y. pseudotuberculosis serotype O:11 OPS. Our results showed that this OPS structure has the same backbone as that of Y. pseudotuberculosis O:1b, but with a 6d-L-Altf side-branch instead of Parf. The 3 end of the gene cluster is the same as that for O:1b and has the genes for synthesis of the backbone and for processing the completed repeat unit. The 5 end has genes for synthesis of 6d-L-Altf and its transfer to the repeating unit backbone. The pathway for the synthesis of the 6d-L-Altf appears to be different from that for 6d-L-Altp in Y. enterocolitica O:3. The chemical structure of the O:11 repeating unit is [structure: see text].
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Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis.
Mol. Microbiol.
PUBLISHED: 01-28-2009
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Biofilm production is thought to be an important step in many enterococcal infections. In several Gram-positive bacteria, membrane glycolipids have been implicated in biofilm formation. We constructed a non-polar deletion mutant of a putative glucosyltransferase designated biofilm-associated glycolipid synthesis A (bgsA) in Enterococcus faecalis 12030. Analysis of major extracted glycolipids by nuclear magnetic resonance spectroscopy revealed that the cell membrane of 12030 Delta bgsA was devoid of diglucosyl-diacylglycerol (DGlcDAG), while monoglucosyl-diacylglycerol was overrepresented. The cell walls of 12030 Delta bgsA contained longer lipoteichoic acid molecules and were less hydrophobic than wild-type bacteria. Inactivation of bgsA in E. faecalis 12030 and E. faecalis V583 led to an almost complete arrest of biofilm formation on plastic surfaces. Overexpression of bgsA, on the other hand, resulted in increased biofilm production. While initial adherence was not affected, bgsA-deficient bacteria did not accumulate in the growing biofilm. Also, adherence of E. faecalis Delta bgsA to Caco-2 cells was impaired. In a mouse bacteraemia model, E. faecalis 12030 Delta bgsA was cleared more rapidly from the bloodstream than the wild-type strain. In summary, BgsA is a glycosyltransferase synthetizing DGlcDAG, a glycolipid and lipoteichoic acid precursor involved in biofilm accumulation, adherence to host cells, and virulence in vivo.
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Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.
Cell. Microbiol.
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Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ?-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.
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The O-specific polysaccharide structure and gene cluster of serotype O:12 of the Yersinia pseudotuberculosis complex, and the identification of a novel L-quinovose biosynthesis gene.
Glycobiology
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A major virulence factor for Yersinia pseudotuberculosis is lipopolysaccharide, including O-polysaccharide (OPS). Currently, the OPS based serotyping scheme for Y. pseudotuberculosis includes 21 known O-serotypes, with genetic and structural data available for 17 of them. The completion of the OPS structures and genetics of this species will enable the visualization of relationships between O-serotypes and allow for analysis of the evolutionary processes within the species that give rise to new serotypes. Here we present the OPS structure and gene cluster of serotype O:12, thus adding one more to the set of completed serotypes, and show that this serotype is present in both Y. pseudotuberculosis and the newly identified Y. similis species. The O:12 structure is shown to include two rares ugars: 4-C[(R)-1-hydroxyethyl]-3,6-dideoxy-D-xylo-hexose(D-yersiniose) and 6-deoxy-L-glucopyranose (L-quinovose).We have identified a novel putative guanine diphosphate(GDP)-L-fucose 4-epimerase gene and propose a pathway for the synthesis of GDP-L-quinovose, which extends the known GDP-L-fucose pathway.
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Structural analysis of the lipoteichoic acids isolated from bovine mastitis Streptococcus uberis 233, Streptococcus dysgalactiae 2023 and Streptococcus agalactiae 0250.
Carbohydr. Res.
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Lipoteichoic acid (LTA) is an amphiphilic polycondensate located in the cell envelope of Gram-positive bacteria. In this study, LTAs were isolated from the three bovine mastitis species Streptococcus uberis 233, Streptococcus dysgalactiae 2023, and Streptococcus agalactiae 0250. Structural investigations of these LTAs were performed applying 1D and 2D nuclear magnetic resonance experiments as well as chemical analyses and mass spectrometry. Compositional analysis revealed the presence of glycerol (Gro), Glc, alanine (Ala), and 16:0, 16:1, 18:0, 18:1. The LTAs of the three Streptococcus strains possessed the same structure, that is, a lipid anchor comprised of ?-Glcp-(1?2)-?-Glcp-(1?3)-1,2-diacyl-sn-Gro and the hydrophilic backbone consisting of poly(sn-Gro-1-phosphate) randomly substituted at O-2 of Gro by d-Ala.
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The structure of the extracellular teichoic acids from the allergy-protective bacterium Lactococcus lactis G121.
Biol. Chem.
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The Gram-positive bacterium Lactococcus lactis G121 is a farm isolate that protects mice from ovalbumin-induced asthma. To understand the molecular mechanisms of such allergy-protective properties, the isolation and characterization of cell envelope constituents is crucial. Here, structural analyses of the extracellular teichoic acid (EC TA) from L. lactis G121 are presented. Extraction with 0.9% saline afforded a crude TA fraction. Consecutive size exclusion chromatography on Biogel P60 and P10 matrix was performed to purify the sample. Chemical component analyses, high-resolution electrospray ionization Fourier-transformed ion cyclotron mass spectrometry, and nuclear magnetic resonance spectroscopy were conducted for structural elucidation. The EC TA was a poly(glycosylglycerol phosphate) molecule with a repeating unit of -6)-[?-D-Glcp-(1?3)-][?-D-GlcpNAc-(1?4)-]?-D-GalpNAc-(1?3)-?-D-GlcpNAc-(1?2)-glycerol-(1-P-).
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The structure of the O-specific polysaccharide of the lipopolysaccharide from Yersinia enterocolitica serotype O:50 strain 3229.
Carbohydr. Res.
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The genus Yersinia represents Gram-negative bacteria that are widely distributed in the environment and possess lipopolysaccharide (LPS) as a major molecule on the surface of the bacterial envelope. LPS is composed of an anchoring lipid, termed lipid A, and a polysaccharide part which not only determines the bacterial serotype but may also serve as a bacteriophage receptor. Here we present the structure of the O-polysaccharide (O-PS) of Yersinia enterocolitica O:50 phage ?R1-37-sensitive strain 3229. The structural characterization was performed utilizing 1D and 2D nuclear magnetic resonance spectroscopy and chemical analyses. The structure of the O-PS biological repeating unit was identified as ?2)-?-L-Rhap-(1?3)-?-L-FucpNAc-(1?3)-?-L-FucpNAc-(1?3)-?-D-GlcpNAc-(1?.
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Secondary cell wall polymers of Enterococcus faecalis are critical for resistance to complement activation via mannose-binding lectin.
J. Biol. Chem.
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The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583?tagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of ?6)[?-l-Rhap-(1?3)]?-D-GalpNAc-(1?5)-Rbo-1-P and ?6) ?-D-Glcp-(1?3) [?-D-Glcp-(1?4)]-?-D-GalpNAc-(1?5)-Rbo-1-P?, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system.
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The bactericidal activity of the C-type lectin RegIII? against Gram-negative bacteria involves binding to lipid A.
J. Biol. Chem.
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RegIII? is a member of the C-type lectin family called RegIII. It is known to bind peptidoglycan, and its bactericidal activity shapes the interactions with commensal and pathogenic gut bacteria. However, little is known about its carbohydrate recognition specificity and the bactericidal mechanism, particularly against Gram-negative bacteria. Here, we show that RegIII? can bind directly to LPS by recognizing the carbohydrate moiety of lipid A via a novel motif that is indispensable for its bactericidal activity. This bactericidal activity of RegIII? could be inhibited by preincubation with LPS, lipid A, or gentiobiose. The latter is a disaccharide composed of two units of ?-(1?6)-linked d-glucose and resembles the carbohydrate moiety of lipid A. Therefore, this structural element may form a key target site recognized by RegIII?. Using point-mutated RegIII? proteins, we found that amino acid residues in two structural motifs termed "loop 1" and "loop 2," are important for peptidoglycan and lipid A binding (Arg-135, Asp-142) and for the bactericidal activity (Glu-134, Asn-136, Asp-142). Thus, the ERN motif and residue Asp-142 in the loop 2 are of critical importance for RegIII? function. This provides novel insights into the carbohydrate recognition specificity of RegIII? and explains its bactericidal activity against Gram-negative bacteria.
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The structure of the wall teichoic acid isolated from Enterococcus faecalis strain 12030.
Carbohydr. Res.
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Wall teichoic acid (WTA) was isolated from Enterococcus faecalis 12030, a clinical isolate and biofilm-producing strain, and analyzed using compositional chemical methods, nuclear magnetic resonance spectroscopy, and mass spectrometry. The repeating units of WTA were composed of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, D-ribitol, and phosphate in a molar ratio 1:2:1:1:1:1, and had the structure given below.
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Bacterial lipopolysaccharides in plant and mammalian innate immunity.
Protein Pept. Lett.
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This mini-review gives a structural view on the lipopolysaccharides (LPSs), the endotoxin from Gram negative bacteria, paying attention on the features that are relevant for their activity as elicitors of the innate immune system of humans, animals and plants.
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Structural analysis of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas veronii bv. sobria strain K49.
Carbohydr. Res.
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The O-specific polysaccharide obtained by mild-acid degradation of the lipopolysaccharide from Aeromonas veronii bv. sobria strain K49 was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy. The sequence of the sugar residues was determined using (1)H,(1)H NOESY and (1)H,(13)C HMBC experiments. The O-specific polysaccharide was found to be a high molecular mass polysaccharide composed of repeating units of the structure: ?2)-?-D-Quip3NAc-(1?3)-?-L-Rhap-(1?3)-?-L-Rhap-(1?2)-?-L-Rhap-(1?3)-?-D-FucpNAc-(1? ESI MS confirmed the pentasaccharide structure of the repeating unit, as the molecular mass peaks seen in the spectrum differed by 812.34 u, a value corresponding to the calculated molecular mass of the O-unit.
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Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis.
Proc. Natl. Acad. Sci. U.S.A.
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WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX(5)GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA.
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