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Sample records for capsular polysaccharide structure

  1. Structurally altered capsular polysaccharides produced by mutant bacteria

    NASA Technical Reports Server (NTRS)

    Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor); Richards, Gil F. (Inventor)

    1995-01-01

    Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

  2. Method for producing capsular polysaccharides

    NASA Technical Reports Server (NTRS)

    Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor); Richards, Gil F. (Inventor)

    1994-01-01

    Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

  3. Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 10 determined by NMR spectroscopy.

    PubMed

    Sheng, S; Cherniak, R

    1997-12-01

    The complete primary structure of the type-specific capsular polysaccharide of Clostridium perfringens Hobbs 10 was determined. The polysaccharide was isolated from C. perfringens Hobbs 10 by cold-water extraction of whole, heavily encapsulated cells. The polysaccharide was purified, by ethanol precipitation, deproteination, selective precipitation with hexadecyltrimethylammonium bromide, ion-exchange chromatography and gel-filtration chromatography. The polysaccharide was comprised of D-glucose, D-galactose, N-acetylgalactosamine, and iduronic acid, in molar ratios of 2:2:1:1. Sequence and linkage assignments of the glycosyl residues were obtained by NMR spectroscopy, specifically by the combination of two-dimensional homonuclear DQF-COSY, TQF-COSY and TOCSY, heteronuclear ¿1H, 13C¿ single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments. The capsular polysaccharide of C. perfringens Hobbs 10 is a polymer composed of a hexasaccharide repeating unit with the following structure: [formula: see text] This structure is novel among bacterial cell-surface polysaccharides, and it is only the second of many serotypically distinct capsular polysaccharides of C. perfringens to be described.

  4. Structural characterization of an all-aminosugar-containing capsular polysaccharide from Colwellia psychrerythraea 34H.

    PubMed

    Casillo, Angela; Ståhle, Jonas; Parrilli, Ermenegilda; Sannino, Filomena; Mitchell, Daniel E; Pieretti, Giuseppina; Gibson, Matthew I; Marino, Gennaro; Lanzetta, Rosa; Parrilli, Michelangelo; Widmalm, Göran; Tutino, Maria L; Corsaro, Maria M

    2017-02-04

    Colwellia psychrerythraea strain 34H, a Gram-negative bacterium isolated from Arctic marine sediments, is considered a model to study the adaptation to cold environments. Recently, we demonstrated that C. psychrerythraea 34H produces two different extracellular polysaccharides, a capsular polysaccharide and a medium released polysaccharide, which confer cryoprotection to the bacterium. In this study, we report the structure of an additional capsular polysaccharide produced by Colwellia grown at a different temperature. The structure was determined using chemical methods, and one- and two-dimensional NMR spectroscopy. The results showed a trisaccharide repeating unit made up of only amino-sugar residues: N-acetyl-galactosamine, 2,4-diacetamido-2,4,6-trideoxy-glucose (bacillosamine), and 2-acetamido-2-deoxyglucuronic acid with the following structure: →4)-β-D-GlcpNAcA-(1 →3)-β-D-QuipNAc4NAc-(1 →3)-β-D-GalpNAc-(1 →. The 3D model, generated in accordance with (1)H,(1)H-NOE NMR correlations and consisting of ten repeating units, shows a helical structure. In contrast with the other extracellular polysaccharides produced from Colwellia at 4 °C, this molecule displays only a low ice recrystallization inhibition activity.

  5. CAPSULAR POLYSACCHARIDE OF AZOTOBACTER AGILIS.

    PubMed

    COHEN, G H; JOHNSTONE, D B

    1964-12-01

    Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Capsular polysaccharide of Azotobacter agilis. J. Bacteriol. 88:1695-1699. 1964.-Capsular polysaccharide from Azotobacter agilis strain 132 was recovered from washed cells by alkaline digestion. The polysaccharide was purified by centrifugation, repeated alcohol precipitation, Sevag deproteinization, and treatment with ribonuclease and charcoal-cellulose. Methods of isolation and purification appeared to provide a polymer showing no evidence of heterogeneity when examined by chemical and physical methods. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed polysaccharide indicated that the polymer contained galactose and rhamnose at a molar ratio of approximately 1.0:0.7. A sialic acid-like component was also present in the polysaccharide. The study shows significant differences in the chemical composition of the extra-cellular polysaccharide of A. agilis and that of A. vinelandii. This adds further biochemical evidence for the right of these species to independent status.

  6. Synthesis of part structures of Cryptococcus neoformans serotype C capsular polysaccharide.

    PubMed

    Guazzelli, Lorenzo; McCabe, Orla; Oscarson, Stefan

    2016-10-04

    Cryptococcus neoformans is a fungal pathogen that can cause life-threatening infections in immunocompromised patients. The development of a vaccine based on the capsular polysaccharide of C. neoformans is still an open challenge due to the heterogeneity of the capsular polysaccharide and the difficulty of identifying protective epitopes. Therefore, construction of structurally defined part structures of the C. neoformans GXM capsule is in great demand. Herein is presented the synthesis of a 3-O-naphthalenylmethyl protected trisaccharide thioglycoside building block which is present in C. neoformans serotype C polysaccharide. Its property as a donor in a glycosylation reaction with a model acceptor has been evaluated together with its behaviour as an acceptor following removal of the temporary protecting group. The heavily branched hexasaccharide was obtained in good yields and excellent α-selectivity. The frame shifted octasaccharide structural triad motif for serotype C was also prepared following the same building block strategy. For the first time this structural motif, which is the most substituted amongst the four C. neoformans serotypes, was prepared. Three synthesized C. neoformans serotype C fragments of varying size, from penta-up to octasaccharide, were deprotected and will be included in unique glycoarrays to further investigate the possibility to develop a synthetic vaccine against this pathogen.

  7. Synthesis of oligosaccharides corresponding to structures found in capsular polysaccharides of Cryptococcus neoformans--II.

    PubMed

    Garegg, P J; Olsson, L; Oscarson, S

    1996-11-01

    Formula 1 depicts a generalized structure of the capsular polysaccharides of four serotypes of the opportunistic microorganism Cryptococcus neoformans, which appears as one of the major infections in the late stages of development of AIDS. Syntheses are now described of two tetrasaccharides with corresponding structures. These are methyl O-alpha-D-mannopyranosyl-(1-->3)-[O-beta-D-xylopyranosyl-(1-->2)] -O-alpha-D-mannopyranosyl-(1-->3)-alpha-D-mannopyranoside and methyl O-alpha-D-mannopyranosyl-(1-->3)-[O-beta-D-glucopyranosyluronic acid-(1-->2)]-O-alpha-D-mannopyranosyl-(1-->3)-alpha-D-mannopyranoside.

  8. Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus.

    PubMed

    Miyafusa, Takamitsu; Caaveiro, Jose M M; Tanaka, Yoshikazu; Tanner, Martin E; Tsumoto, Kouhei

    2013-06-11

    Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein-protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

  9. Structural analysis of the capsular polysaccharide from Campylobacter jejuni RM1221

    USDA-ARS?s Scientific Manuscript database

    The complete genome of Campylobacter jejuni strain RM1221 (Penner serotype HS:53) was reported recently and contains a novel capsular polysaccharide (CPS) biosynthesis locus. Cell surface carbohydrates such as CPS are known to be important for bacterial survival and often contribute to pathogenesis....

  10. Structure elucidation of capsular polysaccharides from Streptococcus pneumoniae serotype 33C, 33D, and revised structure of serotype 33B.

    PubMed

    Lin, Fiona L; Vinogradov, Evgeny; Deng, Chenghua; Zeller, Sandra; Phelan, Lynn; Green, Bruce A; Jansen, Kathrin U; Pavliak, Viliam

    2014-01-13

    We report herein the previously unknown structures of the pneumococcal capsular polysaccharides serotype 33C and 33D, and a revised structure of serotype 33B. The syntenic pair 33B/33D has nearly identical polysaccharide repeat units with the exception of one sugar residue (→2-α-Glcp in 33B and →2-α-Galp in 33D). Serotype 33C is structurally more similar to 33B/33D than 33A/33F, in that it also possesses a backbone ribitol-phosphate group and a →3-β-GalpNAc residue, both of which are absent in the repeat units of 33A/33F. Serotype 33C is notably different from all other serogroup 33 polysaccharides, as there is no →3-β-Glcp residue and the location of the O-acetylation of the →5-β-Galf residue (O-6) differs from the other serogroup 33 polysaccharides (O-2). This completes the structural assignments of polysaccharides within serogroup 33 and provides a framework for understanding the recognition of epitopes by serogroup 33 typing sera based on observed cross-reactivities reported in the literature. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. A novel capsular polysaccharide from Rhizobium rubi strain DSM 30149.

    PubMed

    De Castro, Cristina; Fregolino, Eleonora; Gargiulo, Valentina; Lanzetta, Rosa; Parrilli, Michelangelo

    2008-07-07

    Rhizobium rubi, strain DSM 30149, is a Gram negative phytopathogenic bacterium which produces a linear polysaccharide with the following repeating unit: This new structure was determined by spectroscopical and chemical methods. It presents similar lipophilic features reported for another strain of R. rubi. These contrast with features already known for capsular polysaccharide species from symbiontic members of the Rhizobiaceae family, namely highly anionic polymers.

  12. Structure of the neutral capsular polysaccharide of Acinetobacter baumannii NIPH146 that carries the KL37 capsule gene cluster.

    PubMed

    Arbatsky, Nikolay P; Shneider, Mikhail M; Kenyon, Johanna J; Shashkov, Alexander S; Popova, Anastasiya V; Miroshnikov, Konstantin A; Volozhantsev, Nikolay V; Knirel, Yuriy A

    2015-09-02

    Capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii NIPH146, and the following structure of branched pentasaccharide repeating unit was established by sugar analyses along with 1D and 2D NMR spectroscopy: In comparison to most other known capsular polysaccharides of A. baumannii, the CPS studied is neutral and lacks any specific monosaccharide component. The synthesis, assembly and export of this structure could be attributed to genes in a novel capsule biosynthesis gene cluster, designated KL37, which was found in the NIPH146 genome. The CPS of A. baumannii NIPH146 shares the α-d-Galp-(1→6)-β-d-Glcp-(1→3)-d-GalpNAc-(1→ trisaccharide fragment with the CPS units of several A. baumannii strains, including ATCC 17978 and LUH 5537 that carry the KL3 and KL22 gene clusters, respectively. KL37 contains two genes for glycosyltransferases that are related to two glycosyltransferase genes present in both KL3 and KL22, and the encoded proteins could be tentatively assigned to linkages between sugars in the CPS repeat.

  13. Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions

    DOEpatents

    Stephens, David S [Stone Mountain, GA; Gudlavalleti, Seshu K [Kensington, MD; Tzeng, Yih-Ling [Atlanta, GA; Datta, Anup K [San Diego, CA; Carlson, Russell W [Athens, GA

    2011-02-08

    Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

  14. Structural determination of the K14 capsular polysaccharide from an ST25 Acinetobacter baumannii isolate, D46.

    PubMed

    Kenyon, Johanna J; Hall, Ruth M; De Castro, Cristina

    2015-11-19

    The structure of the capsular polysaccharide (CPS) recovered from D46, an extensively antibiotic resistant ST25 Acinetobacter baumannii clinical isolate, was elucidated. The structure was resolved on the basis of NMR spectroscopy and chemical analyses, and was found to contain a branched neutral pentasaccharide with a backbone composed of GalpNAc and Galp residues, all d configured, and a d-Glcp side group. The KL14 gene cluster found in the D46 genome includes genes for four glycosyltransferases but no modules for synthesis of complex sugars, and this is consistent with the structure of K14. The K14 structure and KL14 sequence clarify the relationship between the structure and K locus sequence for A. nosocomialis isolate LUH5541. The identity of the first sugar of the K14 repeat unit (K unit), and the functions of the four encoded glycosyltransferases and Wzy polymerase were predicted.

  15. A unique capsular polysaccharide structure from the psychrophilic marine bacterium Colwellia psychrerythraea 34H that mimics antifreeze (glyco)proteins.

    PubMed

    Carillo, Sara; Casillo, Angela; Pieretti, Giuseppina; Parrilli, Ermenegilda; Sannino, Filomena; Bayer-Giraldi, Maddalena; Cosconati, Sandro; Novellino, Ettore; Ewert, Marcela; Deming, Jody W; Lanzetta, Rosa; Marino, Gennaro; Parrilli, Michelangelo; Randazzo, Antonio; Tutino, Maria L; Corsaro, M Michela

    2015-01-14

    The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments.

  16. Characterization of the structure and biological functions of a capsular polysaccharide produced by Staphylococcus saprophyticus.

    PubMed

    Park, Sunny; Kelley, Kathryn A; Vinogradov, Evgeny; Solinga, Robert; Weidenmaier, Christopher; Misawa, Yoshiki; Lee, Jean C

    2010-09-01

    Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections in women. S. saprophyticus strain ATCC 15305 carries two staphylococcal cassette chromosome genetic elements, SCC(15305RM) and SCC(15305cap). The SCC(15305cap) element carries 13 open reading frames (ORFs) involved in capsular polysaccharide (CP) biosynthesis, and its G+C content (26.7%) is lower than the average G+C content (33.2%) for the whole genome. S. saprophyticus strain ATCC 15305 capD, capL, and capK (capD(Ssp), capL(Ssp), and capK(Ssp)) are homologous to genes encoding UDP-FucNAc biosynthesis, and gtaB and capI(Ssp) show homology to genes involved in UDP-glucuronic acid synthesis. S. saprophyticus ATCC 15305 CP, visualized by immunoelectron microscopy, was extracted and purified using anionic-exchange and size exclusion chromatography. Analysis of the purified CP by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy and gas-liquid chromatography revealed two types of branched tetrasaccharide repeating units composed of the following: -4)-beta-Glc-(1-3)-Sug-(1-4)-beta-GlcA-(1- | beta-GlcNAc-(1-2) Sug represents two stereoisomers of 2-acetamido-2,6-dideoxy-hexos-4-ulose residues, one of which has an arabino configuration. The encapsulated ATCC 15305 strain was resistant to complement-mediated opsonophagocytic killing by human neutrophils, whereas the acapsular mutant C1 was susceptible. None of 14 clinical isolates reacted with antibodies to the ATCC 15305 CP. However, 11 of the 14 S. saprophyticus isolates were phenotypically encapsulated based on their resistance to complement-mediated opsonophagocytic killing and their failure to hemagglutinate when cultivated aerobically. Ten of the 14 clinical strains carried homologues of the conserved staphylococcal capD gene or the S. saprophyticus gtaB gene, or both. Our results suggest that some strains of S. saprophyticus are encapsulated and that more than one capsular serotype exists.

  17. Cellular immunity to Bacteroides fragilis capsular polysaccharide

    PubMed Central

    1982-01-01

    The polysaccharide capsule of Bacteroides fragilis has been shown to be important in the virulence of the organism. The capsular polysaccharide (CP) of B. fragilis has been extensively purified. Using a murine model of intraabdominal abscess formation, we have been able to demonstrate cellular immunity to the capsular polysaccharide of B. fragilis. Immunization of C57BL/10J mice with the CP over 5 wk prevents abscess formation when the mice are challenged with B. fragilis intraperitoneally. This immunity can be transferred to naive mice with spleen cells from immune animals. The immune cells bear Thy-1.2 and Ly- 2.2 antigens. The immune response has been shown to be antigen specific, but not H-2 restricted. The possibility that these immune cells are suppressor T cells is discussed. The experimental system presented provides a model for the examination of the cellular interactions responsible for abscess formation and the cellular response to bacterial pathogens. PMID:6174672

  18. Masquerading microbial pathogens: Capsular polysaccharides mimic host-tissue molecules

    PubMed Central

    Cress, Brady F.; Englaender, Jacob A.; He, Wenqin; Kasper, Dennis; Linhardt, Robert J.; Koffas, Mattheos A. G.

    2014-01-01

    Summary Bacterial pathogens bearing capsular polysaccharides identical to mammalian glycans benefit from an additional level of protection from host immune response. The increasing prevalence of antibiotic resistant bacteria portends an impending post-antibiotic age, characterized by diminishing efficacy of common antibiotics and routine application of multifaceted, complementary therapeutic approaches to treat bacterial infections, particularly multidrug-resistant organisms. The first line of defense for most bacterial pathogens consists of a physical and immunological barrier known as the capsule, commonly composed of a viscous layer of carbohydrates that are covalently bound to the cell wall in Gram-positive bacteria or often to lipids of the outer membrane in many Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of high molecular weight polysaccharides contributing to virulence of many human pathogens in the gut, respiratory tree, urinary tract, and other host tissues, by hiding cell-surface components that might otherwise elicit host immune response. This review highlights capsular polysaccharides that are structurally identical or similar to polysaccharides found in mammalian tissues, including polysialic acid and glycosaminoglycan capsules hyaluronan, heparosan, and chondroitin. Such non-immunogenic coatings render pathogens insensitive to certain immune responses, effectively increasing residence time in host tissues and enabling pathologically relevant population densities to be reached. Biosynthetic pathways and capsular involvement in immune system evasion are described providing a basis for potential therapies aimed at supplementing or replacing antibiotic treatment. PMID:24372337

  19. A synthetic strategy to xylose-containing thioglycoside tri- and tetrasaccharide building blocks corresponding to Cryptococcus neoformans capsular polysaccharide structures.

    PubMed

    Guazzelli, Lorenzo; Ulc, Rebecca; Rydner, Lina; Oscarson, Stefan

    2015-06-21

    As part of an ongoing project aimed at developing vaccine candidates against Cryptococcus neoformans the preparation of tri- and tetrasaccharide thioglycoside building blocks, to be used in construction of structurally defined part structures of C. neoformans GXM capsular polysaccharide, was investigated. Using a naphthalenylmethyl (NAP) ether as a temporary protecting group and trichloroacetimidate donors in optimized glycosylations the target building blocks, ethyl 6-O-acetyl-2,4-di-O-benzyl-3-O-(2-naphthalenylmethyl)-α-D-mannopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1→2)]-4,6-di-O-benzyl-1-thio-α-D-mannopyranoside (16) and ethyl 2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1→2)-4,6-di-O-benzyl-3-O-(2-naphthalenylmethyl)-α-D-mannopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-β-D-xylopyra-nosyl-(1→2)]-6-O-acetyl-4-O-benzyl-1-thio-α-D-mannopyranoside (21), were efficiently prepared. These synthesized thiosaccharide building blocks were then used as donors in high-yielding (~90%) DMTST promoted glycosylations to a spacer-containing acceptor to, after deprotection, afford GXM polysaccharide part structures ready for protein conjugation to give vaccine candidates. Also, the NAP groups in the building blocks were removed to obtain tri- and tetrasaccharide acceptors suitable for further elongation towards larger thiosaccharide building blocks.

  20. CAPSULAR POLYSACCHARIDE OF AZOTOBACTER AGILIS1

    PubMed Central

    Cohen, Gary H.; Johnstone, Donald B.

    1964-01-01

    Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Capsular polysaccharide of Azotobacter agilis. J. Bacteriol. 88:1695–1699. 1964.—Capsular polysaccharide from Azotobacter agilis strain 132 was recovered from washed cells by alkaline digestion. The polysaccharide was purified by centrifugation, repeated alcohol precipitation, Sevag deproteinization, and treatment with ribonuclease and charcoal-cellulose. Methods of isolation and purification appeared to provide a polymer showing no evidence of heterogeneity when examined by chemical and physical methods. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed polysaccharide indicated that the polymer contained galactose and rhamnose at a molar ratio of approximately 1.0:0.7. A sialic acid-like component was also present in the polysaccharide. The study shows significant differences in the chemical composition of the extra-cellular polysaccharide of A. agilis and that of A. vinelandii. This adds further biochemical evidence for the right of these species to independent status. Images PMID:14240959

  1. Structure Analysis of the Staphylococcus aureus UDP-N-acetyl-mannosamine Dehydrogenase Cap5O Involved in Capsular Polysaccharide Biosynthesis*

    PubMed Central

    Gruszczyk, Jakub; Fleurie, Aurore; Olivares-Illana, Vanesa; Béchet, Emmanuelle; Zanella-Cleon, Isabelle; Moréra, Solange; Meyer, Philippe; Pompidor, Guillaume; Kahn, Richard; Grangeasse, Christophe; Nessler, Sylvie

    2011-01-01

    Bacterial UDP-sugar dehydrogenases are part of the biosynthesis pathway of extracellular polysaccharides. These compounds act as important virulence factors by protecting the cell from opsonophagocytosis and complement-mediated killing. In Staphylococcus aureus, the protein Cap5O catalyzes the oxidation of UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid. Cap5O is crucial for the production of serotype 5 capsular polysaccharide that prevents the interaction of bacteria with both phagocytic and nonphagocytic eukaryotic cells. However, details of its catalytic mechanism remain unknown. We thus crystallized Cap5O and solved the first structure of an UDP-N-acetyl-mannosamine dehydrogenase. This study revealed that the catalytic cysteine makes a disulfide bond that has never been observed in other structurally characterized members of the NDP-sugar dehydrogenase family. Biochemical and mutagenesis experiments demonstrated that the formation of this disulfide bridge regulates the activity of Cap5O. We also identified two arginine residues essential for Cap5O activity. Previous data suggested that Cap5O is activated by tyrosine phosphorylation, so we characterized the phosphorylation site and examined the underlying regulatory mechanism. PMID:21454499

  2. Immunogenic properties of Klebsiella pneumoniae type 2 capsular polysaccharide.

    PubMed Central

    Robert, A; Jouin, H; Fournier, J M

    1986-01-01

    The immunoprotective activity of Klebsiella pneumoniae K2 cell surface preparations and purified capsular polysaccharide was tested in mice. The 50% protective dose (PD50), expressed as capsular polysaccharide content, was 2 ng for cell surface preparations and 50 ng for purified capsular polysaccharide. Both preparations lost their immunoprotective activity after alkali treatment. Immune sera were raised in rabbits immunized with cell surface preparations. The precipitating and hemagglutinating capacity of these antisera was tested against either purified capsular polysaccharide or alkali-treated capsular polysaccharide. No difference was observed between the reactivity of the antisera against each antigen. The protective activity of these sera was tested on mice in passive transfer experiments, before and after absorption with either purified capsular polysaccharide or alkali-treated capsular polysaccharide. The sera lost their protective activity after absorption with purified capsular polysaccharide and after absorption with alkali-treated capsular polysaccharide. These experiments show that the difference in immunoprotective activity of cell surface preparations, purified capsular polysaccharide, and alkali-treated capsular polysaccharide is not due to a difference in their antigenic determinants. Cell surface preparations and purified capsular polysaccharide were fractionated by gel filtration on Sepharose 4B and by ultracentrifugation on cesium chloride density gradients. Three forms of capsular polysaccharide have been characterized. (i) A form of capsular polysaccharide with a very high protective activity (PD50 = 2 ng) that copurified with protein and lipopolysaccharide and was characterized by a low coefficient of distribution (Kd = 0.20) and a low density (1.5 to 1.6 g/cm3). (ii) A form of capsular polysaccharide with an intermediate protective activity (PD50 = 50 ng), contamined by less than 3% protein and 1% lipopolysaccharide, with a Kd of 0.35, and

  3. Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces.

    PubMed

    Washburn, R G; Bryant-Varela, B J; Julian, N C; Bennett, J E

    1991-01-01

    Binding of complement component C3 and Factor B to Cryptococcus neoformans serotypes A through D via the alternative complement pathway was measured in a system containing fresh nonimmune human serum. Serotypes B and C (C. neoformans var. gattii) bound approximately half as many molecules of both complement components as serotypes A and D (C. neoformans var. neoformans). In contrast, removal of xylosyl and glucuronyl side chains from the mannan main chain of capsular polysaccharide by the Smith degradation procedure resulted in binding of similar quantities of C3 to each of the four serotypes. We conclude that the relatively high degree of side chain substitution of capsular polysaccharide from C. neoformans variety gattii contributes to inefficient surface assembly of the alternative pathway C3 convertase. Inefficient binding of alternative pathway complement components to serotypes B and C may contribute to the relative difficulty in successfully treating infections caused by these organisms.

  4. Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp.

    PubMed

    Pan, Yi-Jiun; Lin, Tzu-Lung; Chen, Chun-Tang; Chen, Yi-Yin; Hsieh, Pei-Fang; Hsu, Chun-Ru; Wu, Meng-Chuan; Wang, Jin-Town

    2015-10-23

    A total of 79 capsular types have been reported in Klebsiella spp., whereas capsular polysaccharide synthesis (cps) regions were available in only 22 types. Due to the limitations of serotyping, complete repertoire of cps will be helpful for capsular genotyping. We therefore resolved the rest 57 cps and conducted comparative analysis. Clustering results of 1,515 predicted proteins from cps loci categorized proteins which share similarity into homology groups (HGs) revealing that 77 Wzy polymerases were classified into 56 HGs, which indicate the high specificity of wzy between different types. Accordingly, wzy-based capsular genotyping could differentiate capsule types except for those lacking wzy (K29 and K50), those sharing identical wzy (K22 vs. K37); and should be carefully applied in those exhibited high similarity (K12 vs. K41, K2 vs. K13, K74 vs. K80, K79 vs. KN1 and K30 vs. K69). Comparison of CPS structures in several capsular types that shared similarity in their gene contents implies possible functions of glycosyltransferases. Therefore, our results provide complete set of cps in various types of Klebsiella spp., which enable the understandings of relationship between genes and CPS structures and are useful for identification of documented or new capsular types.

  5. Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp

    PubMed Central

    Pan, Yi-Jiun; Lin, Tzu-Lung; Chen, Chun-Tang; Chen, Yi-Yin; Hsieh, Pei-Fang; Hsu, Chun-Ru; Wu, Meng-Chuan; Wang, Jin-Town

    2015-01-01

    A total of 79 capsular types have been reported in Klebsiella spp., whereas capsular polysaccharide synthesis (cps) regions were available in only 22 types. Due to the limitations of serotyping, complete repertoire of cps will be helpful for capsular genotyping. We therefore resolved the rest 57 cps and conducted comparative analysis. Clustering results of 1,515 predicted proteins from cps loci categorized proteins which share similarity into homology groups (HGs) revealing that 77 Wzy polymerases were classified into 56 HGs, which indicate the high specificity of wzy between different types. Accordingly, wzy-based capsular genotyping could differentiate capsule types except for those lacking wzy (K29 and K50), those sharing identical wzy (K22 vs. K37); and should be carefully applied in those exhibited high similarity (K12 vs. K41, K2 vs. K13, K74 vs. K80, K79 vs. KN1 and K30 vs. K69). Comparison of CPS structures in several capsular types that shared similarity in their gene contents implies possible functions of glycosyltransferases. Therefore, our results provide complete set of cps in various types of Klebsiella spp., which enable the understandings of relationship between genes and CPS structures and are useful for identification of documented or new capsular types. PMID:26493302

  6. Structure of the capsular polysaccharide of Vibrio cholerae O139 synonym Bengal containing D-galactose 4,6-cyclophosphate.

    PubMed

    Knirel, Y A; Paredes, L; Jansson, P E; Weintraub, A; Widmalm, G; Albert, M J

    1995-09-01

    The capsular polysaccharide (CPS) of Vibrio cholerae O139 synonym Bengal, which is thought to carry determinants of O-specificity, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and gel-permeation chromatography. The CPS contained D-galactose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), D-galacturonic acid (D-GalA), and phosphate. The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations, including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48% hydrofluoric acid, which both resulted in complete cleavage of Col. It was concluded that the CPS is built up of hexasaccharide repeating units containing inter alia D-galactose 4,6-cyclophosphate and having the following structure [structure: see text] These data basically confirm the structure of the V. cholerae CPS proposed on the basis of an NMR study [L. M. Preston et al. (1995) J. Bacteriol. 177, 835-838] and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate.

  7. Capsular polysaccharide of Clostridium perfringens Hobbs 10.

    PubMed

    Lee, L; Cherniak, R

    1974-02-01

    A capsular polysaccharide was isolated from a strain of Clostridium perfringens Hobbs 10 type A by cold-water extraction of whole, heavily encapsulated cells. The water-soluble polymer was isolated by alcohol precipitation and purified by treatment with chloroform-butanol, cetytrimethylammonium bromide, and column gel permeation chromatography by using Bio-Gel A-5m agarose. The formation of a single precipitin line, when the isolated polysaccharide was reacted with its homologous antisera by double diffusion in gel, was considered a criterion of immunochemical purity. The purified polymer appeared as a single peak when eluted from diethylaminoethyl-Sephadex with a linear gradient of NaCl. The polysaccharide was composed of glucose, galactose, galactosamine, and iduronic acid in a molar ratio of 4.1:5.1.7:1, respectively. These constituents accounted for 83% of the dry weight. The polysaccharide appeared to have a molecular weight of 40,000 and exhibited aggregation up to 120,000. A trace of peptide material could not be removed during purification.

  8. Capsular Polysaccharide of Clostridium perfringens Hobbs 10

    PubMed Central

    Lee, Linda; Cherniak, Robert

    1974-01-01

    A capsular polysaccharide was isolated from a strain of Clostridium perfringens Hobbs 10 type A by cold-water extraction of whole, heavily encapsulated cells. The water-soluble polymer was isolated by alcohol precipitation and purified by treatment with chloroform-butanol, cetytrimethylammonium bromide, and column gel permeation chromatography by using Bio-Gel A-5m agarose. The formation of a single precipitin line, when the isolated polysaccharide was reacted with its homologous antisera by double diffusion in gel, was considered a criterion of immunochemical purity. The purified polymer appeared as a single peak when eluted from diethylaminoethyl-Sephadex with a linear gradient of NaCl. The polysaccharide was composed of glucose, galactose, galactosamine, and iduronic acid in a molar ratio of 4.1:5.1.7:1, respectively. These constituents accounted for 83% of the dry weight. The polysaccharide appeared to have a molecular weight of 40,000 and exhibited aggregation up to 120,000. A trace of peptide material could not be removed during purification. PMID:4361293

  9. Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H.

    PubMed

    Adlam, C; Knights, J M; Mugridge, A; Lindon, J C; Williams, J M; Beesley, J E

    1985-08-01

    Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.

  10. Genetics of Capsular Polysaccharides and Cell Envelope (Glyco)lipids

    PubMed Central

    Daffé, Mamadou; Crick, Dean C.; Jackson, Mary

    2014-01-01

    This chapter summarizes what is currently known of the structures, physiological roles, involvement in pathogenicity and biogenesis of a variety of non-covalently bound cell envelope lipids and glycoconjugates of Mycobacterium tuberculosis and other Mycobacterium species. Topics addressed in this chapter include phospholipids; phosphatidylinositol mannosides; triglycerides; isoprenoids and related compounds (polyprenyl phosphate, menaquinones, carotenoids, non-carotenoid cyclic isoprenoids); acyltrehaloses (lipooligosaccharides, trehalose mono- and di-mycolates, sulfolipids, di- and poly-acyltrehaloses); mannosyl-beta-1-phosphomycoketides; glycopeptidolipids; phthiocerol dimycocerosates, para-hydroxybenzoic acids and phenolic glycolipids; mycobactins; mycolactones; and capsular polysaccharides. PMID:25485178

  11. Antibiofilm Activity of Actinobacillus pleuropneumoniae Serotype 5 Capsular Polysaccharide

    PubMed Central

    Karwacki, Michael T.; Kadouri, Daniel E.; Bendaoud, Meriem; Izano, Era A.; Sampathkumar, Vandana; Inzana, Thomas J.; Kaplan, Jeffrey B.

    2013-01-01

    Cell-free extracts isolated from colony biofilms of Actinobacillus pleuropneumoniae serotype 5 were found to inhibit biofilm formation by Staphylococcus aureus, S. epidermidis and Aggregatibacter actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself, in a 96-well microtiter plate assay. Physical and chemical analyses indicated that the antibiofilm activity in the extract was due to high-molecular-weight polysaccharide. Extracts isolated from a mutant strain deficient in the production of serotype 5 capsular polysaccharide did not exhibit antibiofilm activity. A plasmid harboring the serotype 5 capsule genes restored the antibiofilm activity in the mutant extract. Purified serotype 5 capsular polysaccharide also exhibited antibiofilm activity against S. aureus. A. pleuropneumoniae wild-type extracts did not inhibit S. aureus growth, but did inhibit S. aureus intercellular adhesion and binding of S. aureus cells to stainless steel surfaces. Furthermore, polystyrene surfaces coated with A. pleuropneumoniae wild-type extracts, but not with capsule-mutant extracts, resisted S. aureus biofilm formation. Our findings suggest that the A. pleuropneumoniae serotype 5 capsule inhibits cell-to-cell and cell-to-surface interactions of other bacteria. A. pleuropneumoniae serotype 5 capsular polysaccharide is one of a growing number of bacterial polysaccharides that exhibit broad-spectrum, nonbiocidal antibiofilm activity. Future studies on these antibiofilm polysaccharides may uncover novel functions for bacterial polysaccharides in nature, and may lead to the development of new classes of antibiofilm agents for industrial and clinical applications. PMID:23691104

  12. Pneumococcus with the "6E" cps Locus Produces Serotype 6B Capsular Polysaccharide.

    PubMed

    Burton, Robert L; Geno, K Aaron; Saad, Jamil S; Nahm, Moon H

    2016-04-01

    Genetic studies of serogroup 6 isolates ofStreptococcus pneumoniaeidentified putative serotype 6E. Although its capsular polysaccharide structure has not been elucidated, putative serotype 6E is described in an increasing number of studies as a potentially new serotype. We show here that SPEC6B, which is widely used as a target strain for serotype 6B opsonophagocytosis assays, has the genetic features of the putative serotype 6E but produces capsular polysaccharide identical to 6B capsular polysaccharide as determined by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR). Thus, putative serotype 6E is a mere genetic variant of serotype 6B. Also, SPEC6B is appropriate as a target strain for serotype 6B opsonophagocytosis assays. This example illustrates the difficulties of assigning new bacterial serotypes based on genetic findings alone.

  13. Serological, chemical, and structural analyses of the Escherichia coli cross-reactive capsular polysaccharides K13, K20, and K23.

    PubMed Central

    Vann, W F; Soderstrom, T; Egan, W; Tsui, F P; Schneerson, R; Orskov, I; Orskov, F

    1983-01-01

    The Escherichia coli K13, K20, and K23 capsular polysaccharide antigens are serologically related. All of these polysaccharides contain ribose and 2-keto-3-deoxyoctonate in equimolar quantities. The K13 and K20 polysaccharides are partially O-acetylated. A comparison of these polysaccharides after O-deacetylation, by nuclear magnetic resonance and permethylation analysis, showed that these polysaccharides contained the disaccharide repeat unit leads to)-beta-ribofuranosyl-(1 leads to 7)-beta-2-keto-3-deoxyoctonate. They differed in the presence and location of an acetyl moiety. The K13 polysaccharide was O-acetylated at C-4 of the 2-keto-3-deoxyoctonate. The K20 antigen was O-acetylated at C-5 of the ribose moiety. The K23 polymer was nonacetylated. The cross-reactivity of these antigens was demonstrated by tandem-crossed immunoelectrophoresis. Antibodies to K23 could be completely absorbed from OK K23 serum by K13, K20, and K23 antigenic extracts. The K13 and K20 antibodies could be completely absorbed from their respective antisera only by homologous antigenic extracts. Monoclonal antibodies were prepared against a protein conjugate of the K13 polysaccharide. Analyses of the reactions of these antibodies with the three polysaccharides suggest that the K13 polysaccharide has at least three antigenic sites, one of which is common to the K13, K20, and K23 polysaccharides. PMID:6187684

  14. Immunochemical characterization of the capsular polysaccharide of Azospirillum irakense KBC1.

    PubMed

    Fedonenko, Yulia P; Burygin, Gennady L; Popova, Irina A; Sigida, Elena N; Surkina, Alina K; Zdorovenko, Evelina L; Konnova, Svetlana A

    2013-08-01

    The repeating unit structure of Azospirillum irakense KBC1 capsular polysaccharide (CPS) was established and was found to be identical to that of the O polysaccharide of A. irakense KBC1 lipopolysaccharide (LPS). The antigenic heterogeneity of the LPS and the CPS was shown to be related to differences in the macromolecular organization of these glycopolymers. After an immune response activation, R-form CPS molecules were found to be predominant.

  15. Combinatorial Library Cloning of Human Antibodies to Streptococcus pneumoniae Capsular Polysaccharides: Variable Region Primary Structures and Evidence for Somatic Mutation of Fab Fragments Specific for Capsular Serotypes 6B, 14, and 23F

    PubMed Central

    Lucas, Alexander H.; Moulton, Karen D.; Tang, Vanessa R.; Reason, Donald C.

    2001-01-01

    Antibodies specific for capsular polysaccharides play a central role in immunity to encapsulated Streptococcus pneumoniae, but little is known about their genetics or the variable (V) region polymorphisms that affect their protective function. To begin to address these issues, we used combinatorial library cloning to isolate pneumococcal polysaccharide (PPS)-specific Fab fragments from two vaccinated adults. We determined complete V region primary structures and performed antigen binding analyses of seven Fab fragments specific for PPS serotype 6B, 14, or 23F. Fabs were of the immunoglobulin G2 or A isotype. Several VHIII gene segments (HV 3-7, 3-15, 3-23, and 3-11) were identified. VL regions were encoded by several κ genes (KV 4-1, 3-15, 2-24, and 2D-29) and a λ gene (LV 1-51). Deviation of the VH and VL regions from their assigned germ line counterparts indicated that they were somatically mutated. Fabs of the same serotype specificity isolated from a single individual differed in affinity, and these differences could be accounted for either by the extent of mutation among clonal relatives or by usage of different V-region genes. Thus, functionally disparate anti-PPS antibodies can arise within individuals both by activation of independent clones and by intraclonal somatic mutation. For one pair of clonally related Fabs, the more extensively mutated VH was associated with lower affinity for PPS 14, a result suggesting that somatic mutation could lead to diminished protective efficacy. These findings indicate that the PPS repertoire in the adult derives from memory B-cell populations that have class switched and undergone extensive hypermutation. PMID:11159978

  16. Structure determination of Streptococcus suis serotype 9 capsular polysaccharide and assignment of functions of the cps locus genes involved in its biosynthesis.

    PubMed

    Vinogradov, Evgueny; Goyette-Desjardins, Guillaume; Okura, Masatoshi; Takamatsu, Daisuke; Gottschalk, Marcelo; Segura, Mariela

    2016-10-04

    Streptococcus suis serotype 9 is the most prevalent S. suis serotype in several European countries. In spite of its pathogenicity for pigs and increasing zoonotic potential, limited information is available on this serotype. Here we determined for the first time the chemical composition and structure of serotype 9 capsular polysaccharide (CPS), a major bacterial virulence factor and the antigen at the origin of S. suis classification into serotypes. Chemical and spectroscopic data gave the repeating unit sequence: [3)Glcol-6-P-3-[D-Gal(α1-2)]D-Gal(β1-3)D-Sug(β1-3)L-Rha(α1-)]n. Compared to previously characterized S. suis CPSs (serotypes 1, 1/2, 2 and 14), serotype 9 CPS does not contain sialic acid but contains a labile 4-keto sugar (2-acetamido-2,6-dideoxy-β-D-xylo-hexopyranos-4-ulose), one particular feature of this serotype. A correlation between S. suis serotype 9 CPS sequence and genes of this serotype cps locus encoding putative glycosyltransferases and polymerase responsible for the biosynthesis of the repeating unit was tentatively established. Knowledge of CPS structure and composition will contribute to better dissect the role of this bacterial component in the pathogenesis of S. suis serotype 9.

  17. Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae.

    PubMed

    Skov Sørensen, Uffe B; Yao, Kaihu; Yang, Yonghong; Tettelin, Hervé; Kilian, Mogens

    2016-11-15

    Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule production distinguishes S. pneumoniae from closely related commensals of the mitis group streptococci. Based on antigenic and genetic analyses of 187 mitis group streptococci, including 90 recognized serotypes of S. pneumoniae, we demonstrated capsule production by the Wzy/Wzx pathway in 74% of 66 S. mitis strains and in virtually all tested strains of S. oralis (subspecies oralis, dentisani, and tigurinus) and S. infantis Additional analyses of genomes of S. cristatus, S. parasanguinis, S. australis, S. sanguinis, S. gordonii, S. anginosus, S. intermedius, and S. constellatus revealed complete capsular biosynthesis (cps) loci in all strains tested. Truncated cps loci were detected in three strains of S. pseudopneumoniae, in 26% of S. mitis strains, and in a single S. oralis strain. The level of sequence identities of cps locus genes confirmed that the structural polymorphism of capsular polysaccharides in S. pneumoniae evolved by import of cps fragments from commensal Streptococcus species, resulting in a mosaic of genes of different origins. The demonstrated antigenic identity of at least eight of the numerous capsular polysaccharide structures expressed by commensal streptococci with recognized serotypes of S. pneumoniae raises concerns about potential misidentifications in addition to important questions concerning the consequences for vaccination and host-parasite relationships both for the commensals and for the pathogen. Expression of a capsular polysaccharide is among the principal virulence factors of Streptococcus pneumoniae and is the basis for successful vaccines against infections caused by this important pathogen. Contrasting with previous

  18. Roles of Lipooligosaccharide and Capsular Polysaccharide in Antimicrobial Resistance and Natural Transformation of Campylobacter jejuni

    USDA-ARS?s Scientific Manuscript database

    Objectives: To investigate the roles of surface polysaccharides, such as capsular polysaccharide (CPS) and lipooligosaccharide (LOS), in modulating natural transformation and antimicrobial resistance in Campylobacter jejuni. Methods: A series of C. jejuni mutants, which are defective in either CPS ...

  19. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  20. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

  1. Purification of capsular polysaccharide from Neisseria meningitidis serogroup C by liquid chromatography.

    PubMed

    Pato, Tânia Pinheiro; Barbosa, Antonio de Pádua R; da Silva Junior, José Godinho

    2006-03-07

    Neisseria meningitidis serogroup C capsular polysaccharide (MenCPS) is an important antigen against meningococcal infection. This paper describes a new purification methodology employing liquid chromatography that resulted in a polysaccharide showing the characteristics recommended by the World Health Organization for vaccine purposes. In this method, steps of the traditional procedure that yield low recovery and use toxic materials were modified. The present process consists in the following steps: (1) continuous flow centrifugation of the culture for removal of the cells; (2) supernatant concentration by tangential filtration (100 kDa cutoff); (3) addition of 0.5% DOC, heating to 55 degrees C during 30 min and tangential filtration (100 kDa cutoff); (4) anion exchange chromatography (Source 15Q) and (5) size exclusion chromatography (Sepharose CL-4B). The polysaccharide C fraction obtained in that way was dialyzed and freeze-dried. The structural identity of the polysaccharide was demonstrated by (1)H-NMR spectrometry.

  2. Vaccination with peptide mimotopes produces antibodies recognizing bacterial capsular polysaccharides.

    PubMed

    Wu, Yang; Zhang, Qibo; Sales, Debra; Bianco, Albert Edward; Craig, Alister

    2010-09-07

    A phage display peptide library was screened using a panel of antibodies to the capsular polysaccharides of Streptococcus agalactiae and Neisseria meningitidis. Mimotopes NPDHPRVPTFMA (2-8), LIPFHKHPHHRG (3-2) and EQEIFTNITDRV (G3) showing the highest binding capacity and strongest ELISA reaction were selected for immunization experiments. These mimotopes were either synthesised as oligodeoxynucleotides for DNA immunization or MAP (multiple antigen peptide) for peptide immunization. Mimotope-DNA vaccination, particularly for G3, induced antibodies recognizing a number of target bacteria. This response was seen after the second boost injection and was significantly enhanced by the 3rd boost injection with a Th1-associated profile, which was dominated by IgG2a, followed by IgG1. Mimotope-MAP immunization also produced strong humoral immune responses to the bacteria. Antibodies from G3 DNA immunization reacted with the surface molecules of S. agalactiae, N. meningitidis and Escherichia coli K5 shown by indirect immunofluorescence staining, indicating a possible localization to the bacterial capsule. Antibodies produced both from DNA/MAP immunization reacted with purified bacterial capsular polysaccharides by ELISA and were of high avidity. We have further characterized peptide G3 by a 'tiling path' study to examine the effect of changing individual residues in the peptide in raising antibodies, which showed that the EIFTN motif in G3 was important in generating antibodies to several capsulated bacteria. We conclude that mimotope immunization with DNA or MAP potentially induces strong antibody responses against encapsulated bacteria. It is suggested that the antibody targets are polysaccharides, and these antibodies may cross react at least among closely related species of bacteria.

  3. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli

    PubMed Central

    Kay, Emily J.; Yates, Laura E.; Terra, Vanessa S.; Cuccui, Jon; Wren, Brendan W.

    2016-01-01

    Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and over 1 million deaths, the majority of them children. The major determinant for pathogenesis is a polysaccharide capsule that is variable and is used to distinguish strains based on their serotype. The capsule forms the basis of the pneumococcal polysaccharide vaccine (PPV23) that contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Purified capsule from S. pneumoniae is required for pneumococcal conjugate vaccine production, and costs can be prohibitively high, limiting accessibility of the vaccine in low-income countries. In this study, we demonstrate the recombinant expression of the capsule-encoding locus from four different serotypes of S. pneumoniae within Escherichia coli. Furthermore, we attempt to identify the minimum set of genes necessary to reliably and efficiently express these capsules heterologously. These E. coli strains could be used to produce a supply of S. pneumoniae serotype-specific capsules without the need to culture pathogenic bacteria. Additionally, these strains could be applied to synthetic glycobiological applications: recombinant vaccine production using E. coli outer membrane vesicles or coupling to proteins using protein glycan coupling technology. PMID:27110302

  4. Gas-chromatographic screening of capsular polysaccharides of Neisseria meningitidis.

    PubMed

    Bryn, K; Frøholm, L O; Holten, E; Bøvre, K

    1983-06-01

    Thirteen systemic strains, i e strains isolated from systemic infections, and 77 carrier isolates of Neisseria meningitidis were serogrouped by agglutination and analyzed by gas chromatography (GC) of phenol extracts. For systemic strains the sugar patterns were in accordance with their group-specific capsular polysaccharides (CPS). Some carrier isolates revealed unexpected GC profiles. Upon immunological retesting with new sera, GC results were generally confirmed. Occasional isolates initially serogrouped as B or Y completely lacked neuraminic acid. Some non-groupable isolates were shown by ultracentrifugation and GC to have significant amounts of this sugar likely to originate from CPS of known composition or from unknown polysaccharides. One such originally non-groupable isolate showed a weak agglutination reaction specifically with group B antiserum when reexamined. Generally, carrier isolates had lower amounts of CPS than systemic strains of the same group. Five successive isolates from one carrier were first serogrouped as X, Z or non-groupable, but they had high amounts of galactosamine and 2-keto-3-deoxy octonate, sugars characterizing CPS of serogroup 29E. These isolates were confirmed by agglutination with recently available group 29E antiserum to be of this serogroup, which has not been reported before in Norway. Ultracentrifugation revealed the presence of unknown polysaccharides containing glucose, galactose or glucosamine, but further purification of these polymers is required to determine their composition and immunological importance.

  5. Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae

    PubMed Central

    Skov Sørensen, Uffe B.; Yao, Kaihu; Yang, Yonghong; Tettelin, Hervé

    2016-01-01

    ABSTRACT Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule production distinguishes S. pneumoniae from closely related commensals of the mitis group streptococci. Based on antigenic and genetic analyses of 187 mitis group streptococci, including 90 recognized serotypes of S. pneumoniae, we demonstrated capsule production by the Wzy/Wzx pathway in 74% of 66 S. mitis strains and in virtually all tested strains of S. oralis (subspecies oralis, dentisani, and tigurinus) and S. infantis. Additional analyses of genomes of S. cristatus, S. parasanguinis, S. australis, S. sanguinis, S. gordonii, S. anginosus, S. intermedius, and S. constellatus revealed complete capsular biosynthesis (cps) loci in all strains tested. Truncated cps loci were detected in three strains of S. pseudopneumoniae, in 26% of S. mitis strains, and in a single S. oralis strain. The level of sequence identities of cps locus genes confirmed that the structural polymorphism of capsular polysaccharides in S. pneumoniae evolved by import of cps fragments from commensal Streptococcus species, resulting in a mosaic of genes of different origins. The demonstrated antigenic identity of at least eight of the numerous capsular polysaccharide structures expressed by commensal streptococci with recognized serotypes of S. pneumoniae raises concerns about potential misidentifications in addition to important questions concerning the consequences for vaccination and host-parasite relationships both for the commensals and for the pathogen. PMID:27935839

  6. Determination of the chemical structure of the capsular polysaccharide of strain B33, a fast-growing soya bean-nodulating bacterium isolated from an arid region of China.

    PubMed Central

    Rodríguez-Carvajal, M A; Tejero-Mateo, P; Espartero, J L; Ruiz-Sainz, J E; Buendía-Clavería, A M; Ollero, F J; Yang, S S; Gil-Serrano, A M

    2001-01-01

    We have determined the structure of a polysaccharide from strain B33, a fast-growing bacterium that forms nitrogen-fixing nodules with Asiatic and American soya bean cultivars. On the basis of monosaccharide analysis, methylation analysis, one-dimensional 1H- and 13C-NMR and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the repeating unit -->6)-4-O-methyl-alpha-D-Glcp-(1-->4)-3-O-methyl-beta-D-GlcpA-(1--> (where GlcpA is glucopyranuronic acid and Glcp is glucopyranose). Strain B33 produces a K-antigen polysaccharide repeating unit that does not have the structural motif sugar-Kdx [where Kdx is 3-deoxy-D-manno-2-octulosonic acid (Kdo) or a Kdo-related acid] proposed for different Sinorhizobium fredii strains, all of them being effective with Asiatic soya bean cultivars but unable to form nitrogen-fixing nodules with American soya bean cultivars. Instead, it resembles the K-antigen of S. fredii strain HH303 (rhamnose, galacturonic acid)n, which is also effective with both groups of soya bean cultivars. Only the capsular polysaccharide from strains B33 and HH303 have monosaccharide components that are also present in the surface polysaccharide of Bradyrhizobium elkanii strains, which consists of a 4-O-methyl-D-glucurono-L-rhamnan. PMID:11439101

  7. Effects of light wavelengths on extracellular and capsular polysaccharide production by Nostoc flagelliforme.

    PubMed

    Han, Pei-pei; Sun, Ying; Jia, Shi-ru; Zhong, Cheng; Tan, Zhi-lei

    2014-05-25

    The influences of different wavelengths of light (red 660nm, yellow 590nm, green 520nm, blue 460nm, purple 400nm) and white light on extracellular polysaccharide (EPS) and capsular polysaccharide (CPS) production by Nostoc flagelliforme in liquid culture were demonstrated in this study. The results showed that, compared with white light, red and blue lights significantly increased both EPS and CPS production while yellow light reduced their production; purple and green lights stimulated EPS production but inhibited CPS formation. Nine constituent monosaccharides and one uronic acid were detected in both EPS and CPS, and their ratios showed significant differences among treatment with different light wavelengths. However, the advanced structure of EPS and CPS from various light conditions did not present obvious difference through Fourier transform infrared spectroscopy and X-ray diffraction characterization. These findings establish a basis for development of high-yielding polysaccharide production process and understanding their regulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. O-METHYL PHOSPHORAMIDATE MODIFICATIONS ON THE CAPSULAR POLYSACCHARIDE OF CAMPYLOBACTER JEJUNI ARE INVOLVED IN SERUM RESISTANCE, INFECTION, AND INSECTICIDAL ACTIVITY

    USDA-ARS?s Scientific Manuscript database

    Campylobacter jejuni is the most commonly reported cause of bacterial foodborne illness in North America. C. jejuni decorates its surface polysaccharides with a variety of variable phosphorylated structures, including O-methyl phosphoramidate (MeOPN) modifications on the capsular polysaccharide. Alt...

  9. Overproduction of Type 8 Capsular Polysaccharide Augments Staphylococcus aureus Virulence

    PubMed Central

    Luong, Thanh T.; Lee, Chia Y.

    2002-01-01

    Type 8 capsular polysaccharide (CP8) is the most prevalent capsule type in clinical isolates of Staphylococcus aureus. However, its role in virulence has not been clearly defined. CP8 strains such as strain Becker produce a small amount of capsule on their surface in vitro. In contrast, CP1 strains such as strain M produce a large amount of capsule, which has been shown to be an important antiphagocytic virulence factor. The cap8 and cap1 operons, required for the synthesis of CP8 and CP1, respectively, have been cloned and sequenced. To test whether CP8 contributes to the pathogenesis of S. aureus, we replaced the weak native promoter of the cap8 operon in strain Becker with the strong constitutive promoter of the cap1 operon of strain M. The resultant strain, CYL770, synthesized cap8-specific mRNA at a level about sevenfold higher than that in the parent strain. Remarkably, the CYL770 strain produced about 80-fold more CP8. In a mouse infection model of bacteremia, the CP8-overproducing strain persisted longer in the bloodstream, the liver, and the spleen in mice than the parent strain. In addition, strain CYL770 was more resistant to ospsonophagocytosis in vitro by human polymorphonuclear leukocytes. These results indicate that CP8 is an antiphagocytic virulence factor of S. aureus. PMID:12065477

  10. Trapped translocation intermediates establish the route for export of capsular polysaccharides across Escherichia coli outer membranes.

    PubMed

    Nickerson, Nicholas N; Mainprize, Iain L; Hampton, Lauren; Jones, Michelle L; Naismith, James H; Whitfield, Chris

    2014-06-03

    The outer membrane (OM) of Gram-negative bacteria is designed to exclude potentially harmful molecules. This property presents a challenge for bacteria that must secrete proteins and large glycoconjugates to grow, divide, and persist. Proteins involved in trafficking such molecules have been identified, but their precise roles are often unresolved due to the difficulty in capturing "snapshots" during the export pathway. Wza is the prototype for the large family of OM polysaccharide export proteins. In Escherichia coli, Wza is essential for the assembly of a capsule, a protective surface coat composed of long-chain polysaccharides. Wza creates an octameric α-helical channel spanning the OM, but the bulk of the protein exists as a large periplasmic structure enclosing an extensive lumen. Residues within the lumen of Wza were targeted for site-specific incorporation of the UV photo-cross-linkable unnatural amino acid p-benzoyl-L-phenylalanine. Using this in vivo photo-cross-linking strategy, we were able to trap polysaccharide translocation intermediates within the lumen of Wza, providing the first unequivocal evidence to our knowledge that nascent capsular polysaccharide chains exit the cell through the Wza portal.

  11. Maternal group B streptococcal immunization: capsular polysaccharide (CPS)-based vaccines and their implications on prevention.

    PubMed

    Palmeiro, Jussara K; De Carvalho, Newton S; Botelho, Ana C N; Fracalanzza, Sérgio E L; Madeira, Humberto M F; Dalla-Costa, Libera M

    2011-05-12

    Group B streptococcal (GBS) capsular polysaccharide (CPS)-based conjugate vaccine, which includes types Ia, Ib, II, III, and V, could potentially prevent neonatal, pediatric, adult, and pregnancy-associated diseases. However, since GBS CPS types included in that vaccine are prevalent serotypes found in North America and Europe, it may not provide the necessary protection for individuals in countries in which other capsular types have been found.

  12. Evidence for Branching in Cryptococcal Capsular Polysaccharides and Consequences on its Biological Activity

    PubMed Central

    Cordero, Radames J.B.; Frases, Susana; Guimaräes, Allan J.; Rivera, Johanna; Casadevall, Arturo

    2011-01-01

    SUMMARY The encapsulated fungus Cryptococcus neoformans is a common cause of life-threatening disease in immunocompromised individuals. Its major virulence determinant is the polysaccharide (PS) capsule. An unsolved problem in cryptococcal biology is whether the PSs composing the capsule are linear or complex branched polymers, as well as the implications of this structural composition in pathogenesis. In this study we approached the problem by combining static and dynamic light scattering, viscosity analysis, and high-resolution microscopy and correlated the findings with biological properties. Analysis of the dependence of capsular PS molecular mass and the radius of gyration provided strong evidence against a simple linear PS configuration. Shape factors calculated from light scattering measurements in solution revealed values consistent with polymer branching. Furthermore, viscosity measurements provided complementary evidence for structural branching. Electron microscopy showed PS spherical-like structures similar to other branched PS. Finally, we show that the capacity of capsular PS to interfere in complement-mediated phagocytosis, inhibit nitric oxide production by macrophage-like cells, protect against reactive oxygen species, antibody reactivity and half-life in serum were influenced by the degree of branching, providing evidence for the notion that PS branching is an important parameter in determining the biological activity of C. neoformans PS. PMID:21208301

  13. Evidence for branching in cryptococcal capsular polysaccharides and consequences on its biological activity.

    PubMed

    Cordero, Radames J B; Frases, Susana; Guimaräes, Allan J; Rivera, Johanna; Casadevall, Arturo

    2011-02-01

    The encapsulated fungus Cryptococcus neoformans is a common cause of life-threatening disease in immunocompromised individuals. Its major virulence determinant is the polysaccharide (PS) capsule. An unsolved problem in cryptococcal biology is whether the PSs composing the capsule are linear or complex branched polymers, as well as the implications of this structural composition in pathogenesis. In this study we approached the problem by combining static and dynamic light scattering, viscosity analysis, and high-resolution microscopy and correlated the findings with biological properties. Analysis of the dependence of capsular PS molecular mass and the radius of gyration provided strong evidence against a simple linear PS configuration. Shape factors calculated from light scattering measurements in solution revealed values consistent with polymer branching. Furthermore, viscosity measurements provided complementary evidence for structural branching. Electron microscopy showed PS spherical-like structures similar to other branched PS. Finally, we show that the capacity of capsular PS to interfere in complement-mediated phagocytosis, inhibit nitric oxide production by macrophage-like cells, protect against reactive oxygen species, antibody reactivity and half-life in serum were influenced by the degree of branching, providing evidence for the notion that PS branching is an important parameter in determining the biological activity of C. neoformans PS.

  14. Acinetobacter baumannii K27 and K44 capsular polysaccharides have the same K unit but different structures due to the presence of distinct wzy genes in otherwise closely related K gene clusters.

    PubMed

    Shashkov, Alexander S; Kenyon, Johanna J; Senchenkova, Sof'ya N; Shneider, Mikhail M; Popova, Anastasiya V; Arbatsky, Nikolay P; Miroshnikov, Konstantin A; Volozhantsev, Nikolay V; Hall, Ruth M; Knirel, Yuriy A

    2016-05-01

    Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D(1)H and(13)C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as anitrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferases were predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 and WzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii.

  15. Latex agglutination: diagnose the early cryptococcus neoformans test of capsular polysaccharide antigen.

    PubMed

    Wang, Huanrong; Yuan, Xueqian; Zhang, Lifeng

    2015-01-01

    This paper aims to discuss the early diagnosis value of latex agglutination test in Cryptococcal meningitis. The cerebrospinal fluid (CSF) of 112 patients with definite Cryptococcal meningitis and 26 patients with tubercular meningitis and virus meningitis were collected, latex agglutination test is adopted to detect Cryptococcal capsular polysaccharide antigen. Then it was compared with fungal culture and direct microscopy method for evaluating the sensitivity and specificity of the diagnosis. The sensitivity of three methods including latex agglutination test, fungal culture and direct microscopy was 91.1%,69.6% and 73.2% respectively. The specificity of latex agglutination test was 96.0%, 100% and 100% respectively. That latex agglutination test to detect Cryptococcal capsular polysaccharide antigen could be taken as the early diagnostic method of Cryptococcus neoformans meningitis.

  16. Surface polysaccharide of Moraxella non-liquefaciens identical to Neisseria meningitidis group B capsular polysaccharide. A chemical and immunological investigation.

    PubMed

    Bøvre, K; Bryn, K; Closs, O; Hagen, N; Frøholm, L O

    1983-06-01

    In whole cell preparations of 27 nonmucoid strains of Moraxella nonliquefaciens neuraminic acid was detected by gas chromatography (GC) in 16 (59%) of the strains. Seven neuraminic-acid-containing strains were tested for agglutination with diagnostic group-specific meningococcal antisera produced in rabbits, and all were positive with group B serum. Counter-immunoelectrophoresis of bacterial suspensions of the three strains with the strongest reaction with such anti-group B serum gave distinct precipitation lines. When tested by double immunodiffusion in agarose with monoclonal antibody to meningococcal group B polysaccharide, suspension of a strain of M nonliquefaciens gave identity reaction with a strain of Neisseria meningitidis, and reacted even more strongly than the latter. Phenol extracts of M nonliquefaciens strains generally contained higher amounts of neuraminic acid than N meningitidis group B strains. Neuraminic-acid-containing polysaccharides of M nonliquefaciens strains sedimented more slowly by ultra-centrifugation than the group-specific B polysaccharide of N meningitidis strains. They also reacted more strongly with a monoclonal anti-group B antiserum than did N meningitidis group B capsular polysaccharide in an antibody binding inhibition test (solid phase radioimmunoassay). Immunological reactivity of the polysaccharides of both species was lost if extraction was performed with unbuffered phenol at 68 degrees C, instead of with neutral phenol at 4 degrees C. The results show that several strains of M nonliquefaciens, often inhabiting the human nose, have high levels of a surface polysaccharide chemically and immunologically closely similar to N meningitidis group B capsular polysaccharide. The cross-reactivity may have immunological implications for meningococcal disease.

  17. [Efficacy of the meningococcal vaccine from Group C capsular polysaccharide].

    PubMed

    González Enríquez, J; García Comas, L; Alcaide Jiménez, J F; Sáenz Calvo, A; Conde Olasagasti, J

    1997-01-01

    This report is a systematic review of the effect intensity and duration of the immune response to meningococcal serogroup C vaccine. The vaccine safety, efficacy and effectiveness are also analyzed. MEDLINE literature search in the period 1970-1996. Meningoccocal polysaccharide vaccine clinical trials and human prospective studies were specifically searched. Quality of the retrieved studies were analyzed. Information available was integrated. Group C meningoccal polysaccharide vaccine is a safe product. Its efficacy is over 85% among adults and children over 5 years old. 70% (CI 95%: 5-91%) under 5 years old, and 55% among children 2-3 years old. The vaccine is not effective under 2 years. The duration of protective antibody levels decrease with age. The proportion of vaccinated children effectively protected one year after vaccination is low. Vaccination does not affect the immune response to ulterior revaccination. Group C meningococcal polysaccharide vaccine is indicated in adults and children over 2 years old to protect them from meningococcal disease due to group C when exposed to high risk of infection. The outbreaks control is the main indication for the use of this vaccine. Routine immunization in not outbreak situation is not recommended due to the small vaccine protection in children under 2 years old, the limited efficacy in children under 5, and the short duration of the immunity in children.

  18. The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 14

    PubMed Central

    ITO, Hiroya

    2015-01-01

    The genetic organization of the gene involved in the capsular polysaccharide (CPS) biosynthesis of Actinobacillus pleuropneumoniae serotype 14 has been determined. The DNA region for the CPS biosynthesis of serotype 14 (cps14) comprised 9 open reading frames, designated as cps14AB1B2B3CDEFG genes, encoding Cps14A to Cps14G protein, respectively. Cps14A was similar to CpsA of A. pleuropneumoniae serotypes 1, 4 and 12; the Cps14B1 and Cps14B2 were similar to CpsB of A. pleuropneumoniae serotypes 1, 4 and 12, suggesting that CPS structure of A. pleuropneumoniae serotype 14 would belong to Group I including A. pleuropneumoniae serotypes 1, 4, 12 and 15. Surprisingly, the overall nucleotide sequence, deduced amino acid sequence, and the genetic organization of the cps14 were nearly identical to those of Actinobacillus suis. This study will provide the molecular basic knowledge for development of diagnostics and vaccine of A. pleuropneumoniae serotype 14. PMID:25648373

  19. The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 14.

    PubMed

    Ito, Hiroya

    2015-05-01

    The genetic organization of the gene involved in the capsular polysaccharide (CPS) biosynthesis of Actinobacillus pleuropneumoniae serotype 14 has been determined. The DNA region for the CPS biosynthesis of serotype 14 (cps14) comprised 9 open reading frames, designated as cps14AB1B2B3CDEFG genes, encoding Cps14A to Cps14G protein, respectively. Cps14A was similar to CpsA of A. pleuropneumoniae serotypes 1, 4 and 12; the Cps14B1 and Cps14B2 were similar to CpsB of A. pleuropneumoniae serotypes 1, 4 and 12, suggesting that CPS structure of A. pleuropneumoniae serotype 14 would belong to Group I including A. pleuropneumoniae serotypes 1, 4, 12 and 15. Surprisingly, the overall nucleotide sequence, deduced amino acid sequence, and the genetic organization of the cps14 were nearly identical to those of Actinobacillus suis. This study will provide the molecular basic knowledge for development of diagnostics and vaccine of A. pleuropneumoniae serotype 14.

  20. Identification of a Group 1-Like Capsular Polysaccharide Operon for Vibrio vulnificus

    PubMed Central

    Wright, Anita C.; Powell, Jan L.; Kaper, James B.; Morris, J. Glenn

    2001-01-01

    Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V. vulnificus CPS locus, which included an upstream ops element, a wza gene (wzaVv), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli. Polar transposon mutations in wzaVv eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wzaVv was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wzaVv and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V. vulnificus. PMID:11598064

  1. Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis.

    PubMed

    Marchetti, Roberta; Dillon, Michael J; Burtnick, Mary N; Hubbard, Mark A; Kenfack, Marielle Tamigney; Blériot, Yves; Gauthier, Charles; Brett, Paul J; AuCoin, David P; Lanzetta, Rosa; Silipo, Alba; Molinaro, Antonio

    2015-10-16

    Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections.

  2. Capsular polysaccharide of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, and its modification with phosphorylcholine.

    PubMed

    Shi, Fang; Harada, Tomoyuki; Ogawa, Yohsuke; Ono, Hiroshi; Ohnishi-Kameyama, Mayumi; Miyamoto, Toru; Eguchi, Masahiro; Shimoji, Yoshihiro

    2012-11-01

    The capsule has been implicated in the virulence of the swine pathogen Erysipelothrix rhusiopathiae, a rod-shaped, intracellular Gram-positive bacterium that has a unique phylogenetic position in the phylum Firmicutes and is a close relative of Mollicutes (mycoplasma species). In this study, we analyzed the genetic locus and composition of the capsular polysaccharide (CPS) of the Fujisawa strain of E. rhusiopathiae. Genome analysis of the Fujisawa strain revealed that the genetic locus for capsular polysaccharide synthesis (cps) is located next to an lic operon, which is involved in the incorporation and expression of phosphorylcholine (PCho). Reverse transcription-PCR analysis showed that cps and lic are transcribed as a single mRNA, indicating that the loci form an operon. Using the cell surface antigen-specific monoclonal antibody (MAb) ER21 as a probe, the capsular materials were isolated from the Fujisawa strain by hot water extraction and treatment with DNase, RNase, pronase, and N-acetylmuramidase SG, followed by anion-exchange and gel filtration chromatography. The materials were then analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The CPS of E. rhusiopathiae is heterogeneous and consists of the major monosaccharides galacturonic acid, galactose, mannose, glucose, arabinose, xylose, and N-acetylglucosamine and some minor monosaccharides containing ribose, rhamnose, and N-acetylgalactosamine. In addition, the capsule is modified by PCho, which comigrates with the capsular materials, as determined by Western immunoblotting, and colocalizes on the cell surface, as determined by immunogold electron microscopy. Virulence testing of PCho-defective mutants in mice demonstrated that PCho is critical for the virulence of this organism.

  3. Capsular Polysaccharide of Erysipelothrix rhusiopathiae, the Causative Agent of Swine Erysipelas, and Its Modification with Phosphorylcholine

    PubMed Central

    Shi, Fang; Harada, Tomoyuki; Ogawa, Yohsuke; Ono, Hiroshi; Ohnishi-Kameyama, Mayumi; Miyamoto, Toru; Eguchi, Masahiro

    2012-01-01

    The capsule has been implicated in the virulence of the swine pathogen Erysipelothrix rhusiopathiae, a rod-shaped, intracellular Gram-positive bacterium that has a unique phylogenetic position in the phylum Firmicutes and is a close relative of Mollicutes (mycoplasma species). In this study, we analyzed the genetic locus and composition of the capsular polysaccharide (CPS) of the Fujisawa strain of E. rhusiopathiae. Genome analysis of the Fujisawa strain revealed that the genetic locus for capsular polysaccharide synthesis (cps) is located next to an lic operon, which is involved in the incorporation and expression of phosphorylcholine (PCho). Reverse transcription-PCR analysis showed that cps and lic are transcribed as a single mRNA, indicating that the loci form an operon. Using the cell surface antigen-specific monoclonal antibody (MAb) ER21 as a probe, the capsular materials were isolated from the Fujisawa strain by hot water extraction and treatment with DNase, RNase, pronase, and N-acetylmuramidase SG, followed by anion-exchange and gel filtration chromatography. The materials were then analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The CPS of E. rhusiopathiae is heterogeneous and consists of the major monosaccharides galacturonic acid, galactose, mannose, glucose, arabinose, xylose, and N-acetylglucosamine and some minor monosaccharides containing ribose, rhamnose, and N-acetylgalactosamine. In addition, the capsule is modified by PCho, which comigrates with the capsular materials, as determined by Western immunoblotting, and colocalizes on the cell surface, as determined by immunogold electron microscopy. Virulence testing of PCho-defective mutants in mice demonstrated that PCho is critical for the virulence of this organism. PMID:22949554

  4. Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

    NASA Astrophysics Data System (ADS)

    Abdelhameed, Ali Saber; Morris, Gordon A.; Almutairi, Fahad; Adams, Gary G.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-10-01

    The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates – in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium “SEDFIT MSTAR” analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), “Conformation Zoning” and Wales-van Holde approaches showed a high degree of flexibility – at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein.

  5. Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

    PubMed Central

    Abdelhameed, Ali Saber; Morris, Gordon A.; Almutairi, Fahad; Adams, Gary G.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-01-01

    The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates – in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium “SEDFIT MSTAR” analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), “Conformation Zoning” and Wales-van Holde approaches showed a high degree of flexibility – at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein. PMID:27782149

  6. In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model

    PubMed Central

    Nualnoi, Teerapat; Kirosingh, Adam; Pandit, Sujata G.; Thorkildson, Peter; Brett, Paul J.; Burtnick, Mary N.; AuCoin, David P.

    2016-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The “gold standard” for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, B. pseudomallei capsular polysaccharide (CPS) was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the in vivo fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis. PMID:27941991

  7. In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model.

    PubMed

    Nualnoi, Teerapat; Kirosingh, Adam; Pandit, Sujata G; Thorkildson, Peter; Brett, Paul J; Burtnick, Mary N; AuCoin, David P

    2016-12-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The "gold standard" for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, B. pseudomallei capsular polysaccharide (CPS) was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the in vivo fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis.

  8. Expression of crossreactive idiotypes by human antibodies specific for the capsular polysaccharide of Hemophilus influenzae B.

    PubMed Central

    Lucas, A H

    1988-01-01

    Human antibodies specific, for polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hemophilus influenzae b, were studied using idiotypic analysis. Antisera were prepared against purified F(ab')2 anti-PRP from two unrelated adults, H.H. and P.T. After repeated absorption with IgG myeloma proteins and with PRP-absorbed normal human Ig and donor Ig, anti-idiotypic (anti-Id) sera were obtained that specifically reacted with anti-PRP antibodies. Anti-IdHH and anti-IdPT reciprocally crossreacted with H.H. and P.T. anti-PRP antibodies and F(ab')2 fragments, and also reacted with the serum anti-PRP antibodies from three additional adults unrelated to P.T. and H.H. Both anti-Id sera partially inhibited anti-PRP paratopes but not anti-tetanus toxoid paratopes. PRP did not inhibit anti-Id recognition of shared or crossreactive idiotypic (CRI) determinants. Naturally occurring and PRP immunization-induced anti-PRP antibodies expressed CRI. While CRI titer increased after immunization, the increase was usually less than the rise in total anti-PRP antibody. Quantitative differences in CRI expression were also apparent between natural and immunization-induced H.H. and P.T. anti-PRP antibodies as shown by their differential inhibitability by anti-Id. Our data demonstrate that anti-PRP antibodies from five unrelated adults express CRI determinants that are probably distant from the PRP combining site. Naturally occurring and immunization-induced anti-PRP antibodies share CRI and therefore appear to be clonally related, although immunization apparently induces the expression CRI-negative antibodies as well. These results, taken with previous studies showing restricted and identical anti-PRP isoelectric focusing spectrotypes in unrelated adults, suggest that some PRP-specific V domains are structurally conserved and probably germ-line encoded. PMID:3257499

  9. Purification and characterisation of anti-pneumococcal capsular polysaccharide IgG immunoglobulins.

    PubMed

    Parker, Antony R; Lock, Emma; Iftikhar, Asma; Barber, Richard; Stubbs, Phil D; Harding, Stephen; Wallis, Gregg L F

    2017-01-01

    The production of reference materials for quantifying pneumococcal antibody concentrations relies upon large scale vaccination. An alternative simple, reproducible protocol has been developed for the affinity purification of 23 serotype anti-pneumococcal capsular polysaccharide (PCP) IgG immunoglobulins. The purification protocol utilised IgG fractionation, capsular polysaccharide (CPS) adsorption, and affinity chromatography using Pneumovax®-Sepharose. Purification efficiency and method reproducibility were assessed by comparison of 4 batches of anti-PCP IgG. Immunoglobulin composition was determined using nephelometry and functionality was evaluated using VaccZyme™ ELISAs. Anti-PCP IgG preparations were ≥95% pure by SDS-PAGE analysis with no contaminating IgA or IgM immunoglobulins or IgG antigen specific antibodies towards haemophilus influenzae b, diphtheria toxoid or tetanus toxoid. The predominant IgG subclass in the preparation was IgG2. This novel purification procedure produced highly specific anti-PCP IgG preparations that compared well to both Lot 89SF and 007sp international serum standards and could be used as an alternative method for the production of reference materials. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  10. CUTANEOUS REACTIONS IN RABBITS TO THE TYPE-SPECIFIC CAPSULAR POLYSACCHARIDES OF PNEUMOCOCCUS

    PubMed Central

    Francis, Thomas; Tillett, William S.

    1931-01-01

    The injection of the type-specific capsular polysaccharides of Pneumococcus Types I, II and III into the skin of rabbits, actively or passively immunized to one of these types of Pneumococcus, elicits a type-specific cutaneous reaction. The form of reaction resembles that described by Arthus. The reaction is produced only when type-specific precipitins for the homologous polysaccharide are demonstrable in the blood of the rabbit. In 84 per cent of actively immunized rabbits, the serum of which contained type-specific precipitins, a reaction was elicited. A positive result was obtained in 100 per cent of rabbits passively immunized with antipneumococcus horse serum whereas, attempts passively to transfer reactivity from immune rabbit to normal rabbit were unsuccessful. The recipients, in the latter group, possessed no demonstrable circulating type-specific precipitins. The reaction produced by specific capsular carbohydrates is always associated with a well grounded type-specific immunity. A brief summary of the relation of hypersensitiveness and immunity to pneumococcus is given. PMID:19869942

  11. HPAEC-PAD method for the analysis of alkaline hydrolyzates of Haemophilus influenzae type b capsular polysaccharide.

    PubMed

    de Haan, Alex; van der Put, Robert M F; Beurret, Michel

    2013-09-01

    A gradient method has been devised for the rapid analysis of alkaline hydrolyzates of Haemophilus influenzae type b (Hib) capsular polysaccharide-based vaccines by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As compared with published procedures, peak shape and sensitivity were significantly improved with this approach, analysis time was short and there was little interference from impurities. The limits of detection and quantification were established with a purified reference polysaccharide. We propose this method as a practical alternative for the analysis of minute amounts of Hib polysaccharide, which can be lower than with the conventional approaches.

  12. The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 15

    PubMed Central

    ITO, Hiroya; SUEYOSHI, Masuo

    2014-01-01

    Nucleotide sequence determination and analysis of the cps gene involved in the capsular polysaccharide biosynthesis of Actinobacillus pleuropneumoniae serotype 15 revealed the presence of three open reading frames, designated as cps15ABC genes. At the protein level, Cps15A and Cps15B showed considerably high homology to CpsA (67.0 to 68.7%) and CpsB (31.7 to 36.8%), respectively, of A. pleuropneumoniae serotypes 1, 4 and 12, revealing the common genetic organization of the cps among serotypes 1, 4, 12 and 15. However, Cps15C showed no homology to any proteins of A. pleuropneumoniae serotypes, indicating that cps15C may be specific to serotype 15. This study will provide the basic molecular knowledge necessary for the development of diagnostics and a vaccine for A. pleuropneumoniae serotype 15. PMID:25502540

  13. The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 15.

    PubMed

    Ito, Hiroya; Sueyoshi, Masuo

    2015-04-01

    Nucleotide sequence determination and analysis of the cps gene involved in the capsular polysaccharide biosynthesis of Actinobacillus pleuropneumoniae serotype 15 revealed the presence of three open reading frames, designated as cps15ABC genes. At the protein level, Cps15A and Cps15B showed considerably high homology to CpsA (67.0 to 68.7%) and CpsB (31.7 to 36.8%), respectively, of A. pleuropneumoniae serotypes 1, 4 and 12, revealing the common genetic organization of the cps among serotypes 1, 4, 12 and 15. However, Cps15C showed no homology to any proteins of A. pleuropneumoniae serotypes, indicating that cps15C may be specific to serotype 15. This study will provide the basic molecular knowledge necessary for the development of diagnostics and a vaccine for A. pleuropneumoniae serotype 15.

  14. The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

    PubMed

    Wangdi, Tamding; Lee, Cheng-Yuk; Spees, Alanna M; Yu, Chenzhou; Kingsbury, Dawn D; Winter, Sebastian E; Hastey, Christine J; Wilson, R Paul; Heinrich, Volkmar; Bäumler, Andreas J

    2014-08-01

    Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

  15. A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate

    PubMed Central

    Abdelhameed, Ali Saber; Adams, Gary G.; Morris, Gordon A.; Almutairi, Fahad M.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-01-01

    Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 106 g.mol−1) compared to the native (Mw ~ 1.2 × 106 g.mol−1). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 106 g.mol−1), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution. PMID:26915577

  16. NMR and molecular dynamics studies of the conformational epitope of the type III group B Streptococcus capsular polysaccharide and derivatives.

    PubMed

    Brisson, J R; Uhrinova, S; Woods, R J; van der Zwan, M; Jarrell, H C; Paoletti, L C; Kasper, D L; Jennings, H J

    1997-03-18

    The conformational epitope of the type III group B Streptococcus capsular polysaccharide (GBSP III) exhibits unique properties which can be ascribed to the presence of sialic acid in its structure and the requirement for an extended binding site. By means of NMR and molecular dynamics studies on GBSP III and its fragments, the extended epitope of GBSP III was further defined. The influence of sialic acid on the conformational properties of GBSP III was examined by performing conformational analysis on desialylated GBSP III, which is identical to the polysaccharide of Streptococcus pneumoniae type 14, and also on oxidized and reduced GBSP III. Conformational changes were gauged by 1H and 13C chemical shift analysis, NOE, 1D selective TOCSY-NOESY experiments, J(HH) and J(CH) variations, and NOE of OH resonances. Changes in mobility were examined by 13C T1 and T2 measurements. Unrestrained molecular dynamics simulations with explicit water using the AMBER force field and the GLYCAM parameter set were used to assess static and dynamic conformational models, simulate the observable NMR parameters and calculate helical parameters. GBSP III was found to be capable of forming extended helices. Hence, the length dependence of the conformational epitope could be explained by its location on extended helices within the random coil structure of GBSP III. The interaction of sialic acid with the backbone of the PS was also found to be important in defining the conformational epitope of GBSP III.

  17. A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate.

    PubMed

    Abdelhameed, Ali Saber; Adams, Gary G; Morris, Gordon A; Almutairi, Fahad M; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E

    2016-02-26

    Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 10(6) g.mol(-1)) compared to the native (Mw ~ 1.2 × 10(6) g.mol(-1)). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 10(6) g.mol(-1)), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.

  18. Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates

    PubMed Central

    2010-01-01

    Background Burkholderia thailandensis is a non-pathogenic environmental saprophyte closely related to Burkholderia pseudomallei, the causative agent of the often fatal animal and human disease melioidosis. To study B. thailandensis genomic variation, we profiled 50 isolates using a pan-genome microarray comprising genomic elements from 28 Burkholderia strains and species. Results Of 39 genomic regions variably present across the B. thailandensis strains, 13 regions corresponded to known genomic islands, while 26 regions were novel. Variant B. thailandensis isolates exhibited isolated acquisition of a capsular polysaccharide biosynthesis gene cluster (B. pseudomallei-like capsular polysaccharide) closely resembling a similar cluster in B. pseudomallei that is essential for virulence in mammals; presence of this cluster was confirmed by whole genome sequencing of a representative variant strain (B. thailandensis E555). Both whole-genome microarray and multi-locus sequence typing analysis revealed that the variant strains formed part of a phylogenetic subgroup distinct from the ancestral B. thailandensis population and were associated with atypical isolation sources when compared to the majority of previously described B. thailandensis strains. In functional assays, B. thailandensis E555 exhibited several B. pseudomallei-like phenotypes, including colony wrinkling, resistance to human complement binding, and intracellular macrophage survival. However, in murine infection assays, B. thailandensis E555 did not exhibit enhanced virulence relative to other B. thailandensis strains, suggesting that additional factors are required to successfully colonize and infect mammals. Conclusions The discovery of such novel variant strains demonstrates how unbiased genomic surveys of non-pathogenic isolates can reveal insights into the development and emergence of new pathogenic species. PMID:20799932

  19. A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice.

    PubMed

    Buchwald, Ulrike K; Lees, Andrew; Steinitz, Michael; Pirofski, Liise-Anne

    2005-01-01

    Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.

  20. Molecular Characterization of Type-Specific Capsular Polysaccharide Biosynthesis Genes of Streptococcus agalactiae Type Ia

    PubMed Central

    Yamamoto, Shin; Miyake, Katsuhide; Koike, Yoichi; Watanabe, Masaki; Machida, Yuichi; Ohta, Michio; Iijima, Shinji

    1999-01-01

    The type-specific capsular polysaccharide (CP) of a group B streptococcus, Streptococcus agalactiae type Ia, is a high-molecular-weight polymer consisting of the pentasaccharide repeating unit 4)-[α-d-NeupNAc-(2→3)-β-d-Galp-(1→4)-β-d-GlcpNAc-(1→3)]-β-d-Galp-(1→4)-β-d-Glcp-(1. Here, cloning, sequencing, and transcription of the type Ia-specific capsular polysaccharide synthesis (cps) genes and functional analysis of these gene products are described. A 26-kb DNA fragment containing 18 complete open reading frames (ORFs) was cloned. These ORFs were designated cpsIaA to cpsIaL, neu (neuraminic acid synthesis gene) A to D, orf1 and ung (uracil DNA glycosylase). The cps gene products of S. agalactiae type Ia were homologous to proteins involved in CP synthesis of S. agalactiae type III and S. pneumoniae serotype 14. Unlike the cps gene cluster of S. pneumoniae serotype 14, transcription of this operon may start from cpsIaA, cpsIaE, and orf1 because putative promoter sequences were found in front of these genes. Northern hybridization, reverse transcription-PCR, and primer extension analyses supported this hypothesis. DNA sequence analysis showed that there were two transcriptional terminators in the 3′ end of this operon (downstream of orf1 and ung). The functions of CpsIaE, CpsIaG, CpsIaI, and CpsIaJ were examined by glycosyltransferase assay by using the gene products expressed in Escherichia coli JM109 harboring plasmids containing various S. agalactiae type Ia cps gene fragments. Enzyme assays suggested that the gene products of cpsIaE, cpsIaG, cpsIaI, and cpsIaJ are putative glucosyltransferase, β-1,4-galactosyltransferase, β-1,3-N-acetylglucosaminyltransferase, and β-1,4-galactosyltransferase, respectively. PMID:10464185

  1. Isolation of a Bacteriophage Specific for a New Capsular Type of Klebsiella pneumoniae and Characterization of Its Polysaccharide Depolymerase

    PubMed Central

    Hsu, Chun-Ru; Lin, Tzu-Lung; Pan, Yi-Jiun; Hsieh, Pei-Fang; Wang, Jin-Town

    2013-01-01

    Background Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed. Methodology/Principal Findings To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS− mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis. Conclusions/Significance Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative

  2. Role of capsular polysaccharide (CPS) in biofilm formation and regulation of CPS production by quorum-sensing in Vibrio vulnificus.

    PubMed

    Lee, Kyung-Jo; Kim, Jeong-A; Hwang, Won; Park, Soon-Jung; Lee, Kyu-Ho

    2013-11-01

    Extracellular polysaccharides, such as lipopolysaccharide and loosely associated exopolysaccharides, are essential for Vibrio vulnificus to form biofilms. The role of another major component of the V. vulnificus extracellular matrix, capsular polysaccharide (CPS), which contributes to colony opacity, has been characterized in biofilm formation. A CPS-deficient mutant, whose wbpP gene encoding UDP-GlcNAc C4-epimerase was knocked out, formed significantly more biofilm than wild type, due to increased hydrophobicity of the cell surface, adherence to abiotic surfaces and cell aggregation. To elucidate the direct effect of CPS on biofilm structure, extracted CPS and a CPS-degrading enzyme, α-N-acetylgalactosaminidase, were added in biofilm assays, resulting in reduction and increment of biofilm sizes respectively. Therefore, it is suggested that CPS play a critical role in determining biofilm size by restricting continual growth of mature biofilms. Since CPS is required after maturation, CPS biosynthesis should be controlled in a cell density-dependent manner, e.g. by quorum-sensing (QS) regulation. Analysing transcription of the CPS gene cluster revealed that it was activated by SmcR, a QS master regulator, via binding to the upstream region of the cluster. Therefore, CPS was produced when biofilm cell density reached high enough to turn on QS regulation and limited biofilms to appropriate sizes.

  3. E. coli Group 1 Capsular Polysaccharide Exportation Nanomachinary as a Plausible Antivirulence Target in the Perspective of Emerging Antimicrobial Resistance

    PubMed Central

    Sachdeva, Shivangi; Palur, Raghuvamsi V.; Sudhakar, Karpagam U.; Rathinavelan, Thenmalarchelvi

    2017-01-01

    Bacteria evolving resistance against the action of multiple drugs and its ability to disseminate the multidrug resistance trait(s) across various strains of the same bacteria or different bacterial species impose serious threat to public health. Evolution of such multidrug resistance is due to the fact that, most of the antibiotics target bacterial survival mechanisms which exert selective pressure on the bacteria and aids them to escape from the action of antibiotics. Nonetheless, targeting bacterial virulence strategies such as bacterial surface associated polysaccharides biosynthesis and their surface accumulation mechanisms may be an attractive strategy, as they impose less selective pressure on the bacteria. Capsular polysaccharide (CPS) or K-antigen that is located on the bacterial surface armors bacteria from host immune response. Thus, unencapsulating bacteria would be a good strategy for drug design, besides CPS itself being a good vaccine target, by interfering with CPS biosynthesis and surface assembly pathway. Gram-negative Escherichia coli uses Wzy-polymerase dependent (Groups 1 and 4) and ATP dependent (Groups 1 and 3) pathways for CPS production. Considering E. coli as a case in point, this review explains the structure and functional roles of proteins involved in Group 1 Wzy dependent CPS biosynthesis, surface expression and anchorage in relevance to drug and vaccine developments. PMID:28217109

  4. Autologous albumin enhances the humoral immune response to capsular polysaccharide covalently co-attached to bacteria-sized latex beads

    PubMed Central

    Colino, Jesus; Duke, Leah; Snapper, Clifford M.

    2014-01-01

    Abundant autologous proteins, like serum albumin, should be immunologically inert. However, individuals with no apparent predisposition to autoimmune disease can develop immune responses to autologous therapeutic proteins. Protein aggregation is a potential major trigger of these responses. Adsorption of proteins to particles provides macromolecular size and may generate structural changes in the protein, resembling aggregation. Using aldehyde/sulfate latex beads coated with murine serum albumin (MSA), we found that mice mounted MSA-specific IgG responses that were dependent on CD4+ T cells. IgG were specific for MSA adsorbed to solid surfaces and non-cross-reactive with human, bovine or pig albumins. T cells induced in response to MSA, augmented the primary and induced boosted secondary IgG and IgM responses specific for the T cell-independent antigen, capsular polysaccharide of Streptococcus pneumoniae type 14 (PPS14), when the latter was attached to the same bead. Similar to the anti-MSA IgG response, the boosted PPS14-specific IgG secondary response was CD4+ T cell-dependent, displayed a typical carrier effect, and was enhanced by, but did not require, Toll-like receptor stimulation. These results provide a potential mechanism for the induction of responses to autoantigens unable to induce specific T cell responses, and provide new insights into polysaccharide-specific immunity. PMID:24481921

  5. Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

    PubMed Central

    Kuo, J S; Doelling, V W; Graveline, J F; McCoy, D W

    1985-01-01

    Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP. Images PMID:3926752

  6. Bacterial capsular polysaccharide prevents the onset of asthma through T-cell activation.

    PubMed

    Johnson, Jenny L; Jones, Mark B; Cobb, Brian A

    2015-04-01

    Over the last four decades, increases in the incidence of immune-mediated diseases in the Western world have been linked to changes in microbial exposure. It is becoming increasingly clear that the normal microbiota in the gut can profoundly alter susceptibility to a wide range of diseases, such as asthma, in which immune homeostasis is disrupted, yet the mechanisms governing this microbial influence remains poorly defined. In this study, we show that gastrointestinal exposure to PSA, a capsular polysaccharide derived from the commensal bacterium Bacteroides fragilis, significantly limits susceptibility to the induction of experimental asthma. We report that direct treatment of mice with PSA generates protection from asthma, and this effect can be given to a naïve recipient by adoptive transfer of CD4(+) T cells from PSA-exposed mice. Remarkably, we found that these PSA-induced T cells are not canonical FoxP3(+) regulatory T cells, but that they potently inhibit both Th1 and Th2 models of asthma in an IL-10-dependent fashion. These findings reveal that bacterial polysaccharides link the microbiota with the peripheral immune system by activating CD4(+)Foxp3(-) T cells upon exposure in the gut, and they facilitate resistance to unnecessary inflammatory responses via the production of IL-10. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b

    SciTech Connect

    Kuo, J.S.; Doelling, V.W.; Graveline, J.F.; McCoy, D.W.

    1985-08-01

    Cells of Haemophilus influenzae type b were grown in a liquid medium containing (TH)palmitate or ( UC)ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( (TH)palmitate and ( UC)ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( (TH)palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with (TH)palmitate or (TH)palmitate and ( UC)ribose followed by chloroform-methanol extraction could most of the TH-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.

  8. Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster

    PubMed Central

    Bertin, Clothilde; Pau-Roblot, Corinne; Courtois, Josiane; Manso-Silván, Lucía; Tardy, Florence; Poumarat, François; Citti, Christine; Sirand-Pugnet, Pascal; Gaurivaud, Patrice

    2014-01-01

    Mycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a β(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β(1→2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides. PMID:25398856

  9. Oxidation of the capsular polysaccharide of pneumoccal type IX by periodate

    PubMed Central

    Das, Amalendu; Higginbotham, John D.; Heidelberger, Michael

    1972-01-01

    1. The pneumococcal type IX polysaccharide (polysaccharide S IX) has been oxidized by sodium metaperiodate and reduced by sodium borohydride. Of the constituent monosaccharides, N-acetylglucosamine and N-acetylmannosamine remain unaltered, whereas 40% of the glucose and 90% of the glucuronic acid are oxidized. 2. The effect of oxidation and subsequent reduction on the precipitation of polysaccharide S IX in anti-(pneumococcal) sera is described and interpreted in structural terms. 3. Oligosaccharides produced by oxidation, reduction and hydrolysis with dilute acid have been isolated and partially characterized. 4. The results in this paper and the preceding one (Higginbotham et al., 1972) are used to postulate a possible structure for polysaccharide S IX. PMID:4403879

  10. Immune suppression induced by Vi capsular polysaccharide is overcome by Vi-DT conjugate vaccine.

    PubMed

    An, So Jung; Yoon, Yeon Kyung; Kothari, Sudeep; Kim, Deok Ryun; Kim, Jeong Ah; Kothari, Neha; Lee, Eugene; Park, Tai Hyun; Carbis, Rodney

    2012-02-01

    The influence pre-exposure of mice to Vi capsular polysaccharide, purified from Salmonella enterica Serovar Typhi, on the subsequent immune response induced by a Vi-diphtheria toxoid (Vi-DT) conjugate was evaluated. Vi induced low anti Vi IgG titers with the dominant subclass being IgG3. The Vi-DT conjugate induced high titers of anti Vi IgG with the dominant subclass being IgG1 but with considerable quantities of IgG2a, IgG2b and IgG3. Priming of mice with Vi suppressed the response to a subsequent dose of conjugate and the suppression was overcome by a second dose of conjugate. Priming with conjugate prevented suppression of the anti Vi response and subsequent dosing with Vi raised titers back to previous levels but did not boost to new higher levels. The anti DT IgG response to one dose of conjugate was relatively strong and protracted and continued to rise for 12 weeks, compared to the response to one dose of DT which was poor and peaked at two weeks. The prolonged anti DT response was most likely due to the slow release of DT from the conjugate lattice as it degrades within the mouse resulting in a continuous stimulation of the immune response. The presence of increasing amounts of un-conjugated Vi, up to 50%, administered with the conjugate resulted in increasingly higher levels of both anti Vi and anti DT. Larger amounts of un-conjugated Vi inhibited the anti Vi response. These findings have implications for vaccine quality and a limit for un-conjugated polysaccharide should not exceed 50% and from a vaccine program perspective if the results presented here translate to humans then a Vi conjugate, once it becomes available, should replace Vi polysaccharide vaccines. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Preparation of pneumococcal capsular polysaccharide-protein conjugate vaccines utilizing new fragmentation and conjugation technologies.

    PubMed

    Pawlowski, A; Källenius, G; Svenson, S B

    2000-03-17

    There is a global urgent need for a new efficient and inexpensive vaccine to combat pneumococcal disease, which should also be affordable in developing countries. In view of this need a simple low-cost technique to prepare such a vaccine was developed. The preparation of serotype 14 and 23F pneumococcal capsular polysaccharide (PnPS)-protein conjugates to be included in a forthcoming multivalent PnPS conjugate vaccine is described. Commercial lots of PnPSs produced according to Good Manufacturing Practice from Streptococcus pneumoniae serotype 14 (PS14) and 23F (PS23F) were partially depolymerized by sonication or irradiation in an electron beam accelerator. The PnPS fragments were conjugated to tetanus toxoid (TT) using a recently developed conjugation chemistry. The application of these new simple, efficient and inexpensive fragmentation and conjugation technologies allowed the synthesis of several PnPS-protein conjugates containing PnPS fragments of preselected sizes and differing in the degree of substitution. The PS14TT and PS23FTT conjugate vaccine candidates were characterized chemically and their immunogenicity was evaluated in rabbits and mice. All PnPS conjugate vaccines, unlike the corresponding plain polysaccharides, produced high IgG titres in both animal species. The PS14TT conjugates tended to be more immunogenic than the PS23FTT conjugates. The immune response to the PS14TT conjugates, but not to the PS23FTT conjugates, was related to the size of the conjugated polysaccharide hapten. Both types of conjugates elicited strong booster effects upon secondary immunizations, resulting in high IgG1, IgG2a and IgG2b titres.

  12. Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants

    SciTech Connect

    Small, J.M.; Mitchell, T.G.

    1986-12-01

    Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with /sup 125/I, and used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal.

  13. Use of an Inhibition Enzyme-Linked Immunosorbent Assay for Quantification of Capsular Polysaccharide or Proteins in Vaccines▿

    PubMed Central

    Inzana, Thomas J.; Champion, Anna

    2007-01-01

    An inhibition enzyme-linked immunosorbent assay (ELISA) is described for quantification of capsular polysaccharide or proteins in vaccines and other samples containing whole cells or extracts of Actinobacillus pleuropneumoniae. The assay can be used to quantify any antigen that can be purified and for which highly specific antibodies are not available. The assay can be carried out by any laboratory capable of performing an ELISA. PMID:17267591

  14. Polysaccharides of the genus Bacillus cross-reactive with the capsular polysaccharides of Diplococcus pneumoniae type 3, Haemophilus influenzae type b, and Neisseria meningitidis group A.

    PubMed

    Myerowitz, R L; Gordon, R E; Robbins, J B

    1973-12-01

    We studied 174 strains of the genus Bacillus for cross-reacting antigens to the capsular polysaccharides of groups A and C meningococcus, types I and III pneumococcus, and Haemophilus influenzae type b. Cross-reactions were detected by immunodiffusion in agarose gel by using type-specific antisera and confirmed by absorption and inhibition experiments. Of 20 Bacillus pumilis strains, six had an antigen cross-reacting with group A meningococcal polysaccharide. Other cross-reactions included one strain of B. pumilis with H. influenzae type b, one of B. cereus var. mycoides with pneumococcus type III, and one of B. alvei with both type b and SIII polysaccharides. These cross-reacting antigens are polysaccharides of vegetative cells and may be extracellular in location. Because these bacilli have antigens cross-reacting with the virulence factors of pyogenic bacteria, they may, as normal flora, be an antigenic stimulus for "natural" serum anti-capsular antibodies to the type b Haemophilus and group A meningococcus polysaccharides.

  15. Polysaccharides of the Genus Bacillus Cross-Reactive with the Capsular Polysaccharides of Diplococcus pneumoniae Type III, Haemophilus influenzae Type b, and Neisseria meningitidis Group A

    PubMed Central

    Myerowitz, Richard L.; Gordon, Ruth E.; Robbins, John B.

    1973-01-01

    We studied 174 strains of the genus Bacillus for cross-reacting antigens to the capsular polysaccharides of groups A and C meningococcus, types I and III pneumococcus, and Haemophilus influenzae type b. Cross-reactions were detected by immunodiffusion in agarose gel by using type-specific antisera and confirmed by absorption and inhibition experiments. Of 20 Bacillus pumilis strains, six had an antigen cross-reacting with group A meningococcal polysaccharide. Other cross-reactions included one strain of B. pumilis with H. influenzae type b, one of B. cereus var. mycoides with pneumococcus type III, and one of B. alvei with both type b and SIII polysaccharides. These cross-reacting antigens are polysaccharides of vegetative cells and may be extracellular in location. Because these bacilli have antigens cross-reacting with the virulence factors of pyogenic bacteria, they may, as normal flora, be an antigenic stimulus for “natural” serum anti-capsular antibodies to the type b Haemophilus and group A meningococcus polysaccharides. Images PMID:4150383

  16. Fine Specificity and Cross-Reactions of Monoclonal Antibodies to Group B Streptococcal Capsular Polysaccharide Type III

    PubMed Central

    Pincus, Seth H.; Moran, Emily; Maresh, Grace; Jennings, Harold J.; Pritchard, David G.; Egan, Marianne L.; Blixt, Ola

    2012-01-01

    Group B streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. Despite aggressive campaigns using antenatal prophylactic antibiotic therapy, infections continue. Developing an effective maternal vaccine is a public health priority. Antibody (Ab) to the capsular polysaccharide (CPS) is considered the dominant “protective” immune mediator. Here we study the fine specificity and potential host reactivity of a panel of well-characterized murine monoclonal Abs against the type III CPS by examining the binding of the Abs to intact and neuraminidase-digested GBS, purified CPS, synthetic carbohydrate structures, and cells. The results showed marked differences in the fine specificity among these mAbs to a single carbohydrate structure. Cross-reactions with synthetic GD3 and GT3 carbohydrates, representing structures found on surfaces of neural and developing cells, were demonstrated using carbohydrate array technology. The anti-CPSIII mAbs did not react with cells expressing GD3 and GT3, nor did mAbs specific for the host carbohydrates cross-react with GBS, raising questions about the physiological relevance of this cross-reaction. But in the process of these investigations, we serendipitously demonstrated cross-reactions of some anti-CPSIII mAbs with antigens, likely carbohydrates, found on human leukocytes. These studies suggest caution in the development of a maternal vaccine to prevent infection by this important human pathogen. PMID:22634296

  17. Differential idiotype utilization for the in vivo type 14 capsular polysaccharide-specific immunoglobulin responses to intact Streptococcus pneumoniae versus a pneumococcal conjugate vaccine

    PubMed Central

    Colino, Jesus; Duke, Leah; Arjunaraja, Swadhinya; Chen, Quanyi; Liu, Leyu; Lucas, Alexander H.; Snapper, Clifford M.

    2012-01-01

    Murine IgG responses specific for the capsular polysaccharide (PPS14) of Streptococcus pneumoniae type 14 (Pn14) induced in response to intact Pn14 or a PPS14-protein conjugate are both dependent on CD4+ T cell help, but appear to utilize marginal zone versus follicular B cells, respectively. Here we identify an idiotype (44.1-Id) that dominates the PPS14-specific IgG, but not IgM, responses to intact Pn14, isolated PPS14 and Group B Streptococcus (strain COH1-11) expressing capsular polysaccharide structurally identical to PPS14. The 44.1-Id is however not expressed in the repertoire of natural PPS14-specific antibodies. In distinct contrast, PPS14-specific IgG responses to a soluble PPS14-protein conjugate exhibits minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all 4 IgG subclasses during both the primary and secondary responses. 44.1-Id usage was linked to the Igha, but not Ighb, allotype and was associated with induction of relatively high total PPS14-specific IgG responses. In contrast to PPS14-protein conjugate, avidity maturation of the 44.1-Id-dominant PPS14-specific IgG responses were limited, even during the highly boosted T cell-dependent PPS14-specific secondary responses to COH1-11. These results indicate that different antigenic forms of the same capsular polysaccharide can recruit distinct B cell clones expressing characteristic idiotypes under genetic control, and suggest that the 44.1-Id is derived from marginal zone B cells. PMID:22706079

  18. Anti-Biofilm Activity: A Function of Klebsiella pneumoniae Capsular Polysaccharide

    PubMed Central

    Dos Santos Goncalves, Marina; Delattre, Cédric; Balestrino, Damien; Charbonnel, Nicolas; Elboutachfaiti, Redouan; Wadouachi, Anne; Badel, Stéphanie; Bernardi, Thierry; Michaud, Philippe; Forestier, Christiane

    2014-01-01

    Competition and cooperation phenomena occur within highly interactive biofilm communities and several non-biocides molecules produced by microorganisms have been described as impairing biofilm formation. In this study, we investigated the anti-biofilm capacities of an ubiquitous and biofilm producing bacterium, Klebsiella pneumoniae. Cell-free supernatant from K. pneumoniae planktonic cultures showed anti-biofilm effects on most Gram positive bacteria tested but also encompassed some Gram negative bacilli. The anti-biofilm non-bactericidal activity was further investigated on Staphylococcus epidermidis, by determining the biofilm biomass, microscopic observations and agglutination measurement through a magnetic bead-mediated agglutination test. Cell-free extracts from K. pneumoniae biofilm (supernatant and acellular matrix) also showed an influence, although to a lesser extend. Chemical analyses indicated that the active molecule was a high molecular weight polysaccharide composed of five monosaccharides: galactose, glucose, rhamnose, glucuronic acid and glucosamine and the main following sugar linkage residues [→2)-α-l-Rhap-(1→]; [→4)-α-l-Rhap-(1→]; [α-d-Galp-(1→]; [→2,3)-α-d-Galp-(1→]; [→3)-β-d-Galp-(1→] and, [→4)-β-d-GlcAp-(1→]. Characterization of this molecule indicated that this component was more likely capsular polysaccharide (CPS) and precoating of abiotic surfaces with CPS extracts from different serotypes impaired the bacteria-surface interactions. Thus the CPS of Klebsiella would exhibit a pleiotropic activity during biofilm formation, both stimulating the initial adhesion and maturation steps as previously described, but also repelling potential competitors. PMID:24932475

  19. Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease

    PubMed Central

    Oh, So-Young; Budzik, Jonathan M.; Garufi, Gabriella; Schneewind, Olaf

    2012-01-01

    Summary Bacillus cereus G9241 causes an anthrax-like respiratory illness in humans, however the molecular mechanisms of disease pathogenesis are not known. Genome sequencing identified two putative virulence plasmids proposed to provide for anthrax toxin (pBCXO1) and/or capsule expression (pBC218). We report here that B. cereus G9241 causes anthrax-like disease in immune-competent mice, which is dependent on each of the two virulence plasmids. pBCXO1 encodes pagA1, the homolog of anthrax protective antigen, as well as hasACB, providing for hyaluronic acid capsule formation, two traits that each contribute to disease pathogenesis. pBC218 harbors bpsX-H, Bacillus cereus exo-polysaccharide, which produce a second capsule. During infection, B. cereus G9241 elaborates both hasACB and bpsX-H capsules, which together are essential for the establishment of anthrax-like disease and the resistance of bacilli to phagocytosis. A single nucleotide deletion causes premature termination of hasA translation in B. anthracis, which is known to escape phagocytic killing by its pXO2 encoded poly-D-γ-glutamic acid (PDGA) capsule. Thus, multiple different gene clusters endow pathogenic bacilli with capsular material, provide for escape from innate host immune responses and aid in establishing the pathogenesis of anthrax-like disease. PMID:21371137

  20. Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease.

    PubMed

    Oh, So-Young; Budzik, Jonathan M; Garufi, Gabriella; Schneewind, Olaf

    2011-04-01

    Bacillus cereus G9241 causes an anthrax-like respiratory illness in humans; however, the molecular mechanisms of disease pathogenesis are not known. Genome sequencing identified two putative virulence plasmids proposed to provide for anthrax toxin (pBCXO1) and/or capsule expression (pBC218). We report here that B. cereus G9241 causes anthrax-like disease in immune-competent mice, which is dependent on each of the two virulence plasmids. pBCXO1 encodes pagA1, the homologue of anthrax protective antigen, as well as hasACB, providing for hyaluronic acid capsule formation, two traits that each contribute to disease pathogenesis. pBC218 harbours bpsX-H, B. cereus exo-polysaccharide, which produce a second capsule. During infection, B. cereus G9241 elaborates both hasACB and bpsX-H capsules, which together are essential for the establishment of anthrax-like disease and the resistance of bacilli to phagocytosis. A single nucleotide deletion causes premature termination of hasA translation in Bacillus anthracis, which is known to escape phagocytic killing by its pXO2 encoded poly-d-γ-glutamic acid (PDGA) capsule. Thus, multiple different gene clusters endow pathogenic bacilli with capsular material, provide for escape from innate host immune responses and aid in establishing the pathogenesis of anthrax-like disease.

  1. Agglutination by anti-capsular polysaccharide antibody is associated with protection against experimental human pneumococcal carriage

    PubMed Central

    Reiné, J; Zangari, T; Owugha, JT; Pennington, SH; Gritzfeld, JF; Wright, AD; Collins, AM; van Selm, S; de Jonge, MI; Gordon, SB; Weiser, JN; Ferreira, DM

    2016-01-01

    The ability of pneumococcal conjugate vaccine (PCV) to decrease transmission by blocking the acquisition of colonization has been attributed to herd immunity. We describe the role of mucosal IgG to capsular polysaccharide (CPS) in mediating protection from carriage, translating our findings from a murine model to humans. We used a flow-cytometric assay to quantify antibody-mediated agglutination demonstrating that hyperimmune sera generated against an unencapsulated mutant was poorly agglutinating. Passive immunization with this antiserum was ineffective to block acquisition of colonization compared to agglutinating antisera raised against the encapsulated parent strain. In the human challenge model samples were collected from PCV and control vaccinated adults. In PCV-vaccinated subjects IgG levels to CPS were increased in serum and nasal wash (NW). IgG to the inoculated strain CPS dropped in NW samples after inoculation suggesting its sequestration by colonizing pneumococci. In post-vaccination NW samples pneumococci were heavily agglutinated compared to pre-vaccination samples in subjects protected against carriage. Our results indicate that pneumococcal agglutination mediated by CPS specific antibodies is a key mechanism of protection against acquisition of carriage. Capsule may be the only vaccine target that can elicit strong agglutinating antibody responses, leading to protection against carriage acquisition and generation of herd immunity. PMID:27579859

  2. Diverse Intestinal Bacteria Contain Putative Zwitterionic Capsular Polysaccharides with Anti-inflammatory Properties.

    PubMed

    Neff, C Preston; Rhodes, Matthew E; Arnolds, Kathleen L; Collins, Colm B; Donnelly, Jody; Nusbacher, Nichole; Jedlicka, Paul; Schneider, Jennifer M; McCarter, Martin D; Shaffer, Michael; Mazmanian, Sarkis K; Palmer, Brent E; Lozupone, Catherine A

    2016-10-12

    Zwitterionic capsular polysaccharides (ZPSs) are bacterial products that modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). However, only a few diverse bacteria are known to modulate the host immune system via ZPS. We present a genomic screen for bacteria encoding ZPS molecules. We identify diverse host-associated bacteria, including commensals and pathogens with known anti-inflammatory properties, with the capacity to produce ZPSs. Human mononuclear cells stimulated with lysates from putative ZPS-producing bacteria induce significantly greater IL-10 production and higher proportions of Tregs than lysates from non-ZPS-encoding relatives or a commensal strain of Bacteroides cellulosilyticus in which a putative ZPS biosynthetic operon was genetically disrupted. Similarly, wild-type B. cellulosilyticus DSM 14838, but not a close relative lacking a putative ZPS, attenuated experimental colitis in mice. Collectively, this screen identifies bacterial strains that may use ZPSs to interact with the host as well as those with potential probiotic properties. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Role of the capsular polysaccharide as a virulence factor for Streptococcus suis serotype 14.

    PubMed

    Roy, David; Auger, Jean-Philippe; Segura, Mariela; Fittipaldi, Nahuel; Takamatsu, Daisuke; Okura, Masatoshi; Gottschalk, Marcelo

    2015-04-01

    Streptococcus suis is an important swine pathogen and a zoonotic agent causing meningitis and septicemia. Although serotype 2 is the most virulent type, serotype 14 is emerging, and understanding of its pathogenesis is limited. To study the role of the capsular polysaccharide (CPS) of serotype 14 as a virulence factor, we constructed knockout mutants devoid of either cps14B, a highly conserved regulatory gene, or neu14C, a gene coding for uridine diphospho-N-acetylglucosamine 2-epimerase, which is involved in sialic acid synthesis. The mutants showed total loss of the CPS with coagglutination assays and electron microscopy. Phagocytosis assays showed high susceptibility of mutant Δcps14B. An in vivo murine model was used to demonstrate attenuated virulence of this non-encapsulated mutant. Despite the difference in the CPS composition of different serotypes, this study has demonstrated for the first time that the CPS of a serotype other than 2 is also an important antiphagocytic factor and a critical virulence factor.

  4. Synthesis of a Glucuronic Acid‐Containing Thioglycoside Trisaccharide Building Block and Its Use in the Assembly of Cryptococcus Neoformans Capsular Polysaccharide Fragments†

    PubMed Central

    Guazzelli, Lorenzo; Ulc, Rebecca

    2015-01-01

    Abstract As part of an ongoing project aimed at identifying protective capsular polysaccharide epitopes for the development of vaccine candidates against the fungal pathogen Cryptococcus neoformans, the synthesis and glycosylation properties of a naphthalenylmethyl (NAP) orthogonally protected trisaccharide thioglycoside, a common building block for construction of serotype B and C capsular polysaccharide structures, were investigated. Ethyl (benzyl 2,3,4‐tri‐O‐benzyl‐β‐d‐glucopyranosyl‐ uronate)‐(1→2)‐[2,3,4‐tri‐O‐benzyl‐β‐d‐xylopyranosyl‐(1→4)]‐6‐O‐benzyl‐3‐O‐(2‐naphthalenylmethyl)‐1‐thio‐α‐d‐mannopyranoside was prepared and used both as a donor and an acceptor in glycosylation reactions to obtain spacer equipped hexa‐ and heptasaccharide structures suitable either for continued elongation or for deprotection and printing onto a glycan array or conjugation to a carrier protein. The glycosylation reactions proceeded with high yields and α‐selectivity, proving the viability of the building block approach also for construction of 4‐O‐xylosyl‐containing C. neoformans CPS structures. PMID:27308199

  5. Reduction of Streptococcus pneumoniae Colonization and Dissemination by a Nonopsonic Capsular Polysaccharide Antibody.

    PubMed

    Doyle, Christopher R; Pirofski, Liise-anne

    2016-02-02

    Streptococcus pneumoniae colonization of the nasopharynx (NP) is a prerequisite for invasive pneumococcal disease (IPD). The marked reduction in IPD that followed the routine use of pneumococcal polysaccharide conjugate vaccines (PCVs) has been linked to reduced NP colonization with vaccine-included serotypes (STs), with the caveat that PCVs are less effective against pneumonia than against IPD. Although PCV-elicited opsonic antibodies that enhance phagocytic killing of the homologous ST are considered a key correlate of PCV-mediated protection, recent studies question this relationship for some STs, including ST3. Studies with monoclonal antibodies (MAbs) to the pneumococcal capsular polysaccharide (PPS) of ST3 (PPS3) have shown that nonopsonic, as well as opsonic, antibodies can each protect mice against pneumonia and sepsis, but the effect of these types of MAbs on NP colonization is unknown. In this study, we determined the effects of protective opsonic and nonopsonic PPS3 MAbs on ST3 NP colonization in mice. Our results show that a nonopsonic MAb reduced early NP colonization and prevented ST3 dissemination to the lungs and blood, but an opsonic MAb did not. Moreover, the opsonic MAb induced a proinflammatory NP cytokine response, but the nonopsonic MAb had an antiinflammatory effect. The effect of the nonopsonic MAb on colonization did not require its Fc region, but its antiinflammatory effect did. Our findings challenge the paradigm that opsonic MAbs are required to prevent NP colonization and suggest that further studies of the activity of nonopsonic antibodies could advance our understanding of mechanisms of PCV efficacy and provide novel correlates of protection. Pneumococcal conjugate vaccines (PCVs) have markedly reduced the incidence of invasive pneumococcal disease (IPD). Vaccine-elicited pneumococcal polysaccharide (PPS) antibodies that enhance in vitro phagocyte killing of vaccine-included serotypes (STs) (opsonic antibodies) have been considered

  6. Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent.

    PubMed Central

    Cywes, C; Hoppe, H C; Daffé, M; Ehlers, M R

    1997-01-01

    The choice of host cell receptor and the mechanism of binding (opsonic versus nonopsonic) may influence the intracellular fate of Mycobacterium tuberculosis. We have identified two substrains of M. tuberculosis H37Rv, designated H37Rv-CC and -HH, that differed in their modes of binding to complement receptor type 3 (CR3) expressed in transfected Chinese hamster ovary (CHO-Mac-1) cells: H37Rv-CC bound nonopsonically, whereas H37Rv-HH bound only after opsonization in fresh serum. H37Rv-CC also bound nonopsonically to untransfected CHO cells, whereas H37Rv-HH binding was enhanced by serum and was mediated by the 1D1 antigen, a bacterial adhesin previously identified as a polar phosphatidylinositol mannoside. H37Rv-CC and -HH had identical IS6110 DNA fingerprint patterns. Of five M. tuberculosis clinical isolates examined, four displayed the same binding phenotype as H37Rv-CC, as did the Erdman strain, whereas one isolate, as well as Mycobacterium smegmatis, behaved like H37Rv-HH. Nonopsonic binding of H37Rv-CC to CHO cell-expressed CR3 was apparently to the beta-glucan lectin site, as it was cation independent and inhibited by laminarin (seaweed beta-glucan) and N-acetylglucosamine; laminarin also inhibited the binding of H37Rv-CC to monocyte-derived macrophages. Further, binding of H37Rv-CC to CHO-Mac-1 cells was inhibited by prior agitation of bacteria with glass beads (which strips outer capsular polysaccharides) and by preincubation with amyloglucosidase, as well as by the presence of capsular D-glucan and D-mannan from M. tuberculosis Erdman, but not by Erdman D-arabino-D-mannan, yeast mannan, or capsular components from H37Rv-HH. Analysis of capsular carbohydrates revealed that H37Rv-CC expressed 5-fold more glucose and 2.5-fold more arabinose and mannose than H37Rv-HH. Flow cytometric detection of surface epitopes indicated that H37Rv-CC displayed twofold less surface-exposed phosphatidylinositol mannoside and bound complement C3 less efficiently than H37Rv

  7. Study of various presentation forms for a peptide mimetic of Neisseria meningitidis serogroup B capsular polysaccharide.

    PubMed

    Garay, Hilda; Menéndez, Tamara; Cruz-Leal, Yoelys; Coizeau, Edelgis; Noda, Jesus; Morera, Vivian; Guillén, Gerardo; Albericio, Fernando; Reyes, Osvaldo

    2011-01-19

    The formulation of a broadly protective vaccine to prevent the serogroup B Neisseria meningitidis (MenB) disease is still an unmet medical need. We have previously reported the induction of bactericidal and protective antibodies against MenB after immunization of mice with a phage-displayed peptide named 4 L-5. This peptide mimics a capsular polysaccharide (CPS) epitope in MenB. With the aim of developing vaccine formulations that could be used in humans, we evaluate in this study various forms of presentation to the immune system of the 4 L-5 sequence, based on synthetic peptides. We synthesized the following: (i) a linear 4 L-5 peptide, (ii) a multiple antigen peptide containing four copies of the 4 L-5 sequence (named MAP), which was then dimerized, and the product named dimeric MAP, and (iii) a second multiple antigen peptide, in this case with two copies of the 4 L-5 sequence and a copy of a T-helper cell epitope of tetanus toxoid, which was then dimerized and the product named MAP-TT. The linear peptide, the MAP, and the dimeric MAP were conjugated to the carrier protein P64K by different conjugation methods. Plain antigens and antigens coupled to P64K were used to immunize BALB/c mice. Of those variants that gave immunogenic results, MAP-TT rendered the highest levels of specific antipeptide IgG antibodies and serum bactericidal activity. These results can find application in the development of meningococcal vaccine candidates and in peptide-based vaccines strategies.

  8. RmpA Regulation of Capsular Polysaccharide Biosynthesis in Klebsiella pneumoniae CG43▿

    PubMed Central

    Cheng, H. Y.; Chen, Y. S.; Wu, C. Y.; Chang, H. Y.; Lai, Y. C.; Peng, H. L.

    2010-01-01

    Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5′ noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur. PMID:20382770

  9. Effectiveness of Vi capsular polysaccharide typhoid vaccine among children: a cluster randomized trial in Karachi, Pakistan.

    PubMed

    Khan, M Imran; Soofi, Sajid Bashir; Ochiai, R Leon; Habib, Mohammad Atif; Sahito, Shah Muhammad; Nizami, S Qamaruddin; Acosta, Camilo J; Clemens, John D; Bhutta, Zulfiqar A

    2012-08-03

    Typhoid fever is endemic in Karachi, with an incidence among children ranging from 170 to 450 per 100,000 child-years. Vaccination strategies are important for prevention, and the Vi capsular polysaccharide (ViCPS) vaccine has been shown to be effective in reducing the burden of typhoid fever. A cluster randomized trial was conducted in three low socioeconomic urban squatter settlements in Karachi, Pakistan between 2002 and 2007. Subsamples were followed up for assessment of immune response and adverse events after vaccination. The study participants were similar in a wide variety of socio-demographic and economic characteristics at baseline. A total of 27,231 individuals of the total target population of 51,965 in 120 clusters either received a ViCPS vaccine (13,238 [52% coverage]) or the control Hepatitis A vaccine (13,993 [53%]). Typhoid fever was diagnosed in 30 ViCPS vaccine recipients and 49 Hepatitis A vaccine recipients with an adjusted total protective effectiveness of 31% (95%CI: -28%, 63%). The adjusted total vaccine protective effectiveness was -38% (95%CI: -192%, 35%) for children aged 2-5 years and 57% (95%CI: 6%, 81%) for children 5-16 years old. The ViCPS vaccine did not confer statistically significant protection to children in the study areas, and there was a decline in antibody response 2 years post-vaccination. However, the ViCPS vaccine showed significant total protection in children 5-16 years of age, which is consistent with other studies of ViCPS vaccine conducted in India, Nepal, China and South Africa. These findings suggest that ViCPS vaccination of school-aged children will protect the children of urban, typhoid endemic areas against typhoid fever. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Cell growth rate regulates expression of group B Streptococcus type III capsular polysaccharide.

    PubMed Central

    Paoletti, L C; Ross, R A; Johnson, K D

    1996-01-01

    The capsular polysaccharide (CPS) of group B streptococci (GBS) is an important virulence factor that also serves to protect cells from nonspecific host defense mechanisms. Expression of CPS by GBS, as with other encapsulated bacterial pathogens, is not constitutive but varies during growth in vitro and in primary cultures isolated from different sites of infection. Despite this understanding, little is known about regulation of this surface-expressed carbohydrate antigen in GBS. Here we report that expression of type III CPS by GBS strain M781 grown in continuous culture with a modified chemically defined medium is regulated by growth rate. Cells in steady state at mass doubling times (tds) of 0.8, 1.4, and 1.6 h expressed an average of sixfold more cell-associated CPS than did cells held at tds of 2.3 and 11 h. Strain M781 grown at a td of 1.4 h repeatedly produced more type III CPS than those held at a td of 11.0 h, even when limited for glucose, pyridoxamine, or thiamine. In our studies, > or = 93% of the total CPS expressed by strain M781 was cell associated. Strain M781 grown at a td of 11.0 h (i.e., lowered CPS expression) was susceptible to in vitro complement-mediated opsonophagocytosis and killing by human peripheral blood leukocytes, whereas cells grown at a td of 1.4 h (i.e., higher CPS expression) were not killed unless type III CPS-specific antibody was present. Factors that allow GBS to asymptomatically colonize women yet cause invasive infection to both mother and infant are poorly understood. Our results shed new light on parameters that regulate the pathogenic potential of GBS and may also serve as a way to discern more fully the genetics and biochemistry of GBS capsule synthesis. PMID:8606082

  11. The efficacy of pneumococcal capsular polysaccharide-specific antibodies to serotype 3 Streptococcus pneumoniae requires macrophages.

    PubMed

    Fabrizio, Kevin; Manix, Catherine; Tian, Haijun; van Rooijen, Nico; Pirofski, Liise-anne

    2010-11-03

    The efficacy of antibody immunity against Streptococcus pneumoniae stems from the ability of opsonic, serotype (ST)-specific antibodies to pneumococcal capsular polysaccharide (PPS) to facilitate killing of the homologous ST by host phagocytes. However, PPS-specific antibodies have been identified that are protective in mice, but do not promote opsonic killing in vitro, raising the question of how they mediate protection in vivo. To probe this question, we investigated the dependence of antibody efficacy against lethal systemic (intraperitoneal, i.p.) infection with Streptococcus pneumoniae serotype 3 (ST3) on macrophages and neutrophils for the following PPS3-specific monoclonal antibodies (MAbs) in survival experiments in mice using a non-opsonic human IgM (A7), a non-opsonic mouse IgG1 (1E2) and an opsonic mouse IgG1 (5F6). The survival of A7- and PPS3-specific and isotype control MAb-treated neutrophil-depleted and neutrophil-sufficient and macrophage-depleted and macrophage-sufficient mice were determined after i.p. challenge with ST3 strains 6303 and WU2. Neutrophils were dispensable for A7 and the mouse MAbs to mediate protection in this model, but macrophages were required for the efficacy of A7 and optimal mouse MAb-mediated protection. For A7-treated mice, macrophage-depleted mice had higher blood CFU, cytokines and peripheral neutrophil levels than macrophage-sufficient mice, and macrophage-sufficient mice had lower tissue bacterial burdens than control MAb-treated mice. These findings demonstrate that macrophages contribute to opsonic and non-opsonic PPS3-specific MAb-mediated protection against ST3 infection by enhancing bacterial clearance and suggest that neutrophils do not compensate for the absence of macrophages in the model used in this study.

  12. A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection.

    PubMed

    Fleuridor, R; Lees, A; Pirofski, L

    2001-01-15

    Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens.

  13. Therapeutic efficacy of a conjugate vaccine containing a peptide mimotope of cryptococcal capsular polysaccharide glucuronoxylomannan.

    PubMed

    Datta, Kausik; Lees, Andrew; Pirofski, Liise-anne

    2008-08-01

    Vaccination with P13, a peptide mimotope of the cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), has been shown to confer protection against a subsequent lethal Cryptococcus neoformans challenge. In this study, we sought to investigate whether P13-based vaccines could be effective in an already-established infection. To address this question, we developed a systemic chronic cryptococcal infection model. We vaccinated chronically infected mice with P13-protein conjugates and monitored their survival. Compared to the controls, the conjugates prolonged the survival of chronically infected mice. The degree of protection was a function of the mouse strain (BALB/c or C57BL/6), the carrier protein (tetanus toxoid or diphtheria toxoid), and the route of infection (intraperitoneal or intravenous). Serum GXM levels were correlated with the day of death, but the correlation was driven by the carrier protein and mouse strain. The passive transfer of heat-treated sera from P13 conjugate-vaccinated mice conferred protection to naïve BALB/c mice, indicating that antibody immunity could contribute to protection. The measurement of peripheral blood cytokine (gamma interferon [IFN-gamma], interleukin-10 [IL-10], and IL-6) gene expression showed that P13 conjugate-vaccinated BALB/c and C57BL/6 mice mounted a strong Th2 (IL-10)-like response relative to the Th1 (IFN-gamma)-like response, with the degree depending on the mouse strain and carrier protein. Taken together, our data suggest that a vaccine could hold promise in the setting of chronic cryptococcosis, and that vaccine efficacy could depend on immunomodulation and augmentation of the natural immune response of the host.

  14. Protection against Streptococcus suis Serotype 2 Infection Using a Capsular Polysaccharide Glycoconjugate Vaccine

    PubMed Central

    Calzas, Cynthia; Shiao, Tze Chieh; Neubauer, Axel; Kempker, Jennifer; Roy, René; Gottschalk, Marcelo

    2016-01-01

    Streptococcus suis serotype 2 is an encapsulated bacterium and one of the most important bacterial pathogens in the porcine industry. Despite decades of research for an efficient vaccine, none is currently available. Based on the success achieved with other encapsulated pathogens, a glycoconjugate vaccine strategy was selected to elicit opsonizing anti-capsular polysaccharide (anti-CPS) IgG antibodies. In this work, glycoconjugate prototypes were prepared by coupling S. suis type 2 CPS to tetanus toxoid, and the immunological features of the postconjugation preparations were evaluated in vivo. In mice, experiments evaluating three different adjuvants showed that CpG oligodeoxyribonucleotide (ODN) induces very low levels of anti-CPS IgM antibodies, while the emulsifying adjuvants Stimune and TiterMax Gold both induced high levels of IgGs and IgM. Dose-response trials comparing free CPS with the conjugate vaccine showed that free CPS is nonimmunogenic independently of the dose used, while 25 μg of the conjugate preparation was optimal in inducing high levels of anti-CPS IgGs postboost. With an opsonophagocytosis assay using murine whole blood, sera from immunized mice showed functional activity. Finally, the conjugate vaccine showed immunogenicity and induced protection in a swine challenge model. When conjugated and administered with emulsifying adjuvants, S. suis type 2 CPS is able to induce potent IgM and isotype-switched IgGs in mice and pigs, yielding functional activity in vitro and protection against a lethal challenge in vivo, all features of a T cell-dependent response. This study represents a proof of concept for the potential of glycoconjugate vaccines in veterinary medicine applications against invasive bacterial infections. PMID:27113360

  15. Hydrolytic stability of pneumococcal group 6 (type 6A and 6B) capsular polysaccharides.

    PubMed Central

    Zon, G; Szu, S C; Egan, W; Robbins, J D; Robbins, J B

    1982-01-01

    The hydrolyses of the immunologically cross-reactive and constitutionally isomeric group 6 pneumococcal polysaccharides, types 6A and 6B, were investigated by 31P nuclear magnetic resonance spectroscopy, gel filtration through Sepharose 4B, reducing-sugar analysis, and rocket immunoelectrophoresis. Phosphorus nuclear magnetic resonance spectroscopy showed that cleavage of the repeating-unit phosphodiester linkages at pH 10, 60 degrees C was considerably faster (greater than 10(3) ) for the type 6A than the type 6B polysaccharide. Under these reaction conditions, 31P nuclear magnetic resonance kinetic measurements showed that the Na+ form of the type 6A polysaccharide underwent phosphodiester-linkage hydrolysis two times slower than the corresponding Ca+2 form; a stoichiometrically excess amount of Ca+2 caused a 30-fold enhancement of the latter hydrolysis rate. The spectroscopic characterization of phosphorus-containing end groups resulting from hydrolysis of the type 6A polymer provided additional mechanistic information. Heating the type 6A and 6B polysaccharides at 56 degrees C for various times led to gel filtration coefficients of distribution (Kd values) which indicated that the type 6A material underwent size reductions considerably faster than did the type 6B antigen; these increased Kd values qualitatively correlated with the loss of immunochemical reactivity measured by rocket immunoelectrophoresis. The application of a statistical theory to the depolymerization of the type 6A and 6B polysaccharides was consistent with random bond cleavage, as evidenced by the calculated versus measured gel filtration patterns. Although the molecular changes causing the size reductions were not fully elaborated, it was established that the acetal linkages of the type 6A and 6B polysaccharides were comparatively resistant to hydrolysis and that depolymerization by hydrolysis of the phosphodiester linkage was a major factor only in the type 6A structure. It was concluded

  16. Hydrolytic stability of pneumococcal group 6 (type 6A and 6B) capsular polysaccharides.

    PubMed

    Zon, G; Szu, S C; Egan, W; Robbins, J D; Robbins, J B

    1982-07-01

    The hydrolyses of the immunologically cross-reactive and constitutionally isomeric group 6 pneumococcal polysaccharides, types 6A and 6B, were investigated by 31P nuclear magnetic resonance spectroscopy, gel filtration through Sepharose 4B, reducing-sugar analysis, and rocket immunoelectrophoresis. Phosphorus nuclear magnetic resonance spectroscopy showed that cleavage of the repeating-unit phosphodiester linkages at pH 10, 60 degrees C was considerably faster (greater than 10(3) ) for the type 6A than the type 6B polysaccharide. Under these reaction conditions, 31P nuclear magnetic resonance kinetic measurements showed that the Na+ form of the type 6A polysaccharide underwent phosphodiester-linkage hydrolysis two times slower than the corresponding Ca+2 form; a stoichiometrically excess amount of Ca+2 caused a 30-fold enhancement of the latter hydrolysis rate. The spectroscopic characterization of phosphorus-containing end groups resulting from hydrolysis of the type 6A polymer provided additional mechanistic information. Heating the type 6A and 6B polysaccharides at 56 degrees C for various times led to gel filtration coefficients of distribution (Kd values) which indicated that the type 6A material underwent size reductions considerably faster than did the type 6B antigen; these increased Kd values qualitatively correlated with the loss of immunochemical reactivity measured by rocket immunoelectrophoresis. The application of a statistical theory to the depolymerization of the type 6A and 6B polysaccharides was consistent with random bond cleavage, as evidenced by the calculated versus measured gel filtration patterns. Although the molecular changes causing the size reductions were not fully elaborated, it was established that the acetal linkages of the type 6A and 6B polysaccharides were comparatively resistant to hydrolysis and that depolymerization by hydrolysis of the phosphodiester linkage was a major factor only in the type 6A structure. It was concluded

  17. Synthesis and conformational analysis of a simplified inositol-model of the Streptococcus pneumoniae 19F capsular polysaccharide repeating unit.

    PubMed

    Catelani, Giorgio; D'Andrea, Felicia; Guazzelli, Lorenzo; Griselli, Alessio; Testi, Nicola; Chiacchio, Maria Assunta; Legnani, Laura; Toma, Lucio

    2017-04-18

    Carbohydrate mimics have been studied for a long time as useful sugar substitutes, both in the investigation of biological events and in the treatment of sugar-related diseases. Here we report further evaluation of the capabilities of inositols as carbohydrate substitutes. The conformational features of an inositol-model of a simplified repeating unit corresponding to the capsular polysaccharide of Streptococcus pneumoniae 19F has been evaluated by computational analysis, and compared to the native repeating unit. The inositol mimic was synthesized, and its experimental spectroscopic data allowed for verification of the theoretical results. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Type 5 and 8 capsular polysaccharides are expressed by Staphylococcus aureus isolates from rabbits, poultry, pigs, and horses.

    PubMed Central

    Poutrel, B; Sutra, L

    1993-01-01

    A total of 103 Staphylococcus aureus isolates from rabbits (n = 37), poultry (n = 33), pigs (n = 27), and horses (n = 6) and 14 Staphylococcus intermedius isolates from wild animals were serotyped for capsular polysaccharide types 5 and 8 by an enzyme-linked immunosorbent assay using polyclonal rabbit antibodies. About 98% of the S. aureus isolates were typeable. Type 5 was predominant in the poultry (75.8%) and pig (66.7%) isolates, whereas type 8 was more frequent among the isolates from rabbits (59.5%) and horses (83.3%). By contrast, none of the 14 S. intermedius isolates was typeable. PMID:8432841

  19. Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni.

    PubMed

    Sahin, Orhan; Terhorst, Samantha A; Burrough, Eric R; Shen, Zhangqi; Wu, Zuowei; Dai, Lei; Tang, Yizhi; Plummer, Paul J; Ji, Ju; Yaeger, Michael J; Zhang, Qijing

    2017-06-01

    Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni. Copyright © 2017 American Society for Microbiology.

  20. Cryptococcus neoformans capsular polysaccharides form branched and complex filamentous networks viewed by high-resolution microscopy.

    PubMed

    Araújo, Glauber R de S; Fontes, Giselle N; Leão, Daniela; Rocha, Gustavo Miranda; Pontes, Bruno; Sant'Anna, Celso; de Souza, Wanderley; Frases, Susana

    2016-01-01

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. Its main virulence factor is an extracellular polysaccharide capsule whose structure, assembly and dynamics remain poorly understood. In this study, we apply improved protocols for sample preparation and recently-developed scanning microscopy techniques to visualize the ultrastructure of the C. neoformans capsule at high-resolution (up to 1 nm) and improved structural preservation. Although most capsule structures in nature consist of linear polymers, we show here that the C. neoformans capsule is a 'microgel-like' structure composed of branched polysaccharides. Moreover, we imaged the capsule-to-cell wall link, which is formed by thin fibers that branch out of thicker capsule filaments, and have one end firmly embedded in the cell wall structure. Together, our findings provide compelling ultrastructural evidence for a branched and complex capsule conformation, which may have important implications for the biological activity of the capsule as a virulence factor. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Levels of Antibodies Specific to Tetanus Toxoid, Haemophilus influenzae Type b, and Pneumococcal Capsular Polysaccharide in Healthy Children and Adults

    PubMed Central

    Schauer, Uwe; Stemberg, Frank; Rieger, Christian H. L.; Büttner, Wolfgang; Borte, Michael; Schubert, Simone; Möllers, Helga; Riedel, Frank; Herz, Udo; Renz, Harald; Herzog, Wilhelm

    2003-01-01

    Antibody levels specific for capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) and for tetanus toxoid were measured in serum samples of 386 age-stratified subjects. The study group consists of healthy adult blood donors and hospitalized children undergoing elective surgery, excluding individuals with a history of infection. In children, anti-tetanus toxoid antibody levels displayed two peaks of 1.20 IU/ml (20.4 mg/liter) and 1.65 IU/ml (28.1 mg/liter) related to the schedule of routine childhood immunization in the first year and at 8 years of age. Eighty percent of the antibodies are of the immunoglobulin G1 (IgG1) isotype. For pneumococcal capsular polysaccharide (PCP), the specific antibody levels represent the acquisition of natural immunity. The initial concentration of 9.2 mg/liter was low in infancy (0.5 to 1 years of age) and remained low until 3 to 4 years of age (14.6 mg/liter). During this period PCP antibodies were almost 100% of the IgG2 subclass. Thereafter, IgG anti-PCP antibody titers increased steadily to adult levels (59.5 mg/liter). The data are intended to provide reference ranges to aid in the interpretation of specific antibody determinations in the clinical setting. PMID:12626443

  2. Levels of antibodies specific to tetanus toxoid, Haemophilus influenzae type b, and pneumococcal capsular polysaccharide in healthy children and adults.

    PubMed

    Schauer, Uwe; Stemberg, Frank; Rieger, Christian H L; Büttner, Wolfgang; Borte, Michael; Schubert, Simone; Möllers, Helga; Riedel, Frank; Herz, Udo; Renz, Harald; Herzog, Wilhelm

    2003-03-01

    Antibody levels specific for capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) and for tetanus toxoid were measured in serum samples of 386 age-stratified subjects. The study group consists of healthy adult blood donors and hospitalized children undergoing elective surgery, excluding individuals with a history of infection. In children, anti-tetanus toxoid antibody levels displayed two peaks of 1.20 IU/ml (20.4 mg/liter) and 1.65 IU/ml (28.1 mg/liter) related to the schedule of routine childhood immunization in the first year and at 8 years of age. Eighty percent of the antibodies are of the immunoglobulin G1 (IgG1) isotype. For pneumococcal capsular polysaccharide (PCP), the specific antibody levels represent the acquisition of natural immunity. The initial concentration of 9.2 mg/liter was low in infancy (0.5 to 1 years of age) and remained low until 3 to 4 years of age (14.6 mg/liter). During this period PCP antibodies were almost 100% of the IgG2 subclass. Thereafter, IgG anti-PCP antibody titers increased steadily to adult levels (59.5 mg/liter). The data are intended to provide reference ranges to aid in the interpretation of specific antibody determinations in the clinical setting.

  3. Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a.

    PubMed Central

    Ward, C K; Inzana, T J

    1997-01-01

    Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens. PMID:9169799

  4. Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in Staphylococcus aureus isolated from bovine milk collected from Brazilian dairy farms.

    PubMed

    Salimena, Alessandra P S; Lange, Carla C; Camussone, Cecilia; Signorini, Marcelo; Calvinho, Luis F; Brito, Maria A V P; Borges, Cristiano A V; Guimarães, Alessandro S; Ribeiro, João B; Mendonça, Letícia C; Piccoli, Roberta H

    2016-12-01

    Staphylococcus aureus is a pathogen that frequently causes mastitis in bovine herds worldwide. This pathogen produces several virulence factors, including cell-associated adhesins, toxic and cytolytic exoproteins, and capsular polysaccharides. The aim of the present study was to test for the presence of genes involved in capsular polysaccharide production and biofilm formation in S. aureus isolated from bovine mastitis samples collected from 119 dairy herds located in three different Brazilian regions, as well as to assay the production of capsular polysaccharides and biofilm, in vitro. The detection of the cap, icaAD, and bap genes was performed using PCR. The detection and quantification of capsular polysaccharide production was performed using ELISA assays. The ability of the isolates to form a biofilm was examined using the polystyrene surface of microtiter plates. All 159 S. aureus isolates investigated harboured the cap gene: 80 % carried the cap5 gene and 20 % carried the cap8 gene. Sixty-nine percent of the isolates expressed capsular polysaccharide (CP) in vitro, 58 % expressed CP5 and 11 % expressed CP8. All of the isolates harboured the icaA and icaD genes, and 95.6 % of the isolates carried the bap gene. Of the 159 isolates analysed, 97.5 % were biofilm producers. A significant association between the capsular genotype and phenotype and the amount of biofilm formation was detected: cap5/CP5 isolates tended to form more biofilm and to produce a thinner CP layer than cap8/CP8 isolates. The results indicate a high potential for pathogenicity among S. aureus isolated from bovine milk collected from three different regions in Brazil.

  5. The contribution of naturally occurring IgM antibodies, IgM cross-reactivity and complement dependency in murine humoral responses to pneumococcal capsular polysaccharides.

    PubMed

    Jones, Hannah E; Taylor, Philip R; McGreal, Eamon; Zamze, Susanne; Wong, Simon Y C

    2009-09-25

    Immunogenicity of 12 capsular polysaccharides (CPS) from Streptococcus pneumoniae did not correlate with pre-existing levels of natural IgM anti-CPS antibodies in mice. Immunization of mice with individual CPS, with the exception of type 14 (the only neutral CPS tested), increased serum IgM that also bound other CPS serotypes independent of structural similarity or commonly known contaminants. Surprisingly only IgM response to type 4 (which has a small immunodominant epitope) was dependent on either complement C3 or complement receptors CD35/CD21. IgG anti-CPS responses were infrequently induced, but critically dependent on complement. Our results have clarified the role of complement in the induction of IgM and IgG anti-CPS antibody responses in mice and have implications for CPS vaccine development.

  6. Recombinant Escherichia coli K5 strain with the deletion of waaR gene decreases the molecular weight of the heparosan capsular polysaccharide.

    PubMed

    Huang, Haichan; Liu, Xiaobo; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

    2016-09-01

    Heparosan, the capsular polysaccharide of Escherichia coli K5 having a carbohydrate backbone similar to that of heparin, has become a potential precursor for bioengineering heparin. In the heparosan biosynthesis pathway, the gene waaR encoding α-1-, 2- glycosyltransferase catalyze s the third glucosyl residues linking to the oligosaccharide chain. In the present study, a waaR deletion mutant of E. coli K5 was constructed. The mutant showed improvement of capsule polysaccharide yield. It is interesting that the heparosan molecular weight of the mutant is reduced and may become more suitable as a precursor for the production of low molecular weight heparin derived from the wild-type K5 capsular polysaccharide.

  7. Genotyping and investigating capsular polysaccharide synthesis gene loci of non-serotypeable Streptococcus suis isolated from diseased pigs in Canada.

    PubMed

    Zheng, Han; Qiu, Xiaotong; Roy, David; Segura, Mariela; Du, Pengchen; Xu, Jianguo; Gottschalk, Marcelo

    2017-02-20

    Streptococcus suis (S. suis) is an important swine pathogen and an emerging zoonotic agent. Most clinical S. suis strains express capsular polysaccharides (CPS), which can be typed by antisera using the coagglutination test. In this study, 79 S. suis strains recovered from diseased pigs in Canada and which could not be typed using antisera were further characterized by capsular gene typing and sequencing. Four patterns of cps locus were observed: (1) fifteen strains were grouped into previously reported serotypes but presented several mutations in their cps loci, when compared to available data from reference strains; (2) seven strains presented a complete deletion of the cps locus, which would result in an inability to synthesize capsule; (3) forty-seven strains were classified in recently described novel cps loci (NCLs); and (4) ten strains carried novel NCLs not previously described. Different virulence gene profiles (based on the presence of mrp, epf, and/or sly) were observed in these non-serotypeable strains. This study provides further insight in understanding the genetic characteristics of cps loci in non-serotypeable S. suis strains recovered from diseased animals. When using a combination of the previously described 35 serotypes and the complete NCL system, the number of untypeable strains recovered from diseased animals in Canada would be significantly reduced.

  8. Antibodies to Staphylococcus aureus capsular polysaccharides 5 and 8 perform similarly in vitro but are functionally distinct in vivo.

    PubMed

    Liu, Bo; Park, Saeyoung; Thompson, Christopher D; Li, Xue; Lee, Jean C

    2016-12-09

    The capsular polysaccharide (CP) produced by Staphylococcus aureus is a virulence factor that allows the organism to evade uptake and killing by host neutrophils. Polyclonal antibodies to the serotype 5 (CP5) and type 8 (CP8) capsular polysaccharides are opsonic and protect mice against experimental bacteremia provoked by encapsulated staphylococci. Thus, passive immunotherapy using CP antibodies has been considered for the prevention or treatment of invasive antibiotic-resistant S. aureus infections. In this report, we generated monoclonal antibodies (mAbs) against S. aureus CP5 or CP8. Backbone specific mAbs reacted with native and O-deacetylated CPs, whereas O-acetyl specific mAbs reacted only with native CPs. Reference strains of S. aureus and a selection of clinical isolates reacted by colony immunoblot with the CP5 and CP8 mAbs in a serotype-specific manner. The mAbs mediated in vitro CP type-specific opsonophagocytic killing of S. aureus strains, and mice passively immunized with CP5 mAbs were protected against S. aureus bacteremia. Neither CP8-specific mAbs or polyclonal antibodies protected mice against bacteremia provoked by serotype 8 S. aureus clinical isolates, although these same antibodies did protect against a serotype 5 S. aureus strain genetically engineered to produce CP8. We detected soluble CP8 in culture supernatants of serotype 8 clinical isolates and in the plasma of infected animals. Serotype 5 S. aureus released significantly less soluble CP5 in vitro and in vivo. The release of soluble CP8 by S. aureus may contribute to the inability of CP8 vaccines or antibodies to protect against serotype 8 staphylococcal infections.

  9. Immunogenicity and efficacy of Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan peptide mimotope-protein conjugates in human immunoglobulin transgenic mice.

    PubMed

    Maitta, Robert W; Datta, Kausik; Lees, Andrew; Belouski, Shelley Sims; Pirofski, Liise-anne

    2004-01-01

    Peptide mimotopes of capsular polysaccharides have been proposed as antigens for vaccines against encapsulated pathogens. In this study, we determined the antibody response to and efficacy of P13, a peptide mimetic of the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM), in mice that produce human antibodies. P13 was conjugated to tetanus toxoid (TT) or diphtheria toxoid (DT) and administered subcutaneously in Alhydrogel with or without CpG to mice transgenic for human immunoglobulin loci (XenoMouse mice) and expressing either immunoglobulin G2 (IgG2) (G2 mice) or IgG4 (G4 mice). Mice were vaccinated and revaccinated two or three times. The serum antibody responses of the mice to GXM and P13 and antibody idiotype expression were analyzed by an enzyme-linked immunosorbent assay. The results showed that both P13-TT and P13-DT were antigenic, inducing a mimetic response to P13 in both G2 and G4 mice, and immunogenic, inducing a mimotope response including VH3 (idiotype)-positive antibodies to GXM in G2 but not G4 mice. CpG led to higher titers of IgG to P13 and GXM in P13-TT-vaccinated G2 mice. C. neoformans challenge of P13-protein conjugate-vaccinated and control G2 mice induced anamnestic IgG- and VH3-positive responses to GXM and was associated with a significantly decreased risk of death and a prolongation of survival in P13-DT-vaccinated mice compared to phosphate-buffered saline-treated or protein carrier-vaccinated mice. These findings reveal that P13 elicited a human antibody response with VH3 expression in human immunoglobulin transgenic mice that has been observed for human antibodies to GXM and support the concept that peptide mimotope-based vaccines may hold promise for the treatment of C. neoformans infections.

  10. Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats.

    PubMed

    TerAvest, Michaela A; He, Zhen; Rosenbaum, Miriam A; Martens, Eric C; Cotta, Michael A; Gordon, Jeffrey I; Angenent, Largus T

    2014-01-01

    Bacteroides thetaiotaomicron is a prominent member of the human distal gut microbiota that specializes in breaking down diet and host-derived polysaccharides. While polysaccharide utilization has been well studied in B. thetaiotaomicron, other aspects of its behavior are less well characterized, including the factors that allow it to maintain itself in the gut. Biofilm formation may be a mechanism for bacterial retention in the gut. Therefore, we used custom GeneChips to compare the transcriptomes of biofilm and planktonic B. thetaiotaomicron during growth in mono-colonized chemostats. We identified 1,154 genes with a fold-change greater than 2, with confidence greater than or equal to 95%. Among the prominent changes observed in biofilm populations were: (i) greater expression of genes in polysaccharide utilization loci that are involved in foraging of O-glycans normally found in the gut mucosa; and (ii) regulated expression of capsular polysaccharide biosynthesis loci. Hierarchical clustering of the data with different datasets, which were obtained during growth under a range of conditions in minimal media and in intestinal tracts of gnotobiotic mice, revealed that within this group of differentially expressed genes, biofilm communities were more similar to the in vivo samples than to planktonic cells and exhibited features of substrate limitation. The current study also validates the use of chemostats as an in vitro "gnotobiotic" model to study gene expression of attached populations of this bacterium. This is important to gut microbiota research, because bacterial attachment and the consequences of disruptions in attachment are difficult to study in vivo. © 2013 Wiley Periodicals, Inc.

  11. Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats

    PubMed Central

    TerAvest, Michaela A.; He, Zhen; Rosenbaum, Miriam A.; Martens, Eric C.; Cotta, Michael A.; Gordon, Jeffrey I.; Angenent, Largus T.

    2014-01-01

    Bacteroides thetaiotaomicron is a prominent member of the human distal gut microbiota that specializes in breaking down diet and host-derived polysaccharides. While polysaccharide utilization has been well studied in B. thetaiotaomicron, other aspects of its behavior are less well characterized, including the factors that allow it to maintain itself in the gut. Biofilm formation may be a mechanism for bacterial retention in the gut. Therefore, we used custom GeneChips to compare the transcriptomes of biofilm and planktonic B. thetaiotaomicron during growth in mono-colonized chemostats. We identified 1154 genes with a fold-change greater than 2, with confidence greater than or equal to 95%. Among the prominent changes observed in biofilm populations were: (i) greater expression of genes in polysaccharide utilization loci that are involved in foraging of O-glycans normally found in the gut mucosa; and (ii) regulated expression of capsular polysaccharide biosynthesis loci. Hierarchical clustering of the data with different datasets, which were obtained during growth under a range of conditions in minimal media and in intestinal tracts of gnotobiotic mice, revealed that within this group of differentially expressed genes, biofilm communities were more similar to the in vivo samples than to planktonic cells and exhibited features of substrate limitation. The current study also validates the use of chemostats as an in vitro ‘gnotobiotic’ model to study gene expression of attached populations of this bacterium. This is important to gut microbiota research, because bacterial attachment and the consequences of disruptions in attachment are difficult to study in vivo. PMID:23996813

  12. Receptor-mediated clearance of Cryptococcus neoformans capsular polysaccharide in vivo.

    PubMed

    Yauch, Lauren E; Mansour, Michael K; Levitz, Stuart M

    2005-12-01

    Cryptococcus neoformans capsular glucuronoxylomannan (GXM) is shed during cryptococcosis and taken up by macrophages. The roles of the putative GXM receptors CD14, CD18, Toll-like receptor 2 (TLR2), and TLR4 in GXM clearance from serum and deposition in the liver and spleen in receptor-deficient mice were studied. While alterations in the kinetics of GXM redistribution were seen in the mutant mice, none of the receptors was absolutely required for serum clearance or hepatosplenic accumulation.

  13. Neisseria meningitidis group B capsular polysaccharide. Bacterial content and release in relation to categories of infection and filtrability of released endotoxin.

    PubMed

    Hoddevik, G M; Baastad, K L; Bukholm, G; Bryn, K; Bøvre, K

    1990-06-01

    A total of 41 Neissera meningitidis isolates were analyzed for capsular polysaccharide (CP) and lipopolysaccharide (LPS) release and filtrability. Twenty-two of these isolates were serogroup B from blood or cerebrospinal fluid of patients, five were group B isolates from healthy throat carriers, and 14 were nongroupable (acapsular) meningococcal isolates from healthy carriers. Filtration of liquid whole-cell cultures through cellulose acetate-nitrate filters resulted in distinctly lower LPS filtrate activity for acapsular than for capsular meningococci (p less than 0.001). On the other hand, when polysulfone membrane filtration was performed, filtrates from acapsular and capsular meningococci contained LPS in similar amounts. These results indicate that LPS-containing particles released from acapsular isolates are larger or more aggregated than corresponding CP- and LPS-containing particles released from capsular isolates. The LPS released from acapsular isolates apparently are more efficiently retained by adsorption to cellulose acetate-nitrate. From the capsular isolates comparatively more CP than LPS appeared to be released, as related to cell-bound amounts. The total amounts of CP and released, filtrable LPS through cellulose acetate-nitrate filters were both relatively low and had similar values for capsular carrier meningococci and systemic isolates from mild meningococcal disease. The amount of CP released and passing this type of membrane was significantly higher for systemic isolates from severe septicemia than from mild meningococcal disease (p = 0.03).

  14. Gene content and diversity of the loci encoding biosynthesis of capsular polysaccharides of the 15 serovar reference strains of Haemophilus parasuis.

    PubMed

    Howell, Kate J; Weinert, Lucy A; Luan, Shi-Lu; Peters, Sarah E; Chaudhuri, Roy R; Harris, David; Angen, Oystein; Aragon, Virginia; Parkhill, Julian; Langford, Paul R; Rycroft, Andrew N; Wren, Brendan W; Tucker, Alexander W; Maskell, Duncan J

    2013-09-01

    Haemophilus parasuis is the causative agent of Glässer's disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3' end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer's disease.

  15. Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

    PubMed Central

    Weinert, Lucy A.; Luan, Shi-Lu; Peters, Sarah E.; Chaudhuri, Roy R.; Harris, David; Angen, Øystein; Aragon, Virginia; Parkhill, Julian; Langford, Paul R.; Rycroft, Andrew N.; Wren, Brendan W.; Tucker, Alexander W.; Maskell, Duncan J.

    2013-01-01

    Haemophilus parasuis is the causative agent of Glässer's disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3′ end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer's disease. PMID:23873912

  16. Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide.

    PubMed

    Mawas, Fatme; Bolgiano, Barbara; Rigsby, Peter; Crane, Dennis; Belgrave, Danielle; Corbel, Michael J

    2007-10-01

    Haemophilus influenzae b conjugate vaccines (Hib) are almost entirely evaluated by physico-chemical methods to ensure the consistency of manufacture of batches. As different assays are employed for the quantification of Hib capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(1-->1)-beta-D-ribose-3-phosphate) in final formulations and bulk components, there was deemed a need for an International Standard of Hib PRP polysaccharide to be made available. Ten laboratories from 8 different countries participated in a collaborative study to determine the PRP content and assess the suitability of a candidate International Standard PRP preparation (02/208). The results illustrate that a reduction in between-laboratory variability could be achieved by use of a common reference preparation and data analysis showed no significant differences in the values obtained by the different assays: ribose, phosphorus, and high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), suggesting the suitability of the proposed reference for use across these assays for quantification of PRP content in Hib vaccines. On the basis of the results of this study, the First International Standard for PRP, NIBSC Code 02/208, has been established by the Expert Committee of Biological Standards of the World Health Organisation, with a content of 4.933+/-0.267mg/ampoule, as determined by the ribose assays carried out by 7 of the participating laboratories.

  17. Variable Region Identical IgA and IgE to Cryptococcus neoformans Capsular Polysaccharide Manifest Specificity Differences*

    PubMed Central

    Janda, Alena; Eryilmaz, Ertan; Nakouzi, Antonio; Pohl, Mary Ann; Bowen, Anthony; Casadevall, Arturo

    2015-01-01

    In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affect the affinity and specificity of antibodies sharing identical variable (V) regions. However, whether the same applies to IgE is unknown. In this study we compared the fine specificity of V region-identical IgE and IgA to Cryptococcus neoformans capsular polysaccharide and found that these differed in specificity from each other. The IgE and IgA paratopes were probed by nuclear magnetic resonance spectroscopy with 15N-labeled peptide mimetics of cryptococcal polysaccharide antigen (Ag). IgE was found to cleave the peptide at a much faster rate than V region-identical IgG subclasses and IgA, consistent with an altered paratope. Both IgE and IgA were opsonic for C. neoformans and protected against infection in mice. In summary, V-region expression in the context of the ϵ constant (C) region results in specificity changes that are greater than observed for comparable IgG subclasses. These results raise the possibility that expression of certain V regions in the context of α and ϵ C regions affects their function and contributes to the special properties of those isotypes. PMID:25778397

  18. A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines.

    PubMed

    Reyes, Fátima; Otero, Oscar; Camacho, Frank; Amin, Nevis; Ramírez, Fidel; Valdés, Yolanda; Acevedo, Reynaldo; García, Luis; Cardoso, Daniel; Cuello, Maribel

    2014-11-01

    A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10(7) M(-1). The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  19. Killing of cryptococcus neoformans by Staphylococcus aureus: the role of cryptococcal capsular polysaccharide in the fungal-bacteria interaction.

    PubMed

    Saito, Fumito; Ikeda, Reiko

    2005-11-01

    Microbes compete for the environmental niche which is their host. To investigate the effects of a pathogenic bacterium on invasion and colonization by a pathogenic yeast, Cryptococcus neoformans was co-cultured with Staphylococcus aureus. We found that the number of colony forming units of C. neoformans was decreased by Staphylococcus aureus. In contrast, the viability of Candida albicans was not affected. Under the microscope, wild-type C. neoformans cells were shown to be surrounded by S. aureus, while cells of a capsuleless mutant of C. neoformans were not. C. neoformans was not killed when a membrane separated it from S. aureus in co-culture. Killing was confirmed by staining with cyanoditolyl tetrazolium chloride: S. aureus stained red, indicating viability, while C. neojormans did not stain, indicating lethality. The in situ terminal deoxynucleotidyl transferase-mediated dUTR nick end labeling (TUNEL) assay indicated cell death with fragmentation of DNA of C. neoformans. Capsular polysaccharide from C. neoformans inhibited the killing. Treatment of the crude polysaccharide with protease increased the inhibition. The protective activity resided in the glucuronoxylomannan (GXM) fraction, although the concentration required for the inhibition was high. These results suggest that S. aureus kills C. neoformans by a process that involves attachment to the cryptococcal capsule.

  20. Capsular polysaccharide typing of domestic mastitis-causing Staphylococcus aureus strains and its potential exploration of bovine mastitis vaccine development. I. Capsular polysaccharide typing, isolation and purification of the strains.

    PubMed

    Han, H R; Pak, S I; Kang, S W; Jong, W S; Youn, C J

    2000-06-01

    One hundred seven isolates of Staphylococcus aureus from bovine mastitis were investigated for colony morphology in serum-soft agar (SSA), autoagglutination in salt, and capsular serotype. Capsular polysaccharide (CP) was purified and quantified from the extracts of clinical isolates. Overall, 89 isolates (83.2%) were diffuse in the SSA, without any difference in the proportion of diffuse colony between type 5 and type 8 strains. Some strains exhibited compact colonies in the SSA and expressed CP as determined by an enzyme-linked immunosorbent assay, indicating that compact morphology does not exclude encapsulation. The majority of the strains (11/12) showed autoagglutination in the salt aggregation test. The serotype 336 accounted for 46.7% of the isolates followed by serotype 5 (12.1%) and serotype 8 (12.1%). Particularly, twenty-six (24.3%) isolates reacted with two serotypes; 7 for type 8/336 and 19 for type 5/336. Five isolates (4.7%) were nontypeable with monoclonal antibodies specific for CP serotype 5, 8, or 336. The CP concentration in culture supernatants varied with the serotypes, and the total amount of CP produced by cells grown in a liquid medium was much less than that produced by cells grown on a solid medium. The Western blotting indicated that the CP bands of S. aureus serotype 5 and 8 were ranged in the molecular mass of 58-84 kilodalton (kDa), with additional bands in the region of approximately >/= 48 or

  1. Homologous overexpression of RfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide.

    PubMed

    Cimini, Donatella; De Rosa, Mario; Carlino, Elisabetta; Ruggiero, Alessandro; Schiraldi, Chiara

    2013-05-09

    Glycosaminoglycans, such as hyaluronic acid, heparin, and chondroitin sulfate, are among the top ranked products in industrial biotechnology for biomedical applications, with a growing world market of billion dollars per year. Recently a remarkable progress has been made in the development of tailor-made strains as sources for the manufacturing of such products. The genetic modification of E. coli K4, a natural producer of chondroitin sulfate precursor, is challenging considering the lack of detailed information on its genome, as well as its mobilome. Chondroitin sulfate is currently used as nutraceutical for the treatment of osteoarthritis, and several new therapeutic applications, spanning from the development of skin substitutes to live attenuated vaccines, are under evaluation. E. coli K4 was used as host for the overexpression of RfaH, a positive regulator that controls expression of the polysaccharide biosynthesis genes and other genes necessary for the virulence of E. coli K4. Various engineering strategies were compared to investigate different types of expression systems (plasmid vs integrative cassettes) and integration sites (genome vs endogenous mobile element). All strains analysed in shake flasks on different media showed a capsular polysaccharide production improved by 40 to 140%, compared to the wild type, with respect to the final product titer. A DO-stat fed-batch process on the 2L scale was also developed for the best performing integrative strain, EcK4r3, yielding 5.3 g ∙ L(-1) of K4 polysaccharide. The effect of rfaH overexpression in EcK4r3 affected the production of lipopolysaccharide and the expression of genes involved in the polysaccharide biosynthesis pathway (kfoC and kfoA), as expected. An alteration of cellular metabolism was revealed by changes of intracellular pools of UDP-sugars which are used as precursors for polysaccharide biosynthesis. The present study describes the identification of a gene target and the application of a

  2. Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate

    PubMed Central

    Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K.; Liu, Peter; Pantua, Homer; Abbas, Alexander R.; Nickerson, Nicholas N.; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min

    2017-01-01

    ABSTRACT Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli. Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro, the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo. Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. PMID:28536290

  3. Polysaccharide capsule of Escherichia coli: microscope study of its size, structure, and sites of synthesis.

    PubMed Central

    Bayer, M E; Thurow, H

    1977-01-01

    This report describes the structure, size, and shape of the uncollapsed polysaccharide capsule of Escherichia coli strain Bi 161/42 [O9:K29(A):H-], its ultrastructural preservation as well as the filamentous components of the isolated capsular material. In a temperature-sensitive mutant, sites were localized at which capsular polysaccharide is "exported" to the cell surface. The highly hydrated capsule of the wild-type cells was visible in the uncollapsed state after freeze-etching, whereas dehydration in greater than or equal to 50% acetone or alcohol caused the capsule to collapse into thick bundles. This was prevented by pretreatment of the cell with capsule-specific immunoglobulin G; the capsule appeared as a homogeneous layer of 250- to 300-nm thickness. The structural preservation depended on the concentration of the anti-capsular immunoglobulin G. Temperature-sensitive mutants, unable to produce capsular antigen at elevated temperatures, showed, 10 to 15 min after shift down to permissive temperature, polysaccharide strands with K29 specificity appearing at the cell surface at roughly 20 sites per cell; concomitantly, capsule-directed antibody started to agglutinate the bacteria. The sites at which the new antigen emerged were found in random distribution over the entire surface of the organism. Spreading of purified polysaccharide was achieved on air-water interfaces; after subsequent shadow casting with heavy metal, filamentous elements were observed with a smallest class of filaments measuring 250 nm in length and 3 to 6 nm in width. At one end these fibers revealed a knoblike structure of about 10-nm diameter. The slimelike polysaccharides from mutants produced filamentous bundles of greater than 100-microns length, with antigenic and phage-receptor properties indistinguishable from those of the wild-type K29 capsule antigen. Images PMID:400798

  4. Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies

    PubMed Central

    Choi, Eun Hwa; Zhang, Fan; Lu, Ying-Jie

    2015-01-01

    The efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes is wzy dependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. The in vitro-released CPS concentrations per 107 CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum from Streptococcus pneumoniae ST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant from S. pneumoniae ST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary. PMID:26677201

  5. Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies.

    PubMed

    Choi, Eun Hwa; Zhang, Fan; Lu, Ying-Jie; Malley, Richard

    2015-12-16

    The efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes is wzy dependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. The in vitro-released CPS concentrations per 10(7) CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum from Streptococcus pneumoniae ST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant from S. pneumoniae ST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary.

  6. Vi Capsular Polysaccharide Produced by Recombinant Salmonella enterica Serovar Paratyphi A Confers Immunoprotection against Infection by Salmonella enterica Serovar Typhi.

    PubMed

    Xiong, Kun; Zhu, Chunyue; Chen, Zhijin; Zheng, Chunping; Tan, Yong; Rao, Xiancai; Cong, Yanguang

    2017-01-01

    Enteric fever is predominantly caused by Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A, and accounts for an annual global incidence of 26.9 millions. In recent years, the rate of S. Paratyphi A infection has progressively increased. Currently licensed vaccines for typhoid fever, live Ty21a vaccine, Vi subunit vaccine, and Vi-conjugate vaccine, confer inadequate cross immunoprotection against enteric fever caused by S. Paratyphi A. Therefore, development of bivalent vaccines against enteric fever is urgently required. The immunogenic Vi capsular polysaccharide is characteristically produced in S. Typhi, but it is absent in S. Paratyphi A. We propose that engineering synthesis of Vi in S. Paratyphi A live-attenuated vaccine may expand its protection range to cover S. Typhi. In this study, we cloned the viaB locus, which contains 10 genes responsible for Vi biosynthesis, and integrated into the chromosome of S. Paratyphi A CMCC 50093. Two virulence loci, htrA and phoPQ, were subsequently deleted to achieve a Vi-producing attenuated vaccine candidate. Our data showed that, despite more than 200 passages, the viaB locus was stably maintained in the chromosome of S. Paratyphi A and produced the Vi polysaccharide. Nasal immunization of the vaccine candidate stimulated high levels of Vi-specific and S. Paratyphi A-specific antibodies in mice sera as well as total sIgA in intestinal contents, and showed significant protection against wild-type challenge of S. Paratyphi A or S. Typhi. Our study show that the Vi-producing attenuated S. Paratyphi A is a promising bivalent vaccine candidate for the prevention of enteric fever.

  7. Vi Capsular Polysaccharide Produced by Recombinant Salmonella enterica Serovar Paratyphi A Confers Immunoprotection against Infection by Salmonella enterica Serovar Typhi

    PubMed Central

    Xiong, Kun; Zhu, Chunyue; Chen, Zhijin; Zheng, Chunping; Tan, Yong; Rao, Xiancai; Cong, Yanguang

    2017-01-01

    Enteric fever is predominantly caused by Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A, and accounts for an annual global incidence of 26.9 millions. In recent years, the rate of S. Paratyphi A infection has progressively increased. Currently licensed vaccines for typhoid fever, live Ty21a vaccine, Vi subunit vaccine, and Vi-conjugate vaccine, confer inadequate cross immunoprotection against enteric fever caused by S. Paratyphi A. Therefore, development of bivalent vaccines against enteric fever is urgently required. The immunogenic Vi capsular polysaccharide is characteristically produced in S. Typhi, but it is absent in S. Paratyphi A. We propose that engineering synthesis of Vi in S. Paratyphi A live-attenuated vaccine may expand its protection range to cover S. Typhi. In this study, we cloned the viaB locus, which contains 10 genes responsible for Vi biosynthesis, and integrated into the chromosome of S. Paratyphi A CMCC 50093. Two virulence loci, htrA and phoPQ, were subsequently deleted to achieve a Vi-producing attenuated vaccine candidate. Our data showed that, despite more than 200 passages, the viaB locus was stably maintained in the chromosome of S. Paratyphi A and produced the Vi polysaccharide. Nasal immunization of the vaccine candidate stimulated high levels of Vi-specific and S. Paratyphi A-specific antibodies in mice sera as well as total sIgA in intestinal contents, and showed significant protection against wild-type challenge of S. Paratyphi A or S. Typhi. Our study show that the Vi-producing attenuated S. Paratyphi A is a promising bivalent vaccine candidate for the prevention of enteric fever. PMID:28484685

  8. Streptococcus suis Capsular Polysaccharide Inhibits Phagocytosis through Destabilization of Lipid Microdomains and Prevents Lactosylceramide-Dependent Recognition

    PubMed Central

    Houde, Mathieu; Gottschalk, Marcelo; Gagnon, Fleur; Van Calsteren, Marie-Rose

    2012-01-01

    Streptococcus suis type 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans. S. suis infects the host through the respiratory route, reaches the bloodstream, and persists until breaching into the central nervous system. The capsular polysaccharide (CPS) of S. suis type 2 is considered a key virulence factor of the bacteria. Though CPS allows S. suis to adhere to the membrane of cells of the immune system, it provides protection against phagocytosis. In fact, nonencapsulated mutants are easily internalized and killed by macrophages and dendritic cells. The objective of this work was to study the molecular mechanisms by which the CPS of S. suis prevents phagocytosis. By using latex beads covalently linked with purified CPS, it was shown that CPS itself was sufficient to inhibit entry of both latex beads and bystander fluorescent beads into macrophages. Upon contact with macrophages, encapsulated S. suis was shown to destabilize lipid microdomains at the cell surface, to block nitric oxide (NO) production during infection, and to prevent lactosylceramide accumulation at the phagocytic cup during infection. In contrast, the nonencapsulated mutant was easily internalized via lipid rafts, in a filipin-sensitive manner, leading to lactosylceramide recruitment and strong NO production. This is the first report to identify a role for CPS in lipid microdomain stability and to recognize an interaction between S. suis and lactosylceramide in phagocytes. PMID:22124659

  9. Capsular polysaccharides facilitate enhanced iron acquisition by the colonial cyanobacterium Microcystis sp. isolated from a freshwater lake.

    PubMed

    Li, Zheng-Ke; Dai, Guo-Zheng; Juneau, Philippe; Qiu, Bao-Sheng

    2016-02-01

    Microcystis sp., especially in its colonial form, is a common dominant species during cyanobacterial blooms in many iron-deficient water bodies. It is still not entirely clear, however, how the colonial forms of Microcystis acclimate to iron-deficient habitats, and the responses of unicellular and colonial forms to iron-replete and iron-deficient conditions were examined here. Growth rates and levels of photosynthetic pigments declined to a greater extent in cultures of unicellular Microcystis than in cultures of the colonial form in response to decreasing iron concentrations, resulting in the impaired photosynthetic performance of unicellular Microcystis as compared to colonial forms as measured by variable fluorescence and photosynthetic oxygen evolution. These results indicate that the light-harvesting ability and photosynthetic capacity of colonial Microcystis was less affected by iron deficiency than the unicellular form. The carotenoid contents and nonphotochemical quenching of colonial Microcystis were less reduced than those of the unicellular form under decreasing iron concentrations, indicating that the colonial morphology enhanced photoprotection and acclimation to iron-deficient conditions. Furthermore, large amounts of iron were detected in the capsular polysaccharides (CPS) of the colonies, and more iron was found to be attached to the colonial Microcystis CPS under decreasing iron conditions as compared to unicellular cultures. These results demonstrated that colonial Microcystis can acclimate to iron deficiencies better than the unicellular form, and that CPS plays an important role in their acclimation advantage in iron-deficient waters.

  10. EDTA Inhibits Biofilm Formation, Extracellular Vesicular Secretion, and Shedding of the Capsular Polysaccharide Glucuronoxylomannan by Cryptococcus neoformans

    PubMed Central

    Robertson, Emma J.; Wolf, Julie M.

    2012-01-01

    The fungal pathogen Cryptococcus neoformans can grow as a biofilm on a range of synthetic and prosthetic materials. Cryptococcal biofilm formation can complicate the placement of shunts used to relieve increased intracranial pressure in cryptococcal meningitis and can serve as a nidus for chronic infection. Biofilms are generally advantageous to pathogens in vivo, as they can confer resistance to antimicrobial compounds, including fluconazole and voriconazole in the case of C. neoformans. EDTA can inhibit biofilm formation by several microbes and enhances the susceptibility of biofilms to antifungal drugs. In this study, we evaluated the effect of sublethal concentrations of EDTA on the growth of cryptococcal biofilms. EDTA inhibited biofilm growth by C. neoformans, and the inhibition could be reversed by the addition of magnesium or calcium, implying that the inhibitory effect was by divalent cation starvation. EDTA also reduced the amount of the capsular polysaccharide glucuronoxylomannan shed into the biofilm matrix and decreased vesicular secretion from the cell, thus providing a potential mechanism for the inhibitory effect of this cation-chelating compound. Our data imply that the growth of C. neoformans biofilms requires the presence of divalent metals in the growth medium and suggest that cations are required for the export of materials needed for biofilm formation, possibly including extracellular vesicles. PMID:22941091

  11. Fur regulation of the capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43.

    PubMed

    Lin, Ching-Ting; Wu, Chien-Chen; Chen, Yu-Sheng; Lai, Yi-Chyi; Chi, Chia; Lin, Jing-Ciao; Chen, Yeh; Peng, Hwei-Ling

    2011-02-01

    The ferric uptake regulator Fur has been reported to repress the expression of rmpA, a regulatory gene for the mucoid phenotype, leading to decreased capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae CG43. Here, quantitative real-time PCR (qRT-PCR) analyses and electrophoretic mobility shift assays showed that Fur also repressed the expression of the CPS regulatory genes rmpA2 and rcsA. Interestingly, deletion of rmpA or rcsA but not rmpA2 from the Δfur strain was able to suppress the deletion effect of Fur. The availability of extracellular iron affected the amount of CPS, suggesting that Fur regulates CPS biosynthesis in an Fe(II)-dependent manner. Increased production of siderophores was observed in the Δfur strain, suggesting that uptake of extracellular iron in K. pneumoniae is regulated by Fur. Fur titration assays and qRT-PCR analyses demonstrated that at least six of the eight putative iron-acquisition systems, identified by a blast search in the contig database of K. pneumoniae CG43, were directly repressed by Fur. We conclude that Fur has a dual role in the regulation of CPS biosynthesis and iron acquisition in K. pneumoniae.

  12. Humoral immune response of a pneumococcal conjugate vaccine: capsular polysaccharide serotype 14-Lysine modified PspA.

    PubMed

    Santamaria, Raquel; Goulart, Cibelly; Perciani, Catia T; Barazzone, Giovana C; Carvalho, Rimenys; Gonçalves, Viviane M; Leite, Luciana C C; Tanizaki, Martha M

    2011-11-03

    Polysaccharide-protein conjugates are so far the current antigens used for pneumococcal vaccines for children under 2 years of age. In this study, pneumococcal surface protein A (PspA) was used as a carrier protein for pneumococcal capsular polysaccharide serotype 14 as an alternative to broaden the vaccine coverage. PspA was modified by reductive amination with formaldehyde in order to improve the specificity of the reaction between protein and polysaccharide, inhibiting polymerization and the gel formation reaction. In the synthesis process, the currently used activator, 1-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloride (EDAC) was substituted for 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). BALB/c mice were immunized with either the PS14-mPspA conjugate or the co-administered components in a three dose regimen and sera from the immunized animals were assayed for immunity induced against both antigens: PS14 and mPspA. Modification of more than 70% of lysine residues from PspA (mPspA) did not interfere in the immune response as evaluated by the anti-PspA titer and C3 complement deposition assay. Sera of mice immunized with conjugated PS14-mPspA showed similar IgG titers, avidity and isotype profile as compared to controls immunized with PspA or mPspA alone. The complement deposition was higher in the sera of mice immunized with the conjugate vaccine and the opsonophagocytic activity was similar for both sera. Conjugation improved the immune response against PS14. The anti PS14 IgG titer was higher in sera of mice immunized with the conjugate than with co-administered antigens and presented an increased avidity index, induction of a predominant IgG1 isotype and increased complement deposition on a bacteria with a surface serotype 14. These results strongly support the use of PspA as carrier in a conjugate vaccine where both components act as antigens.

  13. Comparison of the immunogenicity and safety of two different brands of Salmonella typhi Vi capsular polysaccharide vaccine.

    PubMed

    Sabitha, P; Prabha Adhikari, M R; Chowdary, Abhijit; Prabhu, Malathi; Soofi, Mohammad; Shetty, Meenakshi; Kamath, Asha; Lokaranjan, S S; Bangera, S S

    2004-04-01

    The recent emergence of multi-drug-resistant Salmonella strains highlights the need for better preventive measures, including vaccination. Safe and immunologic vaccines have been developed based on purified Vi polysaccharide. To compare the immune response elicited by two different brands of Salmonella Vi capsular polysaccharide vaccine (ViCPS). Double blind, randomized (3:1), controlled, parallel, phase III study was conducted at two centres in India to compare the safety and immunogenicity of Typbar, the investigational vaccine with an already marketed vaccine "X", in healthy subjects aged between 12 -25 years. A sample size of 184 subjects was calculated. Subjects were randomly distributed in two groups, immunized with single dose of Typbar or Vaccine "X". Serum samples were taken before 7 days and 4 weeks after immunization for the determination of antibodies to Vi polysaccharide, by ELISA method. Safety was assessed by physical examination, laboratory parameters before and after vaccination and by monitoring adverse events. The geometric mean antibody titre (GMT) 4 weeks after vaccination was compared from respective pre-vaccination values by Wilcoxon signed rank test. Geometric mean of antibody levels before and after immunization and the ratio between them (Mann-Whitney test), the Seroconversion rates (Z test of proportions) and the adverse events (Fisher's exact test and Chi square test), were compared between two groups. P value < 0.05 was considered statistically significant. P values and 95% confidence intervals were estimated in two-tailed fashion. 153 subjects (Typbar =116 and Vaccine "X" =37) were studied. 71.6% (95% CI=63.4%-79.8%) and 75.7% (95% CI=64.9% - 89.5%) were the seroconversion rates with Typbar and vaccine "X" respectively. The GMT values for Vi antibodies induced after Typbar and vaccine "X" were 10.23 Typbar and 13.46 mg/mL respectively and these values showed high significance when compared to their respective pre-immunization GMT

  14. Antibody response to pneumococcal capsular polysaccharide vaccine in Down syndrome patients.

    PubMed

    Costa-Carvalho, B T; Martinez, R M A; Dias, A T N; Kubo, C A; Barros-Nunes, P; Leiva, L; Solé, D; Carneiro-Sampaio, M M S; Naspitz, C K; Sorensen, R U

    2006-12-01

    The majority of children with Down syndrome (DS) tend to have frequent bacterial infections including recurrent respiratory infections. Our objective was to evaluate the production of antibodies to pneumococcal polysaccharide antigens after active immunization in DS subjects. IgG antibodies to pneumococcal serotypes (1, 3, 6B, 9V, and 14) were measured before and 6 weeks after immunization with a 23-valent pneumococcal vaccine (Pneumo23, Pasteur-Merrieux) in 6- to 13-year-old DS children (N = 17) and in aged-matched normal controls (N = 30). An adequate response was defined as a 4-fold increase over baseline or a post-immunization level of specific pneumococcal serotype antibody > or = 1.3 microg/mL. After immunization, all DS children had an increase in post-immunization levels against all serotypes analyzed. A 4-fold or more increase was observed in all DS children concerning serotypes 1 and 14, in 90% of subjects for serotypes 3 and 9V, and in 65% for serotype 6B. Regarding this increase, 8 of the 17 DS children had an adequate response to all serotypes analyzed, 8/17 patients to 4 serotypes and 1/17 to 3 serotypes. However, when we compared post-immunization levels between DS children and controls, we observed lower levels in the former group (P < 0.05) for all serotypes except serotype 3. We conclude that pneumococcal polysaccharide immunization could be beneficial for these DS children.

  15. Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus

    PubMed Central

    Renzi, Francesco; Ittig, Simon J.; Sadovskaya, Irina; Hess, Estelle; Lauber, Frederic; Dol, Melanie; Shin, Hwain; Mally, Manuela; Fiechter, Chantal; Sauder, Ursula; Chami, Mohamed; Cornelis, Guy R.

    2016-01-01

    Capnocytophaga canimorsus is a dog’s and cat’s oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus. PMID:27974829

  16. Application of glyco-blotting for identification of structures of polysaccharides causing membrane fouling in a pilot-scale membrane bioreactor treating municipal wastewater.

    PubMed

    Kimura, Katsuki; Nishimura, Shin-Ichiro; Miyoshi, Risho; Hoque, Asiful; Miyoshi, Taro; Watanabe, Yoshimasa

    2015-03-01

    A new approach for the analysis of polysaccharides in membrane bioreactor (MBR) is proposed in this study. Enrichment of polysaccharides by glyco-blotting, in which polysaccharides are specifically collected via interactions between the aldehydes in the polysaccharides and aminooxy groups on glycoblotting beads, enabled MALDI-TOF/MS analysis at a high resolution. Structures of polysaccharides extracted from fouled membranes used in a pilot-scale MBR treating municipal wastewater and those in the supernatant of the mixed liquor suspension in the MBR were investigated. It was found that the overlap between polysaccharides found in the supernatants and those extracted from the fouled membrane was rather limited, suggesting that polysaccharides that dominate in supernatants may not be important in membrane fouling in MBRs. Analysis using a bacterial carbohydrate database suggested that capsular polysaccharides (CPS) and/or lipo-polysaccharides (LPS) produced by gram-negative bacteria are key players in the evolution of membrane fouling in MBRs.

  17. New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors

    PubMed Central

    di Lauro, Irene; Di Nuzzo, Rosaria; De Rosa, Mario; Schiraldi, Chiara

    2017-01-01

    Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 μM·gcdw−1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced. PMID:28104792

  18. Antibodies to Staphylococcus aureus Serotype 8 Capsular Polysaccharide React with and Protect against Serotype 5 and 8 Isolates

    PubMed Central

    Park, Saeyoung; Gerber, Sabina

    2014-01-01

    Most Staphylococcus aureus isolates produce either a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide, and the CP antigens are targets for vaccine development. Since CP5 and CP8 have similar trisaccharide repeating units, it is important to identify an epitope shared by both CP5 and CP8. To characterize cross-reactivity between CP5 and CP8, the immunogenicity of CP5 and CP8 conjugate vaccines in mice and rabbits was evaluated by serological assays. Immune sera were also tested for functional activity by in vitro opsonophagocytic-killing assays and a murine bacteremia model. Antibodies to the CP5-cross-reactive material 197 (CRM197) conjugate vaccine bound only to purified CP5. In contrast, antibodies to the CP8-CRM conjugate vaccine reacted with CP8 and (to a lesser extent) CP5. De-O-acetylation of CP5 increased its reactivity with CP8 antibodies. Moreover, CP8 antibodies bound to Pseudomonas aeruginosa O11 lipopolysaccharide, which has a trisaccharide repeating unit similar to that of the S. aureus CPs. CP8-CRM antibodies mediated in vitro opsonophagocytic killing of S. aureus expressing CP5 or CP8, whereas CP5-CRM antibodies were serotype specific. Passive immunization with antiserum to CP5-CRM or CP8-CRM protected mice against bacteremia induced by a serotype 5 S. aureus isolate, suggesting that CP8-CRM elicits antibodies cross-reactive to CP5. The identification of epitopes shared by CP5 and CP8 may inform the rational design of a vaccine to protect against infections caused by CP5- or CP8-producing strains of S. aureus. PMID:25245803

  19. Sub-MICs of Azithromycin Decrease Biofilm Formation of Streptococcus suis and Increase Capsular Polysaccharide Content of S. suis

    PubMed Central

    Yang, Yan-Bei; Chen, Jian-Qing; Zhao, Yu-Lin; Bai, Jing-Wen; Ding, Wen-Ya; Zhou, Yong-Hui; Chen, Xue-Ying; Liu, Di; Li, Yan-Hua

    2016-01-01

    Streptococcus suis (S. suis) caused serious disease symptoms in humans and pigs. S. suis is able to form thick biofilms and this increases the difficulty of treatment. After growth with 1/2 minimal inhibitory concentration (MIC) of azithromycin, 1/4 MIC of azithromycin, or 1/8 MIC of azithromycin, biofilm formation of S. suis dose-dependently decreased in the present study. Furthermore, scanning electron microscopy analysis revealed the obvious effect of azithromycin against biofilm formation of S. suis. Especially, at two different conditions (1/2 MIC of azithromycin non-treated cells and treated cells), we carried out comparative proteomic analyses of cells by using iTRAQ technology. Finally, the results revealed the existence of 19 proteins of varying amounts. Interestingly, several cell surface proteins (such as ATP-binding cassette superfamily ATP-binding cassette transporter (G7SD52), CpsR (K0FG35), Cps1/2H (G8DTL7), CPS16F (E9NQ13), putative uncharacterized protein (G7SER0), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (G5L259), putative uncharacterized protein (G7S2D6), amino acid permease (B0M0G6), and NsuB (G5L351)) were found to be implicated in biofilm formation. More importantly, we also found that azithromycin affected expression of the genes cps1/2H, cpsR and cps16F. Especially, after growth with 1/2 MIC of azithromycin and 1/4 MIC of azithromycin, the capsular polysaccharide content of S. suis was significantly higher. PMID:27812354

  20. Induction of functional secretory IgA responses in breast milk, by pneumococcal capsular polysaccharides.

    PubMed

    Finn, Adam; Zhang, Qibo; Seymour, Lynn; Fasching, Claudine; Pettitt, Emily; Janoff, Edward N

    2002-11-15

    Capsule-specific secretory IgA (s-IgA) in breast milk may enhance protection against pneumococcal disease in infants. After immunization of 3 lactating mothers with 23-valent polysaccharide vaccine, specific s-IgA, but not IgG, increased by >2-fold in milk of at least 1 subject for 6 of 7 serotypes. The s-IgA was predominantly IgA1, in secretory form, and highly specific with avidity distinct from serum IgA and IgG. Milk whey from 2 immunized women supported dose- and complement-dependent killing of Streptococcus pneumoniae serotypes 19F and 14 by human neutrophils, as did purified s-IgA to serotype 19F. These data reveal that capsule-specific human s-IgA in breast milk can initiate killing of S. pneumoniae, providing proof of concept that vaccine-induced human mucosal s-IgA can support functional bactericidal activity. Determining the biologic role for s-IgA in killing and inhibiting adherence of S. pneumoniae in vivo will contribute to the development of mucosal vaccines against S. pneumoniae.

  1. Capsular polysaccharide from Mycoplasma mycoides subsp. mycoides shows potential for protection against contagious bovine pleuropneumonia.

    PubMed

    Mwirigi, Martin; Nkando, Isabel; Olum, Moses; Attah-Poku, Samuel; Ochanda, Horace; Berberov, Emil; Potter, Andrew; Gerdts, Volker; Perez-Casal, Jose; Wesonga, Hezron; Soi, Reuben; Naessens, Jan

    2016-10-01

    Contagious Bovine Pleuropneumonia (CBPP) is a severe respiratory disease caused by Mycoplasma mycoides subsp. mycoides (Mmm) which is widespread in Africa. The capsule polysaccharide (CPS) of Mmm is one of the few identified virulence determinants. In a previous study, immunization of mice against CPS generated antibodies, but they were not able to prevent multiplication of Mmm in this model animal. However, mice cannot be considered as a suitable animal model, as Mmm does not induce pathology in this species. Our aim was to induce antibody responses to CPS in cattle, and challenge them when they had specific CPS antibody titres similar or higher than those from cattle vaccinated with the live vaccine. The CPS was linked to the carrier protein ovalbumin via a carbodiimide-mediated condensation with 1-ethyl-3(3-imethylaminopropyl) carbodiimide (EDC). Ten animals were immunized twice and challenged three weeks after the booster inoculation, and compared to a group of challenged non-immunized cattle. When administered subcutaneously to adult cattle, the vaccine elicited CPS-specific antibody responses with the same or a higher titre than animals vaccinated with the live vaccine. Pathology in the group of immunized animals was significantly reduced (57%) after challenge with Mmm strain Afadé compared to the non-immunized group, a figure in the range of the protection provided by the live vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Therapeutic properties in Tunisian hot springs: first evidence of phenolic compounds in the cyanobacterium Leptolyngbya sp. biomass, capsular polysaccharides and releasing polysaccharides.

    PubMed

    Trabelsi, Lamia; Mnari, Amira; Abdel-Daim, Mohamed M; Abid-Essafi, Salwa; Aleya, Lotfi

    2016-12-13

    In Tunisia, the use of hot spring waters for both health and recreation is a tradition dating back to Roman times. In fact, thermal baths, usually called "Hammam" are recommended as a therapeutic and prophylactic measure against many types of illness and toxicity. While the chemical concentration of thermal water is admittedly associated with its therapeutic effects, the inclusion in spa waters of efficient bioproduct additives produced by photosynthetic microorganisms and that act against oxidative stress may comprise a significant supplementary value for thermal centers. The aim of this study was to investigate the antioxidant potential of the Tunisian thermophilic cyanobacterium Leptolyngbya sp. and to determine its phytochemical constituents and phenolic profile. BME (Biomass Methanolic Extract), CME (Capsular polysaccharides Methanolic Extract) and RME (Releasing polysaccharides Methanolic Extract) of Leptolyngbya sp. were examined for their antioxidant activities by means of DPPH, hydroxyl radical scavenging and ferrous ion chelating assays. Their total phenols, flavonoids, carotenoids, Mycosporine-like amino acids (MAAs) and vitamin C contents, as well as their phenolic profiles were also determined. BME has the highest content of phenols (139 ± 1.2 mg/g), flavonoids (34.9 ± 0.32 mg CEQ/g), carotenoids (2.03 ± 0.56 mg/g) and vitamin C (15.7 ± 1.55 mg/g), while the highest MAAs content (0.42 ± 0.03 mg/g) was observed in CME. BME presented both the highest DPPH and hydroxyl radical scavenging ability with an IC50 of 0.07 and 0.38 mg/ml, respectively. The highest ferrous chelating capacity was detected in CME with an IC50 = 0.59 mg/ml. Phenolic profiles revealed the presence of 25 phenolic compounds with the existence of hydroxytyrosol, oleuropein, resveratrol and pinoresinol. The study demonstrated that the cyanobacterium Leptolyngbya sp. possesses abundant natural antioxidant products which may have prophylactic and

  3. Synthesis of di- and tri-saccharide fragments of Salmonella typhi Vi capsular polysaccharide and their zwitterionic analogues.

    PubMed

    Fusari, Matteo; Fallarini, Silvia; Lombardi, Grazia; Lay, Luigi

    2015-12-01

    Zwitterionic polysaccharides (ZPS) behave like traditional T cell-dependent antigens, suggesting the design of new classes of vaccines alternative to currently used glycoconjugates and based on the artificial introduction of a zwitterionic charge motif onto the carbohydrate structure of pathogen antigens. Here we report the new synthesis and antigenic evaluation of di-/tri-saccharide fragments of Salmonella typhi Vi polysaccharide, as well as of their corresponding zwitterionic analogues. Our strategy is based on versatile intermediates enabling chain elongation either by iterative single monomer attachment or by faster and more flexible approach using disaccharide donors. The effect of structural modifications of the synthetic compounds on antigenic properties was evaluated by competitive ELISA. All the oligosaccharides were recognized by specific anti-Vi polyclonal antibodies in a concentration-dependent manner, and the introduction of a zwitterionic motif into the synthetic molecules did not prevent the binding.

  4. Impaired Function of Antibodies to Pneumococcal Surface Protein A but Not to Capsular Polysaccharide in Mexican American Adults with Type 2 Diabetes Mellitus

    PubMed Central

    Mathews, Christine E.; Brown, Eric L.; Martinez, Perla J.; Bagaria, Upasana; Nahm, Moon H.; Burton, Robert L.; Fisher-Hoch, Susan P.; McCormick, Joseph B.

    2012-01-01

    The goal of the study was to determine baseline protective titers of antibodies to Streptococcus pneumoniae surface protein A (PspA) and capsular polysaccharide in individuals with and individuals without type 2 diabetes mellitus. A total of 561 individuals (131 individuals with diabetes and 491 without) were screened for antibodies to PspA using a standard enzyme-linked immunosorbent assay (ELISA). A subset of participants with antibodies to PspA were retested using a WHO ELISA to determine titers of antibodies to capsular polysaccharide (CPS) (serotypes 4, 6B, 9V, 14, 18C, 19A, 19F, and 23F). Functional activity of antibodies was measured by assessing their ability to enhance complement (C3) deposition on pneumococci and promote killing of opsonized pneumococci. Titers of antibodies to protein antigens (PspA) were significantly lower in individuals with diabetes than controls without diabetes (P = 0.01), and antibodies showed a significantly reduced complement deposition ability (P = 0.02). Both antibody titers and complement deposition were negatively associated with hyperglycemia. Conversely, titers of antibodies to capsular polysaccharides were either comparable between the two groups or were significantly higher in individuals with diabetes, as was observed for CPS 14 (P = 0.05). The plasma specimens from individuals with diabetes also demonstrated a higher opsonophagocytic index against CPS serotype 14. Although we demonstrate comparable protective titers of antibodies to CPS in individuals with and individuals without diabetes, those with diabetes had lower PspA titers and poor opsonic activity strongly associated with hyperglycemia. These results suggest a link between diabetes and impairment of antibody response. PMID:22761295

  5. Acinetobacter baumannii K11 and K83 capsular polysaccharides have the same 6-deoxy-l-talose-containing pentasaccharide K units but different linkages between the K units.

    PubMed

    Kenyon, Johanna J; Shashkov, Alexander S; Senchenkova, Sof'ya N; Shneider, Mikhail M; Liu, Bin; Popova, Anastasiya V; Arbatsky, Nikolay P; Miroshnikov, Konstantin A; Wang, Lei; Knirel, Yuriy A; Hall, Ruth M

    2017-10-01

    Acinetobacter baumannii produces a variety of capsular polysaccharides (CPS) via genes located at the chromosomal K locus and some KL gene clusters include genes for the synthesis of specific sugars. The structures of K11 and K83 CPS produced by isolates LUH5545 and LUH5538, which carry related KL11a and KL83 gene clusters, respectively, were established by sugar analysis and one- and two-dimensional (1)H and (13)C NMR spectroscopy. Both CPS contain l-rhamnose (l-Rha) and 6-deoxy-l-talose (l-6dTal), and both KL gene clusters include genes for dTDP-l-Rhap synthesis and a tle (talose epimerase) gene encoding an epimerase that converts dTDP-l-Rhap to dTDP-l-6dTalp. The K11 and K83 repeat units are the same pentasaccharide, consisting of d-glucose, l-Rha, l-6dTal, and N-acetyl-d-glucosamine, except that l-6dTal is 2-O-acetylated in K83. However, the K units are linked differently, with l-Rha in the main chain in K11, but as a side-branch in K83. KL11 and KL83 encode unrelated Wzy polymerases that link the K units together and different acetyltransferases, though only Atr8 from KL83 is active. The substrate specificity of each Wzy polymerase was assigned, and the functions of all glycosyltransferases were predicted. The CPS structures produced by three closely related K loci, KL29, KL105 and KL106, were also predicted. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Group B Streptococcus Capsular Polysaccharide-Cholera Toxin B Subunit Conjugate Vaccines Prepared by Different Methods for Intranasal Immunization

    PubMed Central

    Shen, Xuzhuang; Lagergård, Teresa; Yang, Yonghong; Lindblad, Marianne; Fredriksson, Margareta; Holmgren, Jan

    2001-01-01

    Group B Streptococcus (GBS) type III capsular polysaccharide (CPS III) was conjugated to recombinant cholera toxin B subunit (rCTB) using three different methods which employed (i) cystamine and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), (ii) carbodiimide with adipic acid dihydrazide (ADH) as a spacer, or (iii) reductive amination (RA). The CPS III-rCTB conjugates were divided into large- and small-molecular-weight (Mr) fractions, and the immunogenicities of the different preparations after intranasal (i.n.) immunization were studied in mice. Both large- and small-Mr conjugates of CPS III-rCTBRA or CPS III-rCTBADH induced high, almost comparable levels of CPS-specific immunoglobulin G (IgG) in serum, lungs, and vagina that were generally superior to those obtained with CPS III-rCTBSPDP conjugates or a CPS III and rCTB mixture. However, the smaller-Mr conjugates of CPS III-rCTBRA or CPS III-rCTBADH in most cases elicited a lower anti-CPS IgA immune response than the large-Mr conjugates, and the highest anti-CPS IgA titers in both tissues and serum were obtained with the large-Mr CPS III-rCTBRA conjugate. Serum IgG anti-CPS titers induced by the CPS III-rCTBRA conjugate had high levels of specific IgG1, IgG2a, IgG2b, and IgG3 antibodies. Based on the effectiveness of RA for coupling CPS III to rCTB, RA was also tested for conjugating GBS CPS Ia with rCTB. As for the CPS III-rCTB conjugates, the immunogenicity of CPS Ia was greatly increased by conjugation to rCTB. Intranasal immunization with a combination of CPS Ia-rCTB and CPS III-rCTB conjugates was shown to induce anti-CPS Ia and III immune responses in serum and lungs that were fully comparable with the responses to immunization with the monovalent CPS Ia-rCTB or CPS III-rCTB conjugates. These results suggest that the GBS CPS III-rCTB and CPS Ia-rCTB conjugates prepared by the RA method may be used in bivalent and possibly also in multivalent mucosal GBS conjugate vaccines. PMID:11119518

  7. A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge.

    PubMed Central

    Fattom, A I; Sarwar, J; Ortiz, A; Naso, R

    1996-01-01

    The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models. A dose of 2 x 10(5) CFU of S. aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days. Mice passively immunized i.p. 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge. Animals actively immunized with the monovalent type 5 CP-P. aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals. The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively). The IgG was further evaluated in mice challenged i.p. with a sublethal dose of 5 x 10(4) CFU per mouse. Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h. Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids. Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05). These data suggest that S. aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S. aureus infections. PMID:8613375

  8. The capsular polysaccharide of Staphylococcus aureus is attached to peptidoglycan by the LytR-CpsA-Psr (LCP) family of enzymes.

    PubMed

    Chan, Yvonne Gar-Yun; Kim, Hwan Keun; Schneewind, Olaf; Missiakas, Dominique

    2014-05-30

    Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.

  9. Recognition of flavin mononucleotide, Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate.

    PubMed

    Ravi, G; Venkatesh, Yeldur P

    2014-11-01

    D-Ribitol-5-phosphate (Rbt-5-P) is an important metabolite in the pentose phosphate pathway and an integral part of bacterial cell wall polysaccharides, specifically as polyribosyl ribitol phosphate (PRP) in Haemophilus influenzae type b (Hib). The major objective of this study was to investigate whether an antibody specific to Rbt-5-P can recognize the PRP of Hib. D-Ribose-5-phosphate was reacted with proteins in the presence of sodium cyanoborohydride to obtain Rbt-5-P epitopes; 120 h reaction resulted in conjugation of ~30 and ~17 moles of Rbt-5-P/mole of BSA and OVA, respectively, based on decrease in amino groups, MALDI-TOF analyses, an increase in apparent molecular weight (SDS-PAGE) and glycoprotein staining. Immunization of rabbits with Rbt-5-P-BSA conjugate generated antibodies to Rbt-5-P as demonstrated by dot immunoblot and non-competitive ELISA. Homogeneous Rbt-5-P-specific antibody was purified from Rbt-5-P-BSA antiserum subjected to caprylic acid precipitation followed by hapten-affinity chromatography; its affinity constant is 7.1 × 10(8) M(-1). Rbt-5-P antibody showed 100 % specificity to Rbt-5-P, ~230 %, 10 % and 3.4 % cross-reactivity to FMN, riboflavin and FAD, respectively; the antibody showed ~4 % cross-reactivity to D-ribitol and <3 % to other sugars/sugar alcohols. Rbt-5-P-specific antibody recognized Hib conjugate vaccines containing PRP which was inhibited specifically by Rbt-5-P, and also detected Hib cell-surface capsular polysaccharides by immunofluorescence. In conclusion, Rbt-5-P-protein conjugate used as an immunogen elicited antibodies binding to an epitope also present in PRP and Hib bacteria. Rbt-5-P-specific antibody has potential applications in the detection and quantification of free/bound Rbt-5-P and FMN as well as immunological recognition of Hib bacteria and its capsular polysaccharide.

  10. Nucleotide sequence analysis of a DNA region involved in capsular polysaccharide biosynthesis reveals the molecular basis of the nontypeability of two Actinobacillus pleuropneumoniae isolates.

    PubMed

    Ito, Hiroya; Ogawa, Torata; Fukamizu, Dai; Morinaga, Yuiko; Kusumoto, Masahiro

    2016-11-01

    The aim of our study was to reveal the molecular basis of the serologic nontypeability of 2 Actinobacillus pleuropneumoniae field isolates. Nine field strains of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia, were isolated from pigs raised on the same farm and sent to our diagnostic laboratory for serotyping. Seven of the 9 strains were identified as serovar 15 strains by immunodiffusion tests. However, 2 strains, designated FH24-2 and FH24-5, could not be serotyped with antiserum prepared against serovars 1-15. Strain FH24-5 showed positive results in 2 serovar 15-specific PCR tests, whereas strain FH24-2 was only positive in 1 of the 2 PCR tests. The nucleotide sequence analysis of gene clusters involved in capsular polysaccharide biosynthesis of the 2 nontypeable strains revealed that both had been rendered nontypeable by the action of ISApl1, a transposable element of A. pleuropneumoniae belonging to the IS30 family. The results showed that ISApl1 of A. pleuropneumoniae can interfere with both the serologic and molecular typing methods, and that nucleotide sequence analysis across the capsular gene clusters is the best means of determining the cause of serologic nontypeability in A. pleuropneumoniae. © 2016 The Author(s).

  11. Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats

    USDA-ARS?s Scientific Manuscript database

    Bacteroides thetaiotaomicron is a prominent member of the human distal gut microbiota that specializes in breaking down diet and host-derived polysaccharides. While polysaccharide utilization has been well studied in B. thetaiotaomicron, other aspects of its behavior are less well characterized, in...

  12. Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus.

    PubMed

    Sau, S; Lee, C Y

    1996-04-01

    Eleven serotypes of capsular polysaccharide from Staphylococcus aureus have been reported. We have previously cloned a cluster of type 1 capsule (cap1) genes responsible for type 1 capsular polysaccharide biosynthesis in S. aureus M. To clone the type 8 capsule (cap8) genes, a plasmid library of type 8 strain Becker was screened with a labelled DNA fragment containing the cap1 genes under low-stringency conditions. One recombinant plasmid containing a 14-kb insert was chosen for further study and found to complement 14 of the 18 type 8 capsule-negative (Cap8-) mutants used in the study. Additional library screening, subcloning, and complementation experiments showed that all of the 18 Cap8- mutants were complemented by DNA fragments derived from a 20.5-kb contiguous region of the Becker chromosome. The mutants were mapped into six complementation groups, indicating that the cap8 genes are clustered. By Southern hybridization analyses under high-stringency conditions, we found that DNA fragments containing the cap8 gene cluster show extensive homology with all 17 strains tested, including type 1 strains. By further Southern analyses and cloning of the cap8-related homolog from strain M, we show that strain M carries an additional capsule gene cluster different from the cap1 gene cluster. In addition, by using DNA fragments containing different regions of the cap8 gene cluster as probes to hybridize DNA from different strains, we found that the central region of the cap8 gene cluster hybridizes only to DNAs from certain strains tested whereas the flanking regions hybridize to DNAs of all strains tested. Thus, the cap8 gene clusters and its closely related homologs are likely to have organizations similar to those of the encapsulation genes of other bacterial systems.

  13. Tts, a processive beta-glucosyltransferase of Streptococcus pneumoniae, directs the synthesis of the branched type 37 capsular polysaccharide in Pneumococcus and other gram-positive species.

    PubMed

    Llull, D; Garcia, E; Lopez, R

    2001-06-15

    The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating units of [beta-d-Glcp-(1-->2)]-beta-d-Glcp-(1-->3). The elements governing the expression of the tts gene, coding for the glucosyltransferase involved in the synthesis of the type 37 pneumococcal capsular polysaccharide, have been studied. Primer extension analysis and functional tests demonstrated the presence of four new transcriptional start points upstream of the previously reported tts promoter (ttsp). Most interesting, three of these transcriptional start points are located in a RUP element thought to be involved in recombinational events (Oggioni, M. R., and Claverys, J. P. (1999) Microbiology 145, 2647-2653). Transformation experiments using either a recombinant plasmid containing the whole transcriptional unit of tts or chromosomal DNA from a type 37 pneumococcus showed that tts is the only gene required to drive the biosynthesis of a type 37 capsule in S. pneumoniae and other Gram-positive bacteria, namely Streptococcus oralis, Streptococcus gordonii, and Bacillus subtilis. The Tts synthase was overproduced in S. pneumoniae and purified as a membrane-associated enzyme. These membrane preparations used UDP-Glc as substrate to catalyze the synthesis of a high molecular weight polysaccharide immunologically identical to the type 37 capsule. In addition, UDP-Gal was also a substrate to produce type 37 polysaccharide since a strong UDP-Glc-4'-epimerase activity is associated to the membrane fraction of S. pneumoniae. These results indicated that Tts has a dual biochemical activity that leads to the synthesis of the branched type 37 polysaccharide.

  14. Vibrio cholerae O139 Conjugate Vaccines: Synthesis and Immunogenicity of V. cholerae O139 Capsular Polysaccharide Conjugates with Recombinant Diphtheria Toxin Mutant in Mice

    PubMed Central

    Kossaczka, Zuzana; Shiloach, Joseph; Johnson, Virginia; Taylor, David N.; Finkelstein, Richard A.; Robbins, John B.; Szu, Shousun C.

    2000-01-01

    Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to ≤50. Treatment with 2-ME reduced the titers of 17 of 20 patients to ≤50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned. PMID:10948122

  15. Specific and cross-reactive immune response to oral Salmonella Typhi Ty21a and parenteral Vi capsular polysaccharide typhoid vaccines administered concomitantly.

    PubMed

    Pakkanen, Sari H; Kantele, Jussi M; Savolainen, Laura E; Rombo, Lars; Kantele, Anu

    2015-01-09

    Since protective efficacy of the current typhoid vaccines-oral whole-cell Salmonella Typhi Ty21a and parenteral Vi-capsular polysaccharide preparation-is not optimal, and no vaccines are available against paratyphoid or non-typhoidal Salmonella (NTS) serotypes, new approaches deserve to be explored. The immunological mechanisms elicited by the two typhoid vaccines are mainly targeted against different structures. We studied whether these vaccines would enhance S. Typhi-specific immune response and cross-reactivity against other Salmonellae, if administered concomitantly. Volunteers were immunized simultaneously with Ty21a and Vi vaccines (Ty21a+Vi group) or with either of the two singly (Ty21a and Vi groups). All volunteers were investigated for circulating specific and cross-reactive plasmablasts, identified by ELISPOT as IgA, IgG or IgM antibody-secreting cells (ASC) reactive with S. Typhi, S. Paratyphi A/B/C, or selected NTS serotypes (S. Enteritidis, S. Typhimurium). In the Ty21a+Vi group, no specific or cross-reactive plasmablasts were detected before vaccination. After vaccination, the number of S. Typhi-specific plasmablasts (878 ASC/10(6) PBMC, 95%CI 554-1201) proved higher than in the Ty21a (339 ASC/10(6) PBMC; p<0.001) and Vi (149 ASC/10(6) PBMC; p<0.001) groups. Likewise, cross-reactive responses in the Ty21a+Vi group were higher than in the Ty21a and Vi groups (Ty21a+Vi vs Ty21a: ASC against S. Paratyphi A/B, S. Enteritidis and S. Typhimurium p<0.05, against S. Paratyphi C p<0.01; Ty21a+Vi vs Vi: against S. Paratyphi C not significant, others p<0.0001). A gut-directed homing profile was seen among O antigen-specific and a systemic one among Vi antigen-specific plasmablasts. Concomitant administration of Ty21a and Vi vaccines is well tolerated and induces an additive immune response to the two vaccines. Thus it enhances the magnitude of both typhoid-specific plasmablast responses and those cross-reacting with paratyphoid and most important NTS serotypes

  16. Monoclonal antibodies specific for immunorecessive epitopes of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, reduce serotype bias in an immunoassay for cryptococcal antigen.

    PubMed

    Percival, Ann; Thorkildson, Peter; Kozel, Thomas R

    2011-08-01

    Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.

  17. Structural studies of CV-70 polysaccharide.

    PubMed

    Scamparini, A; Mariuzzo, D; Fujihara, H; Jacobusi, R; Vendruscolo, C

    1997-08-01

    The goal of this paper is the characterization of the chemical structure of the water-soluble polysaccharide, CV-70, produced by bacteria Beijerinckia sp. Beijerinckia sp. is a genus of gram-negative, aerobic bacteria, usually found in sugar cane root. The CV-70 polysaccharide was produced in a fermentation medium containing 5% sucrose as the carbon source, tryptose and salts, at 25 degrees C [1]. The polysaccharide was hydrolyzed with 2 N trifluoroacetic acid at 100 degrees C for 16 h, purified, and analyzed by HPLC. Index of refraction was used for the detection of sugars. For GC-MS analysis, the CV-70 polysaccharide was derivatized through methylation and acetylation. Together with the GC-MS data, periodate oxidation studies were used to determine the possible glucosidic linkages. Carbon-13 NMR studies were carried out with hydrolyzed and silylated samples. Glucose, galactose and fucose were identified as the components in the CV-70 polysaccharide, in a 3:1:3 ratio.

  18. Structural analysis of cell wall polysaccharides using PACE

    SciTech Connect

    Mortimer, Jennifer C.

    2017-01-01

    The plant cell wall is composed of many complex polysaccharides. The composition and structure of the polysaccharides affect various cell properties including cell shape, cell function and cell adhesion. Many techniques to characterize polysaccharide structure are complicated, requiring expensive equipment and specialized operators e.g. NMR, MALDI-MS. PACE (Polysaccharide Analysis using Carbohydrate gel Electrophoresis) uses a simple, rapid technique to analyze polysaccharide quantity and structure (Goubet et al. 2002). Whilst the method here describes xylan analysis, it can be applied (by use of the appropriate glycosyl hydrolase) to any cell wall polysaccharide.

  19. Phase II, randomized, multicenter, double-blind, placebo-controlled trial of a polyclonal anti-Staphylococcus aureus capsular polysaccharide immune globulin in treatment of Staphylococcus aureus bacteremia.

    PubMed

    Rupp, Mark E; Holley, H Preston; Lutz, Jon; Dicpinigaitis, Peter V; Woods, Christopher W; Levine, Donald P; Veney, Naomi; Fowler, Vance G

    2007-12-01

    New treatment modalities are needed for the treatment of infections due to multidrug-resistant Staphylococcus aureus. S. aureus capsular polysaccharide immune globulin (Altastaph) is a polyclonal immune globulin preparation that is being developed as adjunctive therapy for persons with S. aureus infections complicated by bacteremia. In a phase II, multicenter, randomized, double-blind, placebo-controlled trial, 40 subjects with documented S. aureus bacteremia received standard therapy plus either Altastaph at 200 mg/kg of body weight in each of two infusions 24 h apart or placebo. During the 42-day observation period, antibody pharmacokinetics and safety were the primary characteristics studied. Information regarding the resolution of bacteremia and fever was also analyzed. Anti-type-5 and anti-type-8 capsular antibody levels peaked after the second infusion at 550 mug/ml and 419 mug/ml, respectively, and remained above 100 mug/ml at day 28. A total of 316 adverse events were noted in 39 of 40 subjects. Infusion-related adverse events in Altastaph recipients were infrequent and similar to those among recipients of commercial intravenously administered immunoglobulin G products. Five of 21 (23%) subjects in the Altastaph group died, whereas 2 of 18 (11%) subjects in the placebo group died (P = 0.42). Compared to the control patients, the Altastaph recipients had a shorter median time to the resolution of fever (2 days and 7 days, respectively; P = 0.09) and a shorter length of hospital stay (9 days and 14 days, respectively; P = 0.03). However, these findings are exploratory, and there were few differences in the other variables measured. High levels of opsonizing antibodies were maintained for the initial 4 weeks. Although the study was not powered to show efficacy, these preliminary findings and safety profile suggest that Altastaph may be an effective adjunct to antibiotics and warrants further investigation (ClinicalTrials.gov number NCT00063089).

  20. Clinical streptococcal isolates, distinct from Streptococcus pneumoniae, but containing the β-glucosyltransferase tts gene and expressing serotype 37 capsular polysaccharide.

    PubMed

    Sheppard, Carmen L; Kapatai, Georgia; Broughton, Karen; Schaefer, Ulf; Hannah, Matthew; Litt, David J; Fry, Norman K

    2017-01-01

    The major virulence factor of the pneumococcus, and target for conjugate vaccines, is the polysaccharide capsule, which is usually encoded by the highly variable cps locus. Serotype 37 is an unusual pneumococcal type in which the single β-glucosyltransferase gene responsible for serotype capsule production (tts) is located outside of the capsular operon region. Using a previously described automated whole genome sequence (WGS)-based serotyping bioinformatics tool, PneumoCaT, we identified and investigated seven clinical isolates (three from blood cultures) of non-pneumococcal streptococci containing a highly homologous tts and included them in a study panel of 20 isolates which included a 11 further clinical isolates of S. pneumoniae serotype 37, a reference strain of serotype 37 and the S. pseudopneumoniae type strain BAA 960(T). The seven non-pneumococcal isolates generated novel alleles at all pneumococcal MLST loci and gave low percentage similarity (<45%) to S. pneumoniae or S. pseudopneumoniae species by comparison of short sequence patterns in genomic data (k-mer analysis). The S. pseudopneumoniae BAA-960(T) isolate generated two novel alleles in the MLST and gave a high similarity (>99%) to the reference sequence for BAA-960(T). Twelve isolates gave high similarity (>77%) to the Streptococcus pneumoniae 5652-06 serotype 19A reference genome sequence and had previously reported MLST alleles. Each of the seven clinical non-pneumococcal strains and all of the 12 S. pneumoniae possessed a β-glycosyltransferase gene (tts) with >95% similarity to the pneumococcal tts reference DNA sequence with 20-22 non-synonymous SNPs. All but two strains in which the tts gene was detected gave positive reactions for serotype 37 in slide agglutination tests with serotype 37 typing sera. Phylogenetic analysis using both SNP and MLST data showed distinct clades corresponding to strains identified as pneumococcus or non-pneumococcus by kmer WGS analysis. Extended k

  1. Clinical streptococcal isolates, distinct from Streptococcus pneumoniae, but containing the β-glucosyltransferase tts gene and expressing serotype 37 capsular polysaccharide

    PubMed Central

    Kapatai, Georgia; Broughton, Karen; Schaefer, Ulf; Hannah, Matthew; Litt, David J.; Fry, Norman K.

    2017-01-01

    The major virulence factor of the pneumococcus, and target for conjugate vaccines, is the polysaccharide capsule, which is usually encoded by the highly variable cps locus. Serotype 37 is an unusual pneumococcal type in which the single β-glucosyltransferase gene responsible for serotype capsule production (tts) is located outside of the capsular operon region. Using a previously described automated whole genome sequence (WGS)-based serotyping bioinformatics tool, PneumoCaT, we identified and investigated seven clinical isolates (three from blood cultures) of non-pneumococcal streptococci containing a highly homologous tts and included them in a study panel of 20 isolates which included a 11 further clinical isolates of S. pneumoniae serotype 37, a reference strain of serotype 37 and the S. pseudopneumoniae type strain BAA 960T. The seven non-pneumococcal isolates generated novel alleles at all pneumococcal MLST loci and gave low percentage similarity (<45%) to S. pneumoniae or S. pseudopneumoniae species by comparison of short sequence patterns in genomic data (k-mer analysis). The S. pseudopneumoniae BAA-960T isolate generated two novel alleles in the MLST and gave a high similarity (>99%) to the reference sequence for BAA-960T. Twelve isolates gave high similarity (>77%) to the Streptococcus pneumoniae 5652-06 serotype 19A reference genome sequence and had previously reported MLST alleles. Each of the seven clinical non-pneumococcal strains and all of the 12 S. pneumoniae possessed a β-glycosyltransferase gene (tts) with >95% similarity to the pneumococcal tts reference DNA sequence with 20–22 non-synonymous SNPs. All but two strains in which the tts gene was detected gave positive reactions for serotype 37 in slide agglutination tests with serotype 37 typing sera. Phylogenetic analysis using both SNP and MLST data showed distinct clades corresponding to strains identified as pneumococcus or non-pneumococcus by kmer WGS analysis. Extended k-mer database

  2. Environmentally triggered genomic plasticity and capsular polysaccharide formation are involved in increased ethanol and acetic acid tolerance in Kozakia baliensis NBRC 16680.

    PubMed

    Brandt, Julia U; Born, Friederike-Leonie; Jakob, Frank; Vogel, Rudi F

    2017-08-10

    tolerance. The polE gene turned out to be involved in the formation of a cell-associated, capsular polysaccharide, which seems to be essential for increased ethanol/acetic tolerance in contrast to the secreted gum-cluster derived heteropolysaccharide. The genetic and morphological switch could represent an adaptive evolutionary step during the development of K. baliensis NBRC 16680 in course of changing environmental conditions.

  3. Thermodynamics and density of binding of a panel of antibodies to high-molecular-weight capsular polysaccharides.

    PubMed

    Harris, Shannon L; Fernsten, Philip

    2009-01-01

    The interaction between antipolysaccharide (anti-PS) antibodies and their antigens was investigated by the use of isothermal titration calorimetry to determine the thermodynamic binding constant (K), the change in the enthalpy of binding (DeltaH), and the binding density (N) to high-molecular-weight PSs. From these values, the change in the entropy of binding (DeltaS) was calculated. The thermodynamic parameters of binding to high-molecular-weight capsular PSs are reported for two monoclonal antibodies (MAbs) with different specificities for meningococcal serogroup C PS, five MAbs specific for different pneumococcal serotypes, and the Fab fragments of two antipneumococcal MAbs. The K values were in the range of 10(6) to 10(7) M(-1), and these values were 1 to 2 orders of magnitude greater than the previously reported K values derived from antibody-oligosaccharide interactions. The DeltaH associated with binding was favorable for each MAb and Fab fragment. The DeltaS associated with binding was also generally favorable for both the MAbs and the Fab fragments, with the exception of the anti-serotype 14 MAb and its Fab fragment. N provides information regarding how densely MAbs or Fabs can bind along PS chains and, as expressed in terms of monosaccharides, was very similar for the seven MAbs, with an average of 12 monosaccharides per bound MAb. The value of N for each Fab was smaller, with five or seven monosaccharides per bound Fab. These results suggest that steric interactions between antibody molecules are a major influence on the values of N of high-affinity MAbs to capsular PSs.

  4. Comparative structural and molecular characterization of ribitol-5-phosphate-containing Streptococcus oralis coaggregation receptor polysaccharides.

    PubMed

    Yang, Jinghua; Ritchey, Mary; Yoshida, Yasuo; Bush, C Allen; Cisar, John O

    2009-03-01

    The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfbeta1-6GalNAcbeta1-3Galalpha1-2ribitol-5-PO(4)(-)-6Galfbeta1-3Galbeta1](n), thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and ribitol-5-phosphate, which is alpha1-2 in strain SK144 versus alpha1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.

  5. Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol.

    PubMed

    Ravi, G; Venkatesh, Yeldur P

    2014-04-01

    D-Ribitol, a five-carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated D-ribose-BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27-30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid-Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol-KLH-Sepharose CL-6B resulted in pure ribitol-specific antibodies (~45-50 μg/mL). The affinity constant of ribitol antibodies was found to be 2.9 × 10(7) M(-1) by non-competitive ELISA. Ribitol antibodies showed 100% specificity towards ribitol, ~800% cross-reactivity towards riboflavin, 10-15% cross-reactivity with sorbitol, xylitol and mannitol, and 5-7% cross-reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4%) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol.

  6. Structure-function relationships of immunostimulatory polysaccharides: A review.

    PubMed

    Ferreira, Sónia S; Passos, Cláudia P; Madureira, Pedro; Vilanova, Manuel; Coimbra, Manuel A

    2015-11-05

    Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and enhance specific mechanisms of the host response. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. The structural features that have been related with such activity are the monosaccharide and glycosidic-linkage composition, conformation, molecular weight, functional groups, and branching characteristics. However, the establishment of structure-function relationships is possible only if purified and characterized polysaccharides are used and selective structural modifications performed. Aiming at contributing to the definition of the structure-function relationships necessary to design immunostimulatory polysaccharides with potential for preventive or therapeutical purposes or to be recognized as health-improving ingredients in functional foods, this review introduces basic immunological concepts required to understand the mechanisms that rule the potential claimed immunostimulatory activity of polysaccharides and critically presents a literature survey on the structural features of the polysaccharides and reported immunostimulatory activity.

  7. Capsular Polysaccharide of Mycoplasma ovipneumoniae Induces Sheep Airway Epithelial Cell Apoptosis via ROS-Dependent JNK/P38 MAPK Pathways

    PubMed Central

    Jiang, Zhongjia; Song, Fuyang; Li, Yanan; Xue, Di; Zhao, Ning; Zhang, Jiamei; Deng, Guangcun; Li, Min

    2017-01-01

    In an attempt to better understand the pathogen-host interaction between invading Mycoplasma ovipneumoniae (M. ovipneumoniae) and sheep airway epithelial cells, biological effects and possible molecular mechanism of capsular polysaccharide of M. ovipneumoniae (CPS) in the induction of cell apoptosis were explored using sheep bronchial epithelial cells cultured in air-liquid interface (ALI). The CPS of M. ovipneumoniae was first isolated and purified. Results showed that CPS had a cytotoxic effect by disrupting the integrity of mitochondrial membrane, accompanied with an increase of reactive oxygen species and decrease of mitochondrial membrane potential (ΔΨm). Of importance, the CPS exhibited an ability to induce caspase-dependent cell apoptosis via both intrinsic and extrinsic apoptotic pathways. Mechanistically, the CPS induced extrinsic cell apoptosis by upregulating FAS/FASL signaling proteins and cleaved-caspase-8 and promoted a ROS-dependent intrinsic cell apoptosis by activating a JNK and p38 signaling but not ERK1/2 signaling of mitogen-activated protein kinases (MAPK) pathways. These findings provide the first evidence that CPS of M. ovipneumoniae induces a caspase-dependent apoptosis via both intrinsic and extrinsic apoptotic pathways in sheep bronchial epithelial cells, which may be mainly attributed by a ROS-dependent JNK and p38 MAPK signaling pathways. PMID:28367270

  8. [Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Salmonella Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in persons from outbreak of typhoid fever].

    PubMed

    Rastawicki, Waldemar; Kałużewski, Stanisław

    2015-01-01

    The laboratory diagnosis of typhoid fever is dependent upon either isolation of S. Typhi from a clinical sample or the detection of raised titers of serum antibodies in the Widal test or the passive hemagglutination assay (PHA). In this study we evaluated the usefulness of ELISA for detection of antibodies to S. Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in the sera of persons from outbreak of typhoid fever. Fifteen serum samples from patients with laboratory confirmed typhoid fever and 140 sera from persons suspected for contact with typhoid fever patients from outbreak in 1974/75 in Poland were tested by ELISA. Additionally, as the control group, we tested 115 sera from blood donors for the presence of S. Typhi anti-LPS and anti-Vi antibodies. Anti-LPS and anti-Vi antibodies were detected in 80% and 53.3% of sera obtained from patients with laboratory confirmed typhoid fever, respectively. The high percentages of positive results in ELISA were also noted in the group of persons suspected for contact with typhoid fever patients (51.4% and 45%) but not in the group of blood donors (7.8% and 6.1%, respectively). The ELISA could be a useful tool for the serological diagnosis of typhoid fever in patients who have clinical symptoms but are culture negative, especially during massive outbreaks of typhoid fever.

  9. Expression of type 8 capsular polysaccharide and production of toxic shock syndrome toxin 1 are associated among vaginal isolates of Staphylococcus aureus.

    PubMed Central

    Lee, J C; Liu, M J; Parsonnet, J; Arbeit, R D

    1990-01-01

    A colony immunoblot method was developed for serotyping the capsular polysaccharides expressed by Staphylococcus aureus isolates. The method was rapid and specific and was performed with either polyclonal or monoclonal antibodies specific for each of the capsule types. S. aureus isolates were obtained from patients with toxic shock syndrome (TSS) or other staphylococcal infections and from asymptomatic women with vaginal colonization. Among the vaginal isolates of S. aureus, expression of the type 8 capsule was significantly (P less than 0.001) more frequent among strains that produced TSS toxin 1 (TSST-1) than it was among TSST-1-negative strains. In contrast, the frequency of type 8 capsule expression was similar among both TSST-1-positive and -negative strains of S. aureus from patients with nonvaginal TSS. When all vaginal and nonvaginal isolates were compared, TSST-1-negative S. aureus strains were equally distributed among the type 5 and 8 and nontypeable capsule groups, whereas TSST-1-positive strains were predominantly capsule type 8. Images PMID:2279990

  10. Comparative characterization of the virulence gene clusters (lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species.

    PubMed

    Richards, Vincent P; Lefébure, Tristan; Pavinski Bitar, Paulina D; Stanhope, Michael J

    2013-03-01

    Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosaccharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain-Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Comparative characterization of the virulence gene clusters (lipooligosacharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species

    PubMed Central

    Richards, Vincent P.; Lefébure, Tristan; Pavinski Bitar, Paulina D.; Stanhope, Michael J.

    2013-01-01

    Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosacharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking. PMID:23279811

  12. ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae

    PubMed Central

    Zheng, Yuqiang; Zhang, Xuemei; Wang, Xiaofang; Wang, Libin; Zhang, Jinghui; Yin, Yibing

    2017-01-01

    The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComED58E) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level. PMID

  13. ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae.

    PubMed

    Zheng, Yuqiang; Zhang, Xuemei; Wang, Xiaofang; Wang, Libin; Zhang, Jinghui; Yin, Yibing

    2017-01-01

    The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComE(D58E)) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level.

  14. Rapid Detection of Contagious Caprine Pleuropneumonia Using a Mycoplasma capricolum subsp. capripneumoniae Capsular Polysaccharide-Specific Antigen Detection Latex Agglutination Test

    PubMed Central

    March, J. B.; Gammack, C.; Nicholas, R.

    2000-01-01

    Latex microspheres (diameter, 8 μm) were coated with anti-Mycoplasma capricolum subsp. capripneumoniae polyclonal immunoglobulin G (IgG) antiserum (anti-F38 biotype). The coated microspheres, when used in a latex agglutination test (LAT), detected M. capricolum subsp. capripneumoniae antigen in the serum of goats with contagious caprine pleuropneumoniae (CCPP). Beads also agglutinated strongly in the presence of purified M. capricolum subsp. capripneumoniae capsular polysaccharide (CPS). Preabsorption of CPS-specific antibodies prior to coating of the beads removed agglutinating activity in the presence of M. capricolum subsp. capripneumoniae, strongly suggesting that CPS is the likely soluble antigen recognized by the test. In addition, the specificity of the LAT exactly mirrored that of an M. capricolum subsp. capripneumoniae CPS-specific monoclonal antibody (WM25): of the 8 other mycoplasma species tested, agglutination was observed only with bovine serogroup 7. The LAT detected all 11 strains of M. capricolum subsp. capripneumoniae examined in this study, with a sensitivity level of 2 ng of CPS, or the equivalent of 1.7 × 104 CFU, in a reaction volume of 0.03 ml of serum. With field sera from goats with CCPP, the results of the LAT exhibited a 67% correlation with the results of the currently used complement fixation test (CFT), with the main discrepancy in diagnosis resulting from the increased sensitivity of the LAT compared to that of CFT. This antigen-detection LAT should prove particularly useful in identifying animals in the earliest stages of CCPP and combines sensitivity and low cost with ease of application in the field, without the need for any specialist training or equipment. PMID:11060083

  15. Design and optimization of a chromatographic purification process for Streptococcus pneumoniae serotype 23F capsular polysaccharide by a Design of Experiments approach.

    PubMed

    Ji, Yu; Tian, Yang; Ahnfelt, Mattias; Sui, Lili

    2014-06-27

    Multivalent pneumococcal vaccines were used worldwide to protect human beings from pneumococcal diseases. In order to eliminate the toxic organic solutions used in the traditional vaccine purification process, an alternative chromatographic process for Streptococcus pneumoniae serotype 23F capsular polysaccharide (CPS) was proposed in this study. The strategy of Design of Experiments (DoE) was introduced into the process development to solve the complicated design procedure. An initial process analysis was given to review the whole flowchart, identify the critical factors of chromatography through FMEA and chose the flowthrough mode due to the property of the feed. A resin screening study was then followed to select candidate resins. DoE was utilized to generate a resolution IV fractional factorial design to further compare candidates and narrow down the design space. After Capto Adhere was selected, the Box-Behnken DoE was executed to model the process and characterize all effects of factors on the responses. Finally, Monte Carlo simulation was used to optimize the process, test the chosen optimal conditions and define the control limit. The results of three scale-up runs at set points verified the DoE and simulation predictions. The final results were well in accordance with the EU pharmacopeia requirements: Protein/CPS (w/w) 1.08%; DNA/CPS (w/w) 0.61%; the phosphorus content 3.1%; the nitrogen 0.315% and the Methyl-pentose percentage 47.9%. Other tests of final pure CPS also met the pharmacopeia specifications. This alternative chromatographic purification process for pneumococcal vaccine without toxic organic solvents was successfully developed by the DoE approach and proved scalability, robustness and suitability for large scale manufacturing.

  16. Sialylation of Group B Streptococcal Capsular Polysaccharide Is Mediated by cpsK and Is Required for Optimal Capsule Polymerization and Expression

    PubMed Central

    Chaffin, D. O.; Mentele, L. M.; Rubens, C. E.

    2005-01-01

    Several bacterial pathogens have evolved the means to escape immune detection by mimicking host cell surface carbohydrates that are crucial for self/non-self recognition. Sialic acid, a terminal residue on these carbohydrates, inhibits activation of the alternate pathway of complement by recruiting the immune modulating molecule factors H, I, and iC3b. Sialylation of capsular polysaccharide (CPS) is important for virulence of group B streptococci (GBS), a significant human pathogen. We previously reported that cpsK, a gene within the cps locus of type III GBS, could complement a sialyltransferase deficient lst mutant of Haemophilus ducreyi, implicating its role in sialylation of the GBS capsule. To explore the function of cpsK in GBS capsule production, we created a mutant in cpsK. Immunoblot analysis and enzyme-linked immunosorbent assay using anti-type III CPS antisera demonstrated that the mutant CPS did not contain sialic acid. This was confirmed by high-performance liquid chromatography after mild acid hydrolysis of the CPS. Although increased CPS chain length was seen for this strain, CPS production was <20% of the parental isolate. An episomal cpsK copy restored synthesis of sialo-CPS to wild-type levels. These data support our hypothesis that cpsK encodes the GBS CPS sialyltransferase and provide further evidence that lack of CPS oligosaccharide sialylation reduces the amount of CPS expressed on the cell surface. These observations also imply that one or more of the components involved in synthesis or transport of oligosaccharide repeating units requires a sialo-oligosaccharide for complete activity. PMID:15968073

  17. Antibodies to capsular polysaccharide and clumping factor A prevent mastitis and the emergence of unencapsulated and small-colony variants of Staphylococcus aureus in mice.

    PubMed

    Tuchscherr, Lorena P N; Buzzola, Fernanda R; Alvarez, Lucía P; Lee, Jean C; Sordelli, Daniel O

    2008-12-01

    The pathogenesis of Staphylococcus aureus infections is influenced by multiple virulence factors that are expressed under variable conditions, and this has complicated the design of an effective vaccine. Clinical trials that targeted the capsule or clumping factor A (ClfA) failed to protect the recipients against staphylococcal infections. We passively immunized lactating mice with rabbit antibodies to S. aureus capsular polysaccharide (CP) serotype 5 (CP5) or CP8 or with monoclonal antibodies to ClfA. Mice immunized with antibodies to CP5 or CP8 or with ClfA had significantly reduced tissue bacterial burdens 4 days after intramammary challenge with encapsulated S. aureus strains. After several passages in mice passively immunized with CP-specific antiserum, increasing numbers of stable unencapsulated variants of S. aureus were cultured from the infected mammary glands. Greater numbers of these unencapsulated S. aureus variants than of the corresponding encapsulated parental strains were internalized in vitro in MAC-T bovine cells. Furthermore, small-colony variants (SCVs) were recovered from the infected mammary glands after several passages in mice passively immunized with CP-specific antiserum. A combination of antibodies effectively sterilized mammary glands in a significant number of passively immunized mice. More importantly, passive immunization with antibodies to both CP and ClfA fully inhibited the emergence of unencapsulated "escape mutants" and significantly reduced the appearance of SCVs. A vaccine formulation comprising CP conjugates plus a surface-associated protein adhesin may be more effective than either antigen alone for prevention of S. aureus infections.

  18. Diversity of Capsular Polysaccharide Gene Clusters in Kpc-Producing Klebsiella pneumoniae Clinical Isolates of Sequence Type 258 Involved in the Italian Epidemic

    PubMed Central

    D’Andrea, Marco Maria; Amisano, Francesco; Giani, Tommaso; Conte, Viola; Ciacci, Nagaia; Ambretti, Simone; Santoriello, Luisa; Rossolini, Gian Maria

    2014-01-01

    Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cpsBO-4 and cps207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cpsBO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cpsBO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cpsBO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps207-2 was only detected in ST258 strains with a more restricted distribution. PMID:24823690

  19. Diversity of capsular polysaccharide gene clusters in Kpc-producing Klebsiella pneumoniae clinical isolates of sequence type 258 involved in the Italian epidemic.

    PubMed

    D'Andrea, Marco Maria; Amisano, Francesco; Giani, Tommaso; Conte, Viola; Ciacci, Nagaia; Ambretti, Simone; Santoriello, Luisa; Rossolini, Gian Maria

    2014-01-01

    Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cpsBO-4 and cps207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cpsBO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cpsBO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cpsBO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps207-2 was only detected in ST258 strains with a more restricted distribution.

  20. USA300 and USA500 Clonal Lineages of Staphylococcus aureus Do Not Produce a Capsular Polysaccharide Due to Conserved Mutations in the cap5 Locus

    PubMed Central

    Li, Xue; Alam, Md Tauqeer; Read, Timothy D.; Sieth, Julia; Cywes-Bentley, Colette; Dobbins, Ginette; David, Michael Z.; Kumar, Neha; Eells, Samantha J.; Miller, Loren G.; Boxrud, David J.; Chambers, Henry F.; Lynfield, Ruth; Lee, Jean C.; Daum, Robert S.

    2015-01-01

    ABSTRACT The surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP−). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP−. Whole-genome sequence analysis of 146 CP− USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates. PMID:25852165

  1. STEPWISE INTRATYPE TRANSFORMATION OF PNEUMOCOCCUS FROM R TO S BY WAY OF A VARIANT INTERMEDIATE IN CAPSULAR POLYSACCHARIDE PRODUCTION

    PubMed Central

    MacLeod, Colin M.; Krauss, Marjorie R.

    1947-01-01

    1. A variant intermediate between the classical R and S forms has been isolated by selective procedures from a rough strain of pneumococcus originally derived from Type II S. 2. The intermediate variant D39/Int53 is avirulent for mice, forms rough colonies, and does not possess a demonstrable capsule. However, it synthesizes SSSII which is immunologically indistinguishable from that produced by fully encapsulated pneumococcus Type II, though in much smaller amount. The polysaccharide is present as a surface component and as it exists in the cell is highly antigenic for rabbits. 3. An extract of the intermediate variant causes the transformation in vitro of an R strain into a variant resembling the intermediate in SSSII production but without any apparent alteration in the colonial characteristics of the R variant. 4. The intermediate variant is convertible in vivo, into a fully encapsulated strain of pneumococcus Type II. Transformation of the intermediate to a heterologous type of pneumococcus (Type III) was unsuccessful. 5. A method is described for the preparation of transforming extracts of pneumococci utilizing the massive growth of the organisms obtained in the presence of a large concentration of glucose. PMID:19871689

  2. Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans

    PubMed Central

    2004-01-01

    UDP-glucose dehydrogenase catalyses the conversion of UDP-glucose into UDP-GlcA, a critical precursor for glycan synthesis across evolution. We have cloned the gene encoding this important enzyme from the opportunistic pathogen Cryptococcus neoformans. In this fungus, UDP-GlcA is required for the synthesis of capsule polysaccharides, which in turn are essential for virulence. The gene was expressed in Escherichia coli and the 51.3-kDa recombinant protein from wild-type and five mutants was purified for analysis. The cryptococcal enzyme is strongly inhibited by UDP-xylose and NADH, has highest activity at pH 7.5 and demonstrates Km (app) values of 0.1 and 1.5 mM for NAD+ and UDP-glucose respectively. Its activity was significantly decreased by mutations in the putative sites of NAD+ and UDP-glucose binding. Unlike previously reported eukaryotic UDP-glucose dehydrogenases, which are hexamers, the cryptococcal enzyme is a dimer. PMID:15030319

  3. The nature of an in vivo anti-capsular polysaccharide response is markedly influenced by the composition and/or architecture of the bacterial subcapsular domain.

    PubMed

    Arjunaraja, Swadhinya; Massari, Paola; Wetzler, Lee M; Lees, Andrew; Colino, Jesus; Snapper, Clifford M

    2012-01-15

    In vivo anti-polysaccharide Ig responses to isolated polysaccharide (PS) are T cell independent, rapid, and fail to generate memory. However, little is known regarding PS-specific Ig responses to intact gram-positive and gram-negative extracellular bacteria. We previously demonstrated that intact heat-killed Streptococcus pneumoniae, a gram-positive bacterium, elicited a rapid primary pneumococcal capsular PS (PPS) response in mice that was dependent on CD4(+) T cells, B7-dependent costimulation, and CD40-CD40L interactions. However, this response was ICOS independent and failed to generate a boosted PPS-specific secondary IgG response. In the current study, we analyzed the murine meningococcal type C PS (MCPS)-specific Ig response to i.p.-injected intact, heat-killed Neisseria meningitidis, serogroup C (MenC), a gram-negative bacterium. In contrast to S. pneumoniae, the IgG anti-MCPS response to MenC exhibited delayed primary kinetics and was highly boosted after secondary immunization, whereas the IgG anti-MCPS response to isolated MCPS was rapid, without secondary boosting, and consisted of only IgG1 and IgG3, as opposed to all four IgG isotypes in response to intact MenC. The secondary, but not primary, IgG anti-MCPS response to MenC was dependent on CD4(+) T cells, CD40L, CD28, and ICOS. The primary and secondary IgG anti-MCPS responses were lower in TLR4-defective (C3H/HeJ) but not TLR2(-/-) or MyD88(-/-) mice, but secondary boosting was still observed. Of interest, coimmunization of S. pneumoniae and MenC resulted in a boosted secondary IgG anti-PPS response to S. pneumoniae. Our data demonstrate that the nature of the in vivo anti-PS response is markedly influenced by the composition and/or architecture of the bacterial subcapsular domain.

  4. Preparation and characterization of a Staphylococcus aureus capsular polysaccharide-protein conjugate prepared by a low cost technique: a proof-of-concept study.

    PubMed

    Pujato, Nazarena; Díaz, Germán; Barbagelata, María Sol; Vicco, Miguel Hernán; Calvinho, Luis Fernando; Marcipar, Iván Sergio

    2015-01-01

    Staphylococcus aureus is a worldwide distributed pathogen that produces several diseases in many species and is the major cause of mastitis in dairy cows. S. aureus capsular polysaccharide 5 (CP5) has been widely proposed as a vaccine candidate since it is expressed in a high proportion of isolates from intramammary infections and is able to induce opsonophagocytic antibodies. However, to reach immunological properties, polysaccharides need to be coupled to carrier proteins. The aim of this study was to evaluate a conjugation method employing p-benzoquinone (PBQ), which was not previously reported for the development of vaccine components. Purified S. aureus CP5 was coupled to human serum albumin (HSA) with high efficiency, reaching a rate PS/protein of 0.5. Mice groups were immunized at days 0, 14, 28, and 42, with the conjugate (CP5-HSAPBQ), free CP5, or PBS, formulated with incomplete Freund adjuvant, and after 3 months, they were challenged with free CP5 to evaluate the memory response. IgG and IgM isotypes were measured on serum samples all along the experiment, and IgG subclasses were determined to analyze the humoral profile. In contrast to the response obtained with free CP5, CP5-HSAPBQ induced IgG titers of 1/238,900 after three doses and a memory response was observed after the challenge. Results indicate that immunization with CP5-HSAPBQ effectively induce a T-dependent immune response against CP5. Moreover, besides IgG2a was the main subtype obtained, the joint production of specific IgG1, IgG2b, and IgG3 types indicated a balanced humoral response. As p-benzoquinone conjugation of CPs to proteins is far less expensive and straightforward than other methods commonly used in vaccine preparations, the robust humoral response obtained using this method points out that this can be an interesting alternative to prepare S. aureus CP5 conjugate vaccines.

  5. Structural diversity of lytic polysaccharide monooxygenases.

    PubMed

    Vaaje-Kolstad, Gustav; Forsberg, Zarah; Loose, Jennifer Sm; Bissaro, Bastien; Eijsink, Vincent Gh

    2017-01-10

    Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds and represent a promising resource for development of industrial enzyme cocktails for biomass processing. LPMOs show high sequence and modular diversity and are known, so far, to cleave insoluble substrates such as cellulose, chitin and starch, as well as hemicelluloses such as beta-glucan, xyloglucan and xylan. All LPMOs share a catalytic histidine brace motif to bind copper, but differ strongly when it comes to the nature and arrangement of residues on the substrate-binding surface. In recent years, the number of available LPMO structures has increased rapidly, including the first structure of an enzyme-substrate complex. The insights gained from these structures is reviewed below.

  6. Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus.

    PubMed

    Byankina Barabanova, A O; Sokolova, E V; Anastyuk, S D; Isakov, V V; Glazunov, V P; Volod'ko, A V; Yakovleva, I M; Solov'eva, T F; Yermak, I M

    2013-10-15

    Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked β-D-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked β-D-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.

  7. Constitutive Expression of the Vi Polysaccharide Capsular Antigen in Attenuated Salmonella enterica Serovar Typhi Oral Vaccine Strain CVD 909

    PubMed Central

    Wang, Jin Yuan; Noriega, Fernando R.; Galen, James E.; Barry, Eileen; Levine, Myron M.

    2000-01-01

    Live oral Ty21a and parenteral Vi polysaccharide vaccines provide significant protection against typhoid fever, albeit by distinct immune mechanisms. Vi stimulates serum immunoglobulin G Vi antibodies, whereas Ty21a, which does not express Vi, elicits humoral and cell-mediated immune responses other than Vi antibodies. Protection may be enhanced if serum Vi antibody as well as cell-mediated and humoral responses can be stimulated. Disappointingly, several new attenuated Salmonella enterica serovar Typhi oral vaccines (e.g., CVD 908-htrA and Ty800) that elicit serum O and H antibody and cell-mediated responses following a single dose do not stimulate serum Vi antibody. Vi expression is regulated in response to environmental signals such as osmolarity by controlling the transcription of tviA in the viaB locus. To investigate if Vi antibodies can be stimulated if Vi expression is rendered constitutive, we replaced PtviA in serovar Typhi vaccine CVD 908-htrA with the constitutive promoter Ptac, resulting in CVD 909. CVD 909 expresses Vi even under high-osmolarity conditions and is less invasive for Henle 407 cells. In mice immunized with a single intranasal dose, CVD 909 was more immunogenic than CVD 908-htrA in eliciting serum Vi antibodies (geometric mean titer of 160 versus 49, P = 0.0007), whereas O antibody responses were virtually identical (geometric mean titer of 87 versus 80). In mice challenged intraperitoneally with wild-type serovar Typhi 4 weeks after a single intranasal immunization, the mortality of those immunized with CVD 909 (3 of 8) was significantly lower than that of control mice (10 of 10, P = 0.043) or mice given CVD 908-htrA (9 of 10, P = 0.0065). PMID:10899868

  8. The K1 Capsular Polysaccharide of Acinetobacter baumannii Strain 307-0294 Is a Major Virulence Factor ▿

    PubMed Central

    Russo, Thomas A.; Luke, Nicole R.; Beanan, Janet M.; Olson, Ruth; Sauberan, Shauna L.; MacDonald, Ulrike; Schultz, L. Wayne; Umland, Timothy C.; Campagnari, Anthony A.

    2010-01-01

    Acinetobacter baumannii is a pathogen of increasing medical importance with a propensity to be multidrug resistant, thereby making treatment challenging. Little is known of virulence traits in A. baumannii. To identify virulence factors and potential drug targets, random transposon (Tn) mutants derived from the A. baumannii strain AB307-0294 were screened to identify genes essential for growth in human ascites fluid in vitro, an inflammatory exudative fluid. These studies led to the identification of two genes that were predicted to be required for capsule polymerization and assembly. The first, ptk, encodes a putative protein tyrosine kinase (PTK), and the second, epsA, encodes a putative polysaccharide export outer membrane protein (EpsA). Monoclonal antibodies used in flow cytometric and Western analyses confirmed that these genes are required for a capsule-positive phenotype. A capsule-positive phenotype significantly optimized growth in human ascites fluid, survival in human serum, and survival in a rat soft tissue infection model. Importantly, the clearance of the capsule-minus mutants AB307.30 (ptk mutant, capsule minus) and AB307.45 (epsA mutant, capsule minus) was complete and durable. These data demonstrated that the K1 capsule from AB307-0294 was an important protectin. Further, these data suggested that conserved proteins, which contribute to the capsule-positive phenotype, are potential antivirulence drug targets. Therefore, the results from this study have important biologic and translational implications and, to the best of our knowledge, are the first to address the role of capsule in the pathogenesis of A. baumannii infection. PMID:20643860

  9. Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus.

    PubMed

    Lin, W S; Cunneen, T; Lee, C Y

    1994-11-01

    We previously cloned a 19.4-kb DNA region containing a cluster of genes affecting type 1 capsule production from Staphylococcus aureus M. Subcloning experiments showed that these capsule (cap) genes are localized in a 14.6-kb region. Sequencing analysis of the 14.6-kb fragment revealed 13 open reading frames (ORFs). Using complementation tests, we have mapped a collection of Cap- mutations in 10 of the 13 ORFs, indicating that these 10 genes are involved in capsule biosynthesis. The requirement for the remaining three ORFs in the synthesis of the capsule was demonstrated by constructing site-specific mutations corresponding to each of the three ORFs. Using an Escherichia coli S30 in vitro transcription-translation system, we clearly identified 7 of the 13 proteins predicted from the ORFs. Homology search between the predicted proteins and those in the data bank showed very high homology (52.3% identity) between capL and vipA, moderate homology (29% identity) between capI and vipB, and limited homology (21.8% identity) between capM and vipC. The vipA, vipB, and vipC genes have been shown to be involved in the biosynthesis of Salmonella typhi Vi antigen, a homopolymer polysaccharide consisting of N-acetylgalactosamino uronic acid, which is also one of the components of the staphylococcal type 1 capsule. The homology between these sets of genes therefore suggests that capL, capI, and capM may be involved in the biosynthesis of amino sugar, N-acetylgalactosamino uronic acid. In addition, the search showed that CapG aligned well with the consensus sequence of a family of acetyltransferases from various prokaryotic organisms, suggesting that CapG may be an acetyltransferase. Using the isogenic Cap- and Cap+ strains constructed in this study, we have confirmed that type 1 capsule is an important virulence factor in a mouse lethality test.

  10. Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus.

    PubMed Central

    Lin, W S; Cunneen, T; Lee, C Y

    1994-01-01

    We previously cloned a 19.4-kb DNA region containing a cluster of genes affecting type 1 capsule production from Staphylococcus aureus M. Subcloning experiments showed that these capsule (cap) genes are localized in a 14.6-kb region. Sequencing analysis of the 14.6-kb fragment revealed 13 open reading frames (ORFs). Using complementation tests, we have mapped a collection of Cap- mutations in 10 of the 13 ORFs, indicating that these 10 genes are involved in capsule biosynthesis. The requirement for the remaining three ORFs in the synthesis of the capsule was demonstrated by constructing site-specific mutations corresponding to each of the three ORFs. Using an Escherichia coli S30 in vitro transcription-translation system, we clearly identified 7 of the 13 proteins predicted from the ORFs. Homology search between the predicted proteins and those in the data bank showed very high homology (52.3% identity) between capL and vipA, moderate homology (29% identity) between capI and vipB, and limited homology (21.8% identity) between capM and vipC. The vipA, vipB, and vipC genes have been shown to be involved in the biosynthesis of Salmonella typhi Vi antigen, a homopolymer polysaccharide consisting of N-acetylgalactosamino uronic acid, which is also one of the components of the staphylococcal type 1 capsule. The homology between these sets of genes therefore suggests that capL, capI, and capM may be involved in the biosynthesis of amino sugar, N-acetylgalactosamino uronic acid. In addition, the search showed that CapG aligned well with the consensus sequence of a family of acetyltransferases from various prokaryotic organisms, suggesting that CapG may be an acetyltransferase. Using the isogenic Cap- and Cap+ strains constructed in this study, we have confirmed that type 1 capsule is an important virulence factor in a mouse lethality test. Images PMID:7961465

  11. Concentration of anti-pneumococcal capsular polysaccharide IgM, IgG and IgA specific antibodies in adult blood donors.

    PubMed

    Parker, Antony R; Allen, Syreeta; Harding, Stephen

    2016-08-01

    Anti-pneumococcal capsular polysaccharide (PCP) IgM, IgG and IgA ELISAs have been developed to aid assessment of the adaptive immune system. The relationship between the concentrations of PCP IgM, IgG, and IgA was investigated. The concentrations of PCP IgM, IgG, and IgA were measured in sera obtained from 231 adult blood donors. Concentrations of each isotype were not normally distributed. The median concentration for PCP IgM was 54 U/mL (range 37-75 U/mL), IgG 40 mg/L (range 26-79 mg/L) and IgA 21 U/mL (range 13-44 U/mL). The median PCP IgM titres decreased with age and were significantly lower in patients aged 81-90 years compared to those aged 18-80 years. By contrast, there was a significantly higher median serum PCP IgG titre in the 61-90 years group compared to those aged 18-60 years and a significantly higher median serum PCP IgA titre in the 51-90 years group compared to those aged 18-50 years. The correlation between PCP IgG and IgA was more significant than between IgM and IgA and between IgM and IgG. Correlation of PCP IgA and IgM concentrations identified four phenotypes: high PCP IgM and IgA; high PCP IgM only; high PCP IgA only; and low PCP IgM and IgA. A significant number of individuals with a PCP IgG concentration >50 mg/L had low PCP IgA and IgM concentrations. The additional measurement of PCP IgA and PCP IgM, alongside PCP IgG, in individuals investigated for a compromised immune system may provide a more detailed antibody profile.

  12. The capsular network of Klebsiella pneumoniae.

    PubMed

    Cassone, A; Garaci, E

    1977-06-01

    Attempts at improving chemical fixation for electron-microscopic observation of the capsule of Klebsiella pneumoniae were made. The capsule was preserved by using alcian blue - lanthanum and tris-(1-aziridinyl) phosphine oxide (TAPO) - aldehyde - osmium procedures. Despite the different retention of the overall capsular material and minor variations in morphological details, in both cases the interpretation of ultrastructural patterns suggested that the capsule be composed of a meshed network of thin polysaccharide fibrils radiating from the cell wall. This organization is in keeping with all recognized chemical properties of bacterial polysaccharide capsules or, at least, does not contradict them. Moreover, an effective preservation of bacterial structures other than capsule has been obtained, mostly in specimens fixed by the TAPO-aldehyde-osmium method, a fact which gives further reliability to the technical approach used for capsule visualization.

  13. Group B Streptococcus and Streptococcus suis Capsular Polysaccharides Induce Chemokine Production by Dendritic Cells via Toll-Like Receptor 2- and MyD88-Dependent and -Independent Pathways

    PubMed Central

    Calzas, Cynthia; Goyette-Desjardins, Guillaume; Lemire, Paul; Gagnon, Fleur; Lachance, Claude; Van Calsteren, Marie-Rose

    2013-01-01

    Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1β (IL-1β), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner. PMID:23774593

  14. The KL24 gene cluster and a genomic island encoding a Wzy polymerase contribute genes needed for synthesis of the K24 capsular polysaccharide by the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51.

    PubMed

    Kenyon, Johanna J; Kasimova, Anastasiya A; Shneider, Mikhail M; Shashkov, Alexander S; Arbatsky, Nikolay P; Popova, Anastasiya V; Miroshnikov, Konstantin A; Hall, Ruth M; Knirel, Yuriy A

    2017-03-01

    The whole-genome sequence of the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51 belonging to sequence type ST103 (Institut Pasteur scheme) revealed that the set of genes at the capsule locus, KL24, includes four genes predicted to direct the synthesis of 3-acetamido-3,6-dideoxy-d-galactose (d-Fuc3NAc), and this sugar was found in the capsular polysaccharide (CPS). One of these genes, fdtE, encodes a novel bifunctional protein with an N-terminal FdtA 3,4-ketoisomerase domain and a C-terminal acetyltransferase domain. KL24 lacks a gene encoding a Wzy polymerase to link the oligosaccharide K units to form the CPS found associated with isolate RCH51, and a wzy gene was found in a small genomic island (GI) near the cpn60 gene. This GI is in precisely the same location as another GI carrying wzy and atr genes recently found in several A. baumannii isolates, but it does not otherwise resemble it. The CPS isolated from RCH51, studied by sugar analysis and 1D and 2D 1H and 13C NMR spectroscopy, revealed that the K unit has a branched pentasaccharide structure made up of Gal, GalNAc and GlcNAc residues with d-Fuc3NAc as a side branch, and the K units are linked via a β-d-GlcpNAc-(1→3)-β-d-Galp linkage formed by the Wzy encoded by the GI. The functions of the glycosyltransferases encoded by KL24 were assigned to formation of specific bonds. A correspondence between the order of the genes in KL24 and other KL and the order of the linkages they form was noted, and this may be useful in future predictions of glycosyltransferase specificities.

  15. The KL24 gene cluster and a genomic island encoding a Wzy polymerase contribute genes needed for synthesis of the K24 capsular polysaccharide by the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51.

    PubMed

    Kenyon, Johanna J; Kasimova, Anastasiya A; Shneider, Mikhail; Shashkov, Alexander S; Arbatsky, Nikolay P; Popova, Anastasiya V; Miroshnikov, Konstantin A; Hall, Ruth M; Knirel, Yuriy A

    2017-01-17

    The whole genome sequence of the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51 belonging to sequence type ST103 (Institut Pasteur scheme) revealed that the set of genes at the capsule locus, KL24, includes four genes predicted to direct the synthesis of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) and this sugar was found in the capsular polysaccharide (CPS). One of these genes, fdtE, encodes a novel bifunctional protein with an N-terminal FdtA 3,4-ketoisomerase domain and a C-terminal acetyltransferase domain. KL24 lacks a gene encoding a Wzy polymerase to link the oligosaccharide K units to form the CPS found associated with isolate RCH51, and a wzy gene was found in a small genomic island (GI) near cpn60 gene. This GI is in precisely the same location as a GI carrying wzy and atr genes recently found in several A. baumannii isolates, but does not otherwise resemble it. The CPS isolated from RCH51, studied by sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopy, revealed that the K unit has a branched pentasaccharide structure made up of Gal, GalNAc and GlcNAc residues with D-Fuc3NAc as a side branch, and the K units are linked via a β-D-GlcpNAc-(1→3)-β-D-Galp linkage formed by the Wzy encoded by the GI. The functions of the glycosyltransferases encoded by KL24 were assigned to formation of specific bonds. A correspondence between the order of the genes in KL24 and other KL and the order of the linkages they form was noted, and this may be useful in future predictions of glycosyltransferase specificities.

  16. Structural modification of polysaccharides: A biochemical-genetic approach

    NASA Technical Reports Server (NTRS)

    Kern, Roger G.; Petersen, Gene R.

    1991-01-01

    Polysaccharides have a wide range of industrial and biomedical applications. An industry trend is underway towards the increased use of bacteria to produce polysaccharides. Long term goals of this work are the adaptation and enhancement of saccharide properties for electronic and optic applications. In this report we illustrate the application of enzyme-bearing bacteriophage on strains of the enteric bacterium Klebsiella pneumoniae, which produces a polysaccharide with the relatively rare rheological property of drag-reduction. This has resulted in the production of new polysaccharides with enhanced rheological properties. Our laboratory is developing techniques for processing and structurally modifying bacterial polysaccharides and oligosaccharides which comprise their basic polymeric repeat units. Our research has focused on bacteriophage which produce specific polysaccharide degrading enzymes. This has lead to the development of enzymes generated by bacteriophage as tools for polysaccharide modification and purification. These enzymes were used to efficiently convert the native material to uniform-sized high molecular weight polymers, or alternatively into high-purity oligosaccharides. Enzyme-bearing bacteriophage also serve as genetic selection tools for bacteria that produce new families of polysaccharides with modified structures.

  17. Synthesis and immunological properties of conjugates composed of group B streptococcus type III capsular polysaccharide covalently bound to tetanus toxoid.

    PubMed Central

    Lagergard, T; Shiloach, J; Robbins, J B; Schneerson, R

    1990-01-01

    A synthetic scheme for covalently binding group B streptococcus type III to tetanus toxoid (TT), using adipic acid dihydrazide as a spacer, is described. Type III alone or as a conjugate with TT was injected subcutaneously into laboratory mice, and the type-specific and TT antibody responses elicited by these immunogens were assayed. Type III-TT elicited significantly higher levels of type-specific antibodies after each immunization than did the type III alone. These levels were related to the dosage of the conjugate, enhanced by Freund adjuvant, and exhibited booster responses. Type III alone elicited only immunoglobulin M (IgM) antibodies in Swiss albino mice and mostly IgM and low levels of IgG antibodies of the IgG3 subclass in BALB/c mice. Type III-TT conjugates, in contrast, elicited mostly IgG antibodies in both strains of mice. IgA type III antibodies were not detected. The first two immunizations with the conjugates elicited type III antibodies in the IgG1 and in the IgG3 subclasses. Low levels of IgG2a type III antibodies were detected after a third injection of type III-TT. Conjugate-induced antibodies facilitated opsonization of group B streptococcus type III organisms and did not react with the structurally related pneumococcus type 14. TT alone or as a component of type III-TT induced mostly antibodies of the IgG class: IgG1 levels were the highest of the four subclasses. No IgA TT antibodies were detected. The conjugation procedure, therefore, enhanced the immunogenicity of and conferred T-cell dependent properties to the type III while preserving the immunogenicity of the TT component. The T-cell dependent properties of the conjugates were responsible for stimulating IgG type III antibodies which could be boosted. Evaluation of type III-TT conjugates in antibody-negative women of child-bearing age is planned. PMID:2407652

  18. Chemical Structures and Bioactivities of Sulfated Polysaccharides from Marine Algae

    PubMed Central

    Jiao, Guangling; Yu, Guangli; Zhang, Junzeng; Ewart, H. Stephen

    2011-01-01

    Sulfated polysaccharides and their lower molecular weight oligosaccharide derivatives from marine macroalgae have been shown to possess a variety of biological activities. The present paper will review the recent progress in research on the structural chemistry and the bioactivities of these marine algal biomaterials. In particular, it will provide an update on the structural chemistry of the major sulfated polysaccharides synthesized by seaweeds including the galactans (e.g., agarans and carrageenans), ulvans, and fucans. It will then review the recent findings on the anticoagulant/antithrombotic, antiviral, immuno-inflammatory, antilipidemic and antioxidant activities of sulfated polysaccharides and their potential for therapeutic application. PMID:21566795

  19. The structural properties of the lateral retinaculum and capsular complex of the knee

    PubMed Central

    Merican, Azhar M.; Sanghavi, Sanjay; Iranpour, Farhad; Amis, Andrew A.

    2009-01-01

    Although lateral retinacular releases are not uncommon, there is very little scientific knowledge about the properties of these tissues, on which to base a rationale for the surgery. We hypothesised that we could identify specific tissue bands and measure their structural properties. Eight fresh-frozen knees were dissected, and the lateral soft tissues prepared into three distinct structures: a broad tissue band linking the iliotibial band (ITB) to the patella, and two capsular ligaments: patellofemoral and patellomeniscal. These were individually tensile tested to failure by gripping the patella in a vice jaw and the soft tissues in a freezing clamp. Results: the ITB–patellar band was strongest, at a mean of 582 N, and stiffest, at 97 N/mm. The patellofemoral ligament failed at 172 N with 16 N/mm stiffness; the patellomeniscal ligament failed at 85 N, with 13 N/mm stiffness. These structural properties suggest that most of the load in-vivo is transmitted to the patella by the transverse fibres that originate from the ITB. PMID:19647256

  20. Combined meningococcal serogroup A and W135 outer-membrane vesicles activate cell-mediated immunity and long-term memory responses against non-covalent capsular polysaccharide A.

    PubMed

    Romeu, Belkis; Lastre, Miriam; García, Luis; Cedré, Bárbara; Mandariote, Aleida; Fariñas, Mildrey; Oliva, Reynaldo; Rosenqvist, Einar; Pérez, Oliver

    2014-01-01

    Outer-membrane vesicles (OMVs) have inherent adjuvant properties, and many vaccines use OMV as vaccine components. Utilizing the adjuvant properties of OMV could lead to the formulation of vaccines that are less expensive and potentially more immunogenic than covalently conjugated polysaccharide vaccines. We evaluated the adjuvant effect in Balb/c mice of combinations of OMV from Neisseria meningitidis serogroup A and W135 as compared to that of the non-covalently conjugated capsular polysaccharide A. Both antigens were adsorbed onto aluminum hydroxide. The mice were given a booster dose of plain polysaccharide A to stimulate an immunologic memory response. Subclasses determination and cytokine assays demonstrated the capacity of OMV to induce a IgG2a/IgG2b isotype profile and IFN-γ production, suggesting the induction of a Th1 pattern immune response. Lymphoproliferative responses to OMVs were high, with affinity maturation of antibodies observed. Bactericidal titers after the booster dose were also observed. Memory B cells and long-term memory T cells were also detected. The results of this study indicate that combined meningococcal serogroup A and W135 OMV can activate cell-mediated immunity and induce a long-term memory response.

  1. Correlation Between Chain Architecture and Hydration Water Structure in Polysaccharides

    NASA Astrophysics Data System (ADS)

    Grossutti, Michael; Dutcher, John

    The physical properties of confined water can differ dramatically from those of bulk water. Hydration water associated with polysaccharides provides a particularly important example of confined water, with differences in polysaccharide structure providing different spatially confined environments for water adsorption. We have used attenuated total reflection infrared (ATR-IR) spectroscopy to investigate the structure of hydration water in films of three different polysaccharides under controlled relative humidity (RH) conditions. We compare the results obtained for films of highly branched, monodisperse phytoglycogen nanoparticles to those obtained for two unbranched polysaccharides, hyaluronic acid (HA) and chitosan. We find similarities between water structuring in the two linear polysaccharides, and significant differences for phytoglycogen. In particular, the phytoglycogen nanoparticles exhibited high network water connectivity, and a large increase in the fraction of multimer water clusters with increasing RH, whereas the water structure for HA and chitosan was found to be insensitive to changes in RH. These measurements provide unique insight into the relationship between the chain architecture and hydration of polysaccharides.

  2. Structural and functional comparison of polysaccharide-degrading enzymes.

    PubMed

    Jedrzejas, M J

    2000-01-01

    Sugar molecules as well as enzymes degrading them are ubiquitously present in physiological systems, especially for vertebrates. Polysaccharides have at least two aspects to their function, one due to their mechanical properties and the second one involves multiple regulatory processes or interactions between molecules, cells, or extracellular space. Various bacteria exert exogenous pressures on their host organism to diversity glycans and their structures in order for the host organism to evade the destructive function of such microbes. Many bacterial organism produce glycan-degrading enzymes in order to facilitate their invasion of host tissues. Such polysaccharide degrading enzymes utilize mainly two modes of polysaccharide-degradation, a hydrolysis and a beta-elimination process. The three-dimensional structures of several of these enzymes have been elucidated recently using X-ray crystallography. There are many common structural motifs among these enzymes, mainly the presence of an elongated cleft transversing these molecules which functions as a polysaccharide substrate binding site as well as the catalytic site for these enzymes. The detailed structural information obtained about these enzymes allowed formulation of proposed mechanisms of their action. The polysaccharide lyases utilize a proton acceptance and donation mechanism (PAD), whereas polysaccharide hydrolases use a direct double displacement (DD) mechanism to degrade their substrates.

  3. Differential Regulation of IgG Anti-Capsular Polysaccharide and Antiprotein Responses to Intact Streptococcus pneumoniae in the Presence of Cognate CD4+ T Cell Help

    DTIC Science & Technology

    2004-01-01

    polysaccharide (PPS14), the phosphorylcholine determinant of the cell wall C-polysaccharide, and the cell wall protein, pneumococcal surface protein A...a more rapid delivery of CD4 T cell help. In contrast, the IgG anti- phosphorylcholine response, although also dependent on CD4 T cells, is TCR...and/or IgG isotypes specific for the phosphorylcholine (PC) determinant of the cell wall C-polysaccharide (teichoic acid) and for the cell wall

  4. Anticorrosive Microbial Polysaccharides: Structure-Function Relationships

    USDA-ARS?s Scientific Manuscript database

    Water-soluble microbial polysaccharides are often implicated in biofilm formation and are believed to mediate cell-cell aggregation and adhesion to surfaces. Generally, biofilm formation is considered harmful or undesirable, as it leads to increased drag, plugging of pores, dimished heat transfer, ...

  5. Synthetic trimer and tetramer of 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys.

    PubMed Central

    Peeters, C C; Evenberg, D; Hoogerhout, P; Käyhty, H; Saarinen, L; van Boeckel, C A; van der Marel, G A; van Boom, J H; Poolman, J T

    1992-01-01

    Synthetic oligosaccharides derived from the capsular polysaccharide (PRP) of Haemophilus influenzae type b were conjugated to carrier proteins via a thioether linkage. Conjugates were made of trimeric and tetrameric ribose-ribitol-phosphate and tetanus toxoid or diphtheria toxin. All conjugates elicited anti-PRP antibody responses with an increasing immunoglobulin G/immunoglobulin M ratio in adult mice and monkeys. Trimer conjugates elicited lower anti-PRP antibody responses compared with tetramer conjugates. Adult monkeys responded equally well to the tetrameric oligosaccharide-tetanus toxoid conjugate as to the oligosaccharide-CRM197 conjugate (HbOC), which elicits protective levels of serum antibodies in human infants after two or three injections. PMID:1563770

  6. Hypolipidemic effects of crude green tea polysaccharide on rats, and structural features of tea polysaccharides isolated from the crude polysaccharide.

    PubMed

    Nakamura, Michiko; Miura, Sayaka; Takagaki, Akiko; Nanjo, Fumio

    2017-05-01

    Crude tea polysaccharide (crude TPS) was prepared from instant green tea by ethanol precipitation followed by ultrafiltration membrane treatment and its effects on blood lipid, liver lipid, and fecal lipid levels were examined with Sprague-Dawley rats fed a high-fat diet. Although crude TPS showed no effects on the serum lipid levels, it suppressed the liver lipid accumulation and increased the fecal excretion of dietary fat. Then, the structural features of crude TPS were investigated. After separation of crude TPS by DEAE-cellulose and gel-filtration column chromatography, two kinds of neutral tea polysaccharides (NTPS-LP and NTPS-HH) and an acidic polysaccharide (ATPS-MH) were obtained. According to monosaccharide composition, methylation, and NMR analyses, NTPS-LP, NPTS-HH, and ATPS-MH were presumed to be starch, arabinogalactan with β-1,3-linked galactosyl backbone blanched at position 6 and with 1,5-linked arabinofuranosyl residues, and α-1,4-linked galacturonic acid backbone with arabinogalactan region, respectively.

  7. Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.

    PubMed Central

    Pazzani, C; Rosenow, C; Boulnois, G J; Bronner, D; Jann, K; Roberts, I S

    1993-01-01

    The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters. Images PMID:8397187

  8. Structural characterization of polysaccharides from bamboo

    NASA Astrophysics Data System (ADS)

    Kamil, Ruzaimah Nik Mohamad; Yusuf, Nur'aini Raman; Yunus, Normawati M.; Yusup, Suzana

    2014-10-01

    The alkaline and water soluble polysaccharides were isolate by sequential extractions with distilled water, 60% ethanol containing 1%, 5% and 8% NaOH. The samples were prepared at 60 °C for 3 h from local bamboo. The functional group of the sample were examined using FTIR analysis. The most precipitate obtained is from using 60% ethanol containing 8% NaOH with yield of 2.6%. The former 3 residues isolated by sequential extractions with distilled water, 60% ethanol containing 1% and 5% NaOH are barely visible after filtering with cellulose filter paper. The FTIR result showed that the water-soluble polysaccharides consisted mainly of OH group, CH group, CO indicates the carbohydrate and sugar chain. The sample weight loss was slightly decreased with increasing of temperature.

  9. Current status of meningococcal group B vaccine candidates: capsular or noncapsular?

    PubMed Central

    Diaz Romero, J; Outschoorn, I M

    1994-01-01

    Meningococcal meningitis is a severe, life-threatening infection for which no adequate vaccine exists. Current vaccines, based on the group-specific capsular polysaccharides, provide short-term protection in adults against serogroups A and C but are ineffective in infants and do not induce protection against group B strains, the predominant cause of infection in western countries, because the purified serogroup B polysaccharide fails to elicit human bactericidal antibodies. Because of the poor immunogenicity of group B capsular polysaccharide, different noncapsular antigens have been considered for inclusion in a vaccine against this serogroup: outer membrane proteins, lipooligosaccharides, iron-regulated proteins, Lip, pili, CtrA, and the immunoglobulin A proteases. Alternatively, attempts to increase the immunogenicity of the capsular polysaccharide have been made by using noncovalent complexes with outer membrane proteins, chemical modifications, and structural analogs. Here, we review the strategies employed for the development of a vaccine for Neisseria meningitidis serogroup B; the difficulties associated with the different approaches are discussed. PMID:7834605

  10. Structural characteristics of a bioactive polysaccharide from Sorghum arundinaceum.

    PubMed

    da Silva, Bernadete P; Silva, Graziela M; Mendes, Tatiana P; Parente, José P

    2003-01-01

    A polysaccharide, an alpha-D-glucan with an apparent molecular weight of 6.85 x 10(4), called PSa glucan, was isolated from fresh seeds of Sorghum arundinaceum by fractionation on Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that it has a highly branched glucan type structure composed of alpha-(1-->4) linked D-glucopyranose residues with (1-->3), (1-->6) branching points, and a significant amount of alpha-(1-->6) branching to alpha-(1-->3) linked D-glucopyranose residues. The anti-inflammatory activity of the polysaccharide was performed using the capillary permeability assay.

  11. Composition and Partial Structure Characterization of Tremella Polysaccharides

    PubMed Central

    2009-01-01

    Heteropolysaccharides isolated from liquid cultures of nine Tremella species contained 0.3 to 1.2% protein, 2.7 to 5% ash, 0.9 to 3.4% acetyl groups, 76.5 to 84.2% carbohydrates and trace amounts of starch. The polysaccharides in aqueous solution were slightly acidic (pH 5.1 to 5.6). They consisted of the following monomeric sugars: fucose, ribose, xylose, arabinose, mannose, galactose, glucose and glucuronic acid. The backbones of the polysaccharide structures consisted of α-(1→3)-links while the side chains were β-linked. PMID:23983549

  12. Structure of pectic polysaccharides from sunflower salts-soluble fraction

    USDA-ARS?s Scientific Manuscript database

    The manuscript discusses the structural features of pectin polysaccharides extracted from seedless sunflower head residues. The analysis using 1H, 13C and two-dimensional gHSQC NMR showed various numbers of methyl and hydroxyl groups attached to the anomeric carbons in the pectin backbone at differe...

  13. Polysaccharide-Based Vaccines

    NASA Astrophysics Data System (ADS)

    Santana, Violeta Fernández; Balbin, Yury Valdés; Calderón, Janoi Chang; Icart, Luis Peña; Verez-Bencomo, Vicente

    Capsular polysaccharides (CPS) and lipopolysaccharides from bacteria are employed for the production of vaccines against human diseases. Initial development of CPS as a vaccine was followed by the development and introduction of conjugate polysaccharide-protein vaccines. The principles leading to both developments are reviewed.

  14. Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism.

    PubMed Central

    Hannesson, H H; Hagner-McWhirter, A; Tiedemann, K; Lindahl, U; Malmström, A

    1996-01-01

    The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ([GlcA(beta 1-->3)GalNAc(beta 1-->4)]n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate). Vmax was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed. PMID:8573097

  15. [Antigenic bacterial polysaccharides. 24. The structure of the O-specific polysaccharide chain of Salmonella arizonae 063 (Arizona 08) lipopolysaccharide].

    PubMed

    Vinogradov, E V; Knirel', Iu A; Lipkind, G M; Shashkov, A S; Kochetkov, N K

    1987-10-01

    The O-specific polysaccharide chain of the Salmonella arizonae O63 lipopolysaccharide is composed of D-glucose, D-galactose, N-acetyl-D-galactosamine, and 3-acetamido-3,6-dideoxy-D-galactose (Fuc3NAc) residues in the ratio 1:1:2:1. On the basis of methylation analysis and calculations of 13C-NMR-spectra of the polysaccharide and of the product of its selective cleavage with anhydrous hydrogen fluoride, the linear polymer lacking 3-acetamido-3,6-dideoxygalactose, it was concluded that the polysaccharide has the following structure: (Formula: see text).

  16. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure.

    PubMed

    Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé; Garajova, Sona; Foucat, Loïc; Falourd, Xavier; Saake, Bodo; Berrin, Jean-Guy; Cathala, Bernard

    2017-01-10

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers.

  17. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

    PubMed Central

    Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé; Garajova, Sona; Foucat, Loïc; Falourd, Xavier; Saake, Bodo; Berrin, Jean-Guy; Cathala, Bernard

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers. PMID:28071716

  18. Functional and Structural Characterization of Polysaccharide Co-polymerase Proteins Required for Polymer Export in ATP-binding Cassette Transporter-dependent Capsule Biosynthesis Pathways*

    PubMed Central

    Larue, Kane; Ford, Robert C.; Willis, Lisa M.; Whitfield, Chris

    2011-01-01

    Neisseria meningitidis serogroup B and Escherichia coli K1 bacteria produce a capsular polysaccharide (CPS) that is composed of α2,8-linked polysialic acid (PSA). Biosynthesis of PSA in these bacteria occurs via an ABC (ATP-binding cassette) transporter-dependent pathway. In N. meningitidis, export of PSA to the surface of the bacterium requires two proteins that form an ABC transporter (CtrC and CtrD) and two additional proteins, CtrA and CtrB, that are proposed to form a cell envelope-spanning export complex. CtrA is a member of the outer membrane polysaccharide export (OPX) family of proteins, which are proposed to form a pore to mediate export of CPSs across the outer membrane. CtrB is an inner membrane protein belonging to the polysaccharide co-polymerase (PCP) family. PCP proteins involved in other bacterial polysaccharide assembly systems form structures that extend into the periplasm from the inner membrane. There is currently no structural information available for PCP or OPX proteins involved in an ABC transporter-dependent CPS biosynthesis pathway to support their proposed roles in polysaccharide export. Here, we report cryo-EM images of purified CtrB reconstituted into lipid bilayers. These images contained molecular top and side views of CtrB and showed that it formed a conical oligomer that extended ∼125 Å from the membrane. This structure is consistent with CtrB functioning as a component of an envelope-spanning complex. Cross-complementation of CtrA and CtrB in E. coli mutants with defects in genes encoding the corresponding PCP and OPX proteins show that PCP-OPX pairs require interactions with their cognate partners to export polysaccharide. These experiments add further support for the model of an ABC transporter-PCP-OPX multiprotein complex that functions to export CPS across the cell envelope. PMID:21454677

  19. Nasal immunization of mice with AFCo1 or AFPL1 plus capsular polysaccharide Vi from Salmonella typhi induces cellular response and memory B and T cell responses.

    PubMed

    Romeu, Belkis; Lastre, Miriam; Reyes, Laura; González, Elizabeth; Borrero, Yusnaby; Lescaille, Diandra; Pérez, Rocmira; Nuñez, Darzy; Pérez, Oliver

    2014-12-05

    The response to infection against Salmonella involves both B and T cell mediated immunity. An effective immunization can activate an adequate immune response capable to control the primary infection and protect against a secondary infection. Mucosal vaccination, by inducing local pathogen-specific immune responses, has the potential to counter mucosally transmitted pathogens at the portal of entry, thereby increasing the efficacy of vaccines. The aim of this work was to explore the efficacy of AFCo1 or AFPL1, as mucosal adjuvants to stimulate cell immunity and memory responses against Vi polysaccharide antigen of Salmonella typhi (PsVi). Mice immunized with 3 intranasal doses exhibited high levels of PsVi-specific IgG (p<0.05), IgG2a and IgG2c subclasses. Also, an amplified recall response after a booster immunization with a plain polysaccharide vaccine was induced. Avidities index were higher in mice immunized with adjuvanted formulations at different chaotropic concentrations. Furthermore, IL-12 and IFN-γ levels in nasally vaccinated mice with both adjuvants were induced. Moreover, priming with 3 doses followed by booster immunization with VaxTyVi(®) resulted in high levels of anti-Vi specific IgG, IgG subclasses and antibody avidity. Long lived plasma cells in bone marrow, memory B cells and long-term memory T cells after booster dose were induced. The combined formulation of Vi polysaccharide with mucosal adjuvants provides an improved immunogenicity, in particular with regard to cellular responses and long lasting cells responses.

  20. Structural elucidation of polysaccharide fractions from brown seaweed Sargassum pallidum.

    PubMed

    Ye, Hong; Zhou, Chunhong; Li, Wei; Hu, Bing; Wang, Xiaoqing; Zeng, Xiaoxiong

    2013-09-12

    The structural characteristics of two purified fractions of polysaccharides from Sargassum pallidum (SPS) were investigated in the present study. As results, the molecular weights of the two polysaccharide fractions, SPS-3-1 and SPS-3-2, were determined to be 5.87 and 7.25 kDa, respectively. SPS-3-1 was composed of glucose, mannose and galactose in a molar ratio of 11.18:1.00:0.96, while SPS-3-2 was composed of fucose, xylose, mannose, glucose and galactose in a molar ratio of 2.53:0.61:1.00:0.46:0.92. Both SPS-3-1 and SPS-3-2 exhibited the characteristics of polysaccharide in the frequency range of 4000-400 cm(-1) based on their Fourier-transform infrared spectra. Furthermore, the results of periodic acid oxidation, Smith degradation, methylation analysis and nuclear magnetic resonance spectroscopic analysis suggested that SPS-3-2 was composed of (1→4)-linked fucopyranosyl backbone and (1→3)-linked galactopyranosyl, (1→3)-linked mannopyranosyl, (1→2)-linked xylopyranosyl and (1→6)-linked glucopyranosyl branch chains.

  1. Antibacterial Activity of Cold Atmospheric Pressure Argon Plasma against 78 Genetically Different (mecA, luk-P, agr or Capsular Polysaccharide Type) Staphylococcus aureus Strains.

    PubMed

    Matthes, Rutger; Lührman, Anne; Holtfreter, Silva; Kolata, Julia; Radke, Dörte; Hübner, Nils-Olaf; Assadian, Ojan; Kramer, Axel

    2016-01-01

    Previous studies on the antimicrobial activity of cold atmospheric pressure argon plasma showed varying effects against mecA+ or mecA-Staphylococcus aureus strains. This observation may have important clinical and epidemiological implications. Here, the antibacterial activity of argon plasma was investigated against 78 genetically different S. aureus strains, stratified by mecA, luk-P, agr1-4, or the cell wall capsule polysaccharide types 5 and 8. kINPen09® served as the plasma source for all experiments. On agar plates, mecA+luk-P-S. aureus strains showed a decreased susceptibility against plasma compared to other S. aureus strains. This study underlines the high complexity of microbial defence against antimicrobial treatment and confirms a previously reported strain-dependent susceptibility of S. aureus to plasma treatment. © 2016 S. Karger AG, Basel.

  2. The structure of mushroom polysaccharides and their beneficial role in health.

    PubMed

    Huang, Xiaojun; Nie, Shaoping

    2015-10-01

    Mushroom is a kind of fungus that has been popular for its special flavour and renowned biological values. The polysaccharide contained in mushroom is regarded as one of the primary bioactive constituents and is beneficial for health. The structural features and bioactivities of mushroom polysaccharides have been studied extensively. It is believed that the diverse biological bioactivities of polysaccharides are closely related to their structure or conformation properties. In this review, the structural characteristics, conformational features and bioactivities of several mushroom polysaccharides are summarized, and their beneficial mechanisms and the relationships between their structure and bioactivities are also discussed.

  3. Structural studies of the O-specific polysaccharide(s) from the lipopolysaccharide of Azospirillum brasilense type strain Sp7.

    PubMed

    Sigida, Elena N; Fedonenko, Yuliya P; Shashkov, Alexander S; Zdorovenko, Evelina L; Konnova, Svetlana A; Ignatov, Vladimir V; Knirel, Yuriy A

    2013-10-18

    Lipopolysaccharide was obtained by phenol-water extraction from dried bacterial cells of Azospirillum brasilense type strain Sp7. Mild acid hydrolysis of the lipopolysaccharide followed by GPC on Sephadex G-50 resulted in a polysaccharide mixture, which was studied by composition and methylation analyses, Smith degradation and (1)H and (13)C NMR spectroscopy. The following polysaccharide structures were established, where italics indicate a non-stoichiometric (∼40%) 2-O-methylation of l-rhamnose. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. [Antigenic bacterial polysaccharides. 23. The structure of the O-specific polysaccharide chain of Salmonella arizonae 059 lipopolysaccharide].

    PubMed

    Vinogradov, E V; Knirel', Iu A; Lipkind, G M; Shashkov, A S; Kochetkov, N K

    1987-09-01

    The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.

  5. EXTRACELLULAR POLYSACCHARIDES OF AZOTOBACTER VINELANDII1

    PubMed Central

    Cohen, Gary H.; Johnstone, Donald B.

    1964-01-01

    Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Extracellular polysaccharides of Azotobacter vinelandii. J. Bacteriol. 88:329–338. 1964.—Extracellular polysaccharides synthetized by Azotobacter vinelandii strains 155, 102, and 3A were shown to be carboxylic acid heteropolysaccharides of apparent high molecular weight. Cells were grown in a nitrogen-free, mineral broth medium with 2% sucrose. Extracellular slime was recovered by centrifugation and purified by repeated alcohol precipitation and Sevag deproteinization. Capsular polysaccharide was recovered from washed cells by mild alkaline digestion. Methods of isolation and purification appeared to provide polysaccharide showing no evidence of heterogeneity when examined by chemical and physical methods. Infrared analysis of purified slime from the three strains suggested fundamental structural similarities. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed slime polysaccharide indicated that the polymers contained in common galacturonic acid, [α] d-glucose, and rhamnose at a ratio of approximately 43:2:1, as well as a hexuronic acid lactone, probably mannurono-lactone. However, as shown by chemical and infrared analysis, minor differences did exist; namely, slime from strain 155 and 102 contained o-acetyl groups, whereas slime from strain 3A contained none. A sialic acid-like component (1.5% of dry weight of the polysaccharide, calculated as N-acetyl neuraminic acid), was found only in the slime of strain 155. Capsular polysaccharide composition closely resembled that for slime. It is of interest that the major slime components were identical whether the energy source provided for the cells was sucrose, glucose, fructose, or ethanol. PMID:14203348

  6. Observation of the helical structure of the bacterial polysaccharide acetan by atomic force microscopy.

    PubMed Central

    Kirby, A R; Gunning, A P; Morris, V J; Ridout, M J

    1995-01-01

    A method has been developed that has been found to give reproducible images of uncoated polysaccharides by Atomic Force Microscopy (AFM). Aqueous solutions of the polysaccharide are deposited as drops onto freshly cleaved mica surfaces, air dried, and then imaged under butanol. The method has been used to obtain images of the bacterial polysaccharide acetan. In regions within the deposited sample, where the molecules are aligned side-by-side, it has been possible to observe a periodic structure along the polysaccharide chain, attributable to the helical structure of acetan. Images FIGURE 1 FIGURE 3 FIGURE 4 PMID:7711262

  7. Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.

    PubMed

    Sani, Musa; Houben, Edith N G; Geurtsen, Jeroen; Pierson, Jason; de Punder, Karin; van Zon, Maaike; Wever, Brigitte; Piersma, Sander R; Jiménez, Connie R; Daffé, Mamadou; Appelmelk, Ben J; Bitter, Wilbert; van der Wel, Nicole; Peters, Peter J

    2010-03-05

    The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.

  8. Spatial structure of plant cell wall polysaccharides and its functional significance.

    PubMed

    Gorshkova, T A; Kozlova, L V; Mikshina, P V

    2013-07-01

    Plant polysaccharides comprise the major portion of organic matter in the biosphere. The cell wall built on the basis of polysaccharides is the key feature of a plant organism largely determining its biology. All together, around 10 types of polysaccharide backbones, which can be decorated by different substituents giving rise to endless diversity of carbohydrate structures, are present in cell walls of higher plants. Each of the numerous cell types present in plants has cell wall with specific parameters, the features of which mostly arise from the structure of polymeric components. The structure of polysaccharides is not directly encoded by the genome and has variability in many parameters (molecular weight, length, and location of side chains, presence of modifying groups, etc.). The extent of such variability is limited by the "functional fitting" of the polymer, which is largely based on spatial organization of the polysaccharide and its ability to form supramolecular complexes of an appropriate type. Consequently, the carrier of the functional specificity is not the certain molecular structure but the certain type of the molecules having a certain degree of heterogeneity. This review summarizes the data on structural features of plant cell wall polysaccharides, considers formation of supramolecular complexes, gives examples of tissue- and stage-specific polysaccharides and functionally significant carbohydrate-carbohydrate interactions in plant cell wall, and presents approaches to analyze the spatial structure of polysaccharides and their complexes.

  9. Optimization of polysaccharides extraction from watermelon rinds: Structure, functional and biological activities.

    PubMed

    Romdhane, Molka Ben; Haddar, Anissa; Ghazala, Imen; Jeddou, Khawla Ben; Helbert, Claire Boisset; Ellouz-Chaabouni, Semia

    2017-02-01

    In the present work, optimization of hot water extraction, structural characteristics, functional properties, and biological activities of polysaccharides extracted from watermelon rinds (WMRP) were investigated. The physicochemical characteristics and the monosaccharide composition of these polysaccharides were then determined using chemical composition analysis, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and gas chromatography-flame ionization detection (GC-FID). SEM images showed that extracted polysaccharides had a rough surface with many cavities. GC-FID results proved that galactose was the dominant sugar in the extracted polysaccharides, followed by arabinose, glucose, galacturonic acid, rhamnose, mannose, xylose and traces of glucuronic acid. The findings revealed that WMRP displayed excellent antihypertensive and antioxidant activities. Those polysaccharides had also a protection effect against hydroxyl radical-induced DNA damage. Functional properties of extracted polysaccharides were also evaluated. WMRP showed good interfacial dose-dependent proprieties. Overall, the results suggested that WMRP presents a promising natural source of antioxidants and antihypertensive agents.

  10. [The structure of the glycerophosphate-containing O-specific polysaccharide from Escherichia coli 0130].

    PubMed

    Perepelov, A V; Lu, B; Sebchenkova, S N; Shevelev, S D; Wang, V; Shashkov, A S; Feng, L; Wang, L; Knirel', Iu A

    2007-01-01

    A phosphorylated O-specific polysaccharide was obtained by mild acidic degradation of the lipopolysaccharide from the intestinal bacterium Escherichia coli 0130 and characterized by the methods of chemical analysis, including dephosphorylation, and 1H and 13C NMR spectroscopy. The polysaccharide was shown to be composed of branched tetrasaccharide repeating units containing two N-acetyl-D-galactosamine residues, D-galactose, D-glucose, and glycerophosphate residues (one of each). The polysaccharide has the following structure, which is unique among the known bacterial polysaccharides.

  11. Structural characteristics and antioxidant activities of polysaccharides from longan seed.

    PubMed

    Jiang, Guoxiang; Wen, Lingrong; Chen, Feng; Wu, Fuwang; Lin, Sen; Yang, Bao; Jiang, Yueming

    2013-01-30

    Ultrasound-assisted extraction was employed to extract polysaccharides from longan seed (LSP), with aids of a Box-Behnken statistical design to investigate the effects of ultrasonic power, time and liquid/solid ratio on the extraction recovery of the LSP. The structural analysis indicated that arabinose, galactose, glucose and mannose were major components of LSP, with →6)-Gal-(→1, Glc-(→1 and →6)-Glc-(1→ glycosidic linkages. In an in vitro antioxidant activity of the 1,1-diphenyl-2-picryldydrazyl radical-scavenging assay, LSP exhibited a dose-dependent property within the concentration range tested. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. The Effect of a BSA Conjugate of a Synthetic Hexasaccharide Related to the Fragment of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 on the Activation of Innate and Adaptive Immune Responses

    PubMed Central

    Akhmatova, Nelli K.; Kurbatova, Ekaterina A.; Akhmatov, Elvin A.; Egorova, Nadezhda B.; Logunov, Denis Yu.; Gening, Marina L.; Sukhova, Elena V.; Yashunsky, Dmitry V.; Tsvetkov, Yury E.; Nifantiev, Nikolay E.

    2016-01-01

    We report the effect of a bovine serum albumin (BSA) conjugate of a synthetic hexasaccharide (HS) related to the fragment of the capsular polysaccharide (PS) of Streptococcus pneumoniae type 14 on the stimulation of innate immune system and the subsequent development of a PS-specific antibody response. Glycoconjugate (GC) in the presence (GC + AL) or absence of aluminum hydroxide was administered to mice twice. GC increased the number of TLR2-expressing cells and induced the maturation of dendritic cells (CD11c+, CD80+ and, MHCII+), which secreted IL-1β, IL-6, and TNFα into the culture medium. The level of IL-1β, IL-10, IFNγ, and TNFα in the blood increased within 24 h after the single GC administration to mice. On day 7, the numbers of splenic CD4+ and CD8+ T lymphocytes and B lymphocytes increased. After the second immunization, the levels of CD4+ and CD8+ T lymphocytes were lower than in the control, whereas the B cell, NK cell, and MHC class II-expressing cell numbers remained enhanced. However, of the presence of anti-PS, IgG antibodies were not detected. The addition of aluminum hydroxide to GC stimulated the production of GM-CSF, IL-1β, IL-5, IL-6, IL-10, IL-17, IFNγ, and TNFα. Anti-PS IgG1 antibody titers 7 days after the second immunization were high. During that period, normal levels of splenic CD4+ T lymphocytes were maintained, whereas reduced CD8+ T lymphocyte numbers and increased levels of B lymphocytes, NK cells, and MHC class II-expressing cell numbers were observed. Anti-PS IgG levels diminished until day 92. A booster immunization with GC + AL stimulated the production of anti-PS IgG memory antibodies, which were determined within 97 days. The elucidation of specific features of the effect of the synthetic HS conjugate on the stimulation of innate, cell-mediated immunity, and antibody response can favor the optimization of GC vaccine design. PMID:27446078

  13. Structural insights into antibody recognition of mycobacterial polysaccharides.

    PubMed

    Murase, Tomohiko; Zheng, Ruixiang Blake; Joe, Maju; Bai, Yu; Marcus, Sandra L; Lowary, Todd L; Ng, Kenneth K S

    2009-09-18

    Mycobacteria are major human pathogens responsible for such serious and widespread diseases as tuberculosis and leprosy. Among the evolutionary adaptations essential for pathogenicity in mycobacteria is a complex carbohydrate-rich cell-wall structure that contains as a major immunomodulatory molecule the polysaccharide lipoarabinomannan (LAM). We report here crystal structures of three fragments from the non-reducing termini of LAM in complex with a murine antibody Fab fragment (CS-35Fab). These structures reveal for the first time the three-dimensional structures of key components of LAM and the molecular basis of LAM recognition at between 1.8- and 2.0-A resolution. The antigen-binding site of CS-35Fab forms three binding pockets that show a high degree of complementarity to the reducing end, the branch point and one of the non-reducing ends of the Y-shaped hexasaccharide moiety found at most of the non-reducing termini of LAM. Structures of CS-35Fab bound to two additional tetrasaccharides confirm the general mode of binding seen in the hexasaccharide and indicate how different parts of LAM are recognized. Altogether, these structures provide a rational basis for understanding the overall architecture of LAM and identify the key elements of an epitope that may be exploited for the development of novel and more effective anti-mycobacterial vaccines. Moreover, this study represents the first high-resolution X-ray crystallographic investigation of oligofuranoside-protein recognition.

  14. Structural peculiarities of the O-specific polysaccharides of Azospirillum bacteria of serogroup III.

    PubMed

    Fedonenko, Yu P; Boiko, A S; Zdorovenko, E L; Konnova, S A; Shashkov, A S; Ignatov, V V; Knirel, Yu A

    2011-07-01

    Lipopolysaccharides and O-specific polysaccharides were isolated from the outer membrane of bacterial cells of three strains belonging to two Azospirillum species, and their structures were established by monosaccharide analysis including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy. It was shown that while having the identical composition, the O-polysaccharides have different branched tetrasaccharide repeating units. Two neutral polysaccharides were found in the lipopolysaccharide of A. brasilense 54, and the structure for the predominant O-polysaccharide was determined. The structural data, together with results of serological studies, enabled assignment of strains examined to a novel serogroup, III. The chemical basis for the serological relatedness among the azospirilla of this serogroup is presumably the presence of a common →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→ oligosaccharide motif in their O-polysaccharides.

  15. Split of Chiral Degeneracy in Mechanical and Structural Properties of Oligopeptide-Polysaccharide Biomaterials

    SciTech Connect

    Taraban, Marc B.; Hyland, Laura L.; Yu, Y. Bruce

    2013-09-23

    Enantiomeric biomaterials which are mirror images of each other are characterized by chiral degeneracy—identical structural characteristics and bulk material properties. The addition of another chiral component, d-polysaccharide, has been shown to split such degeneracy and result in two distinct biomaterials. Dynamic oscillatory rheometry and small-angle X-ray scattering demonstrate that the natural biochirality combination of l-peptides and d-polysaccharides assembles faster, has higher elastic moduli (G'), and is structurally more beneficial as opposed to the alternative d-peptide and d-polysaccharide combination. Chemical modifications of the OH-groups in α-d-glucose units in d-polysaccharides weaken such splitting of chiral degeneracy. These findings form a basis to design novel biomaterials and provide additional insight on why proteins and polysaccharides have oppoiste chirality in the biological world.

  16. Sequence diversity within the capsular genes of Streptococcus pneumoniae serogroup 6 and 19.

    PubMed

    Elberse, Karin; Witteveen, Sandra; van der Heide, Han; van de Pol, Ingrid; Schot, Corrie; van der Ende, Arie; Berbers, Guy; Schouls, Leo

    2011-01-01

    The main virulence factor of Streptococcus pneumoniae is the capsule. The polysaccharides comprising this capsule are encoded by approximately 15 genes and differences in these genes result in different serotypes. The aim of this study was to investigate the sequence diversity of the capsular genes of serotypes 6A, 6B, 6C, 19A and 19F and to explore a possible effect of vaccination on variation and distribution of these serotypes in the Netherlands. The complete capsular gene locus was sequenced for 25 serogroup 6 and for 20 serogroup 19 isolates. If one or more genes varied in 10 or more base pairs from the reference sequence, it was designated as a capsular subtype. Allele-specific PCRs and specific gene sequencing of highly variable capsular genes were performed on 184 serogroup 6 and 195 serogroup 19 isolates to identify capsular subtypes. This revealed the presence of 6, 3 and a single capsular subtype within serotypes 6A, 6B and 6C, respectively. The serotype 19A and 19F isolates comprised 3 and 4 capsular subtypes, respectively. For serogroup 6, the genetic background, as determined by multi locus sequence typing (MLST) and multiple-locus variable number of tandem repeat analysis (MLVA), seemed to be closely related to the capsular subtypes, but this was less pronounced for serogroup 19 isolates. The data also suggest shifts in the occurrence of capsular subtypes within serotype 6A and 19A after introduction of the 7-valent pneumococcal vaccine. The shifts within these non-vaccine serotypes might indicate that these capsular subtypes are filling the niche of the vaccine serotypes. In conclusion, there is considerable DNA sequence variation of the capsular genes within pneumococcal serogroup 6 and 19. Such changes may result in altered polysaccharides or in strains that produce more capsular polysaccharides. Consequently, these altered capsules may be less sensitive for vaccine induced immunity.

  17. Structure and organization of phospholipid/polysaccharide nanoparticles

    NASA Astrophysics Data System (ADS)

    Gerelli, Y.; Di Bari, M. T.; Deriu, A.; Cantù, L.; Colombo, P.; Como, C.; Motta, S.; Sonvico, F.; May, R.

    2008-03-01

    In recent years nanoparticles and microparticles composed of polymeric or lipid material have been proposed as drug carriers for improving the efficacy of encapsulated drugs. For the production of these systems different materials have been proposed, among them phospholipids and polysaccharides due to their biocompatibility, biodegradability, low cost and safety. We report here a morphological and structural investigation, performed using cryo-TEM, static light scattering and small angle neutron and x-ray scattering, on phospholipid/saccharide nanoparticles loaded with a lipophilic positively charged drug (tamoxifen citrate) used in breast cancer therapy. The lipid component was soybean lecithin; the saccharide one was chitosan that usually acts as an outer coating increasing vesicle stability. The microscopy and scattering data indicate the presence of two distinct nanoparticle families: uni-lamellar vesicles with average radius 90 Å and multi-lamellar vesicles with average radius 440 Å. In both families the inner core is occupied by the solvent. The presence of tamoxifen gives rise to a multi-lamellar structure of the lipid outer shell. It also induces a positive surface charge into the vesicles, repelling the positively charged chitosan molecules which therefore do not take part in nanoparticle formation.

  18. Advances in antitumor polysaccharides from phellinus sensu lato: Production, isolation, structure, antitumor activity, and mechanisms.

    PubMed

    Yan, Jing-Kun; Pei, Juan-Juan; Ma, Hai-Le; Wang, Zhen-Bin; Liu, Yuan-Shuai

    2017-04-13

    Edible and medicinal fungi (mushrooms) are widely applied to functional foods and nutraceutical products because of their proven nutritive and medicinal properties. Phellinus sensu lato is a well-known medicinal mushroom that has long been used in preventing ailments, including gastroenteric dysfunction, diarrhea, hemorrhage, and cancers, in oriental countries, particularly in China, Japan, and Korea. Polysaccharides represent a major class of bioactive molecules in Phellinus s. l., which have notable antitumor, immunomodulatory, and medicinal properties. Polysaccharides that were isolated from fruiting bodies, cultured mycelia, and filtrates of Phellinus s. l. have not only activated different immune responses of the host organism but have also directly suppressed tumor growth and metastasis. Studies suggest that polysaccharides from Phellinus s. l. are promising alternative anticancer agents or synergizers for existing antitumor drugs. This review summarizes the recent development of polysaccharides from Phellinus s. l., including polysaccharide production, extraction and isolation methods, chemical structure, antitumor activities, and mechanisms of action.

  19. Structural characterization and biomedical properties of sulfated polysaccharide from the gladius of Sepioteuthis lessoniana (Lesson, 1831).

    PubMed

    Seedevi, Palaniappan; Moovendhan, Meivelu; Vairamani, Shanmugam; Shanmugam, Annian

    2016-04-01

    Sulfated polysaccharide was extracted from the internal shell (gladius) of Sepioteuthis lessoniana. The sulfated polysaccharide contained 61.3% of carbohydrate, 0.8% of protein, 28.2% of ash and 1.33% of moisture respectively. The elemental composition was analyzed using CHNS/O analyzer. The molecular weight of sulfated polysaccharide determined through PAGE was found to be as 66 kDa. Monosaccharides analysis revealed that sulfated polysaccharide was composed of rhamnose, galactose, xylose and glucose. The structural features of sulfated polysaccharide were analyzed by FT-IR and NMR spectroscopy. Further the sulfated polysaccharide was evaluated for its antibacterial activity against selected human clinical pathogens, namely Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Vibrio cholerae, Klebsiella oxytoca, Escherichia coli, Salmonella paratyphi, Proteus mirabilis, Vibrio parahaemolyticus and Streptococcus pyogenes using agar well diffusion method. The polysaccharide has showed good antibacterial activity and MIC and MBC have also been evaluated. The anticancer activity was tested against HeLa cell line by MTT assay. The Cytotoxic Concentration (CC50) was observed as 700 μg/ml and the maximum anticancer activity of 62.89% was recorded at 200 μg/ml; whereas, the lowest of 9.87% was observed at 25 μg/ml. In conclusion, the sulfated polysaccharide is an alternate, non-toxic and cheap source of substance that showed good antibacterial and anticancer acitivity.

  20. New structure for the O-polysaccharide of Providencia alcalifaciens O27 and revised structure for the O-polysaccharide of Providencia stuartii O43.

    PubMed

    Ovchinnikova, Olga G; Bushmarinov, Ivan S; Kocharova, Nina A; Toukach, Filip V; Wykrota, Marianna; Shashkov, Alexander S; Knirel, Yuriy A; Rozalski, Antoni

    2007-06-11

    The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O27 and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments. It was found that the polysaccharide is built up of linear partially O-acetylated tetrasaccharide repeating units and has the following structure: [structure: see text] where Qui4NFo stands for 4-formamido-4,6-dideoxyglucose (4-formamido-4-deoxyquinovose). The O-polysaccharide structure of Providencia stuartii O43 established earlier was revised with respect to the configuration of the constituent 4-amino-4,6-dideoxyhexose (from Rha4N to Qui4N).

  1. Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O33.

    PubMed

    Ovchinnikova, Olga G; Shashkov, Alexander S; Chizhov, Alexander O; Moryl, Magdalena; Rozalski, Antoni; Knirel, Yuriy A

    2014-05-22

    Mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O33 resulted in an O-polysaccharide along with core and O-unit-bearing core oligosaccharides. Composition of the oligosaccharides was inferred by ESI mass spectrometry. Based on sugar and methylation analyses, Smith degradation and (1)H and (13)C NMR spectroscopy data, the following structure of the tetrasaccharide O-unit of the O-polysaccharide was established: Another O-polysaccharide structure has been reported earlier for Providencia stuartii О33 but later found to belong to a P. stuartii О52 strain.

  2. Regioselective sulfation of Artemisia sphaerocephala polysaccharide: Characterization of chemical structure.

    PubMed

    Wang, Junlong; Yang, Wen; Wang, Jiancheng; Wang, Xia; Wu, Fang; Yao, Jian; Zhang, Ji; Lei, Ziqiang

    2015-11-20

    The biological activities of sulfated polysaccharides are related to the substitution positions of functional groups. In this study, regioselective sulfation of Artemisia sphaerocephala polysaccharides (SRSASP) was prepared by using triphenylchloromethane (TrCl) as protecting precursor. FT-IR spectra and X-ray photoelectron spectroscopy (XPS) showed that SO(3-) group (S(6+), high binding energy of 168.7eV) was widely present in sulfated polysaccharides. (13)C NMR spectroscopy showed that C-2 and C-3 substitution was occurred but not fully sulfation. Meanwhile, C-6 substituted signals near 65ppm were not observed. The degree of substitution varied from 0.44 to 0.63 in SRSASP which could be attributed to the low reactivity at secondary hydroxyl. Monosaccharide composition result showed a decrease in the ratio of mannose/glucose, indicating the change of chemical composition in sulfated polysaccharides. In size-exclusion chromatograph analysis, a decrease in molecular weight and broadening of molecular weight distribution of sulfated polysaccharides was also observed. It could be attributed to the hydrolysis of polysaccharide in the sulfated reaction.

  3. Effect of extraction method on structure and antioxidant activity of Hohenbuehelia serotina polysaccharides.

    PubMed

    Li, Xiaoyu; Wang, Lu

    2016-02-01

    The impacts of four extraction methods (hot water, enzyme assistance, ultrasonic assistance and ultrasonic-enzyme assistance) on the extraction yields, preliminary structure and antioxidant activities of the Hohenbuehelia serotina polysaccharides (HW-HSP, EA-HSP, UA-HSP and UEA-HSP) were systematically investigated. The yield of the polysaccharides (20.70±0.17%) obtained by ultrasonic-enzyme assistance was higher than the polysaccharides by other methods'. Four kinds of polysaccharides possessed the different preliminary structural characteristics including molecular weight distributions, monosaccharide compositions, crystallization and spiral structures, while different surface morphology. Through the measurements of antioxidant activities in vitro, UEA-HSP exhibited the most significant scavenging capacities on non-physiological ABTS free radicals and physiological hydroxyl radicals. These data showed that ultrasonic-enzyme assistance was more beneficial to enhance the extraction yields of the polysaccharides, and obtain higher bioactive polysaccharides. The results also suggested that H. serotina polysaccharides possessed potential healthcare application in food field due to their antioxidant activities.

  4. Structure of complex cell wall polysaccharides isolated from Trichoderma and Hypocrea species.

    PubMed

    Prieto, A; Leal, J A; Poveda, A; Jiménez-Barbero, J; Gómez-Miranda, B; Domenech, J; Ahrazem, O; Bernabé, M

    1997-11-28

    The structure of fungal polysaccharides isolated from the cell wall of Trichoderma reesei, T. koningii, and Hypocrea psychrophila, have been investigated by means of chemical analyses and 1D and 2D NMR spectroscopy. The polysaccharides have an irregular structure, idealized as follows: [formula: see text] The proportions of the different side chains vary from a species to another, being n above some three times larger in H. psychrophila than in T. reesei or T. koningii.

  5. The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O47.

    PubMed

    Ovchinnikova, Olga G; Kocharova, Nina A; Bakinovskiy, Leon V; Torzewska, Agnieszka; Shashkov, Alexander S; Knirel, Yuriy A; Rozalski, Antoni

    2004-10-20

    The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O47:H4, strain 3646/51. Studies by sugar and methylation analyses along with Smith degradation and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY and H-detected 1H,13C HSQC and HMBC experiments, showed that the polysaccharide has a branched hexasaccharide repeating unit with the following structure: [carbohydrate structure: see text

  6. Factors affecting capsular volume changes and association with outcomes after Bankart repair and capsular shift.

    PubMed

    Park, Jin-Young; Chung, Seok Won; Kumar, Gurudeo; Oh, Kyung-Soo; Choi, Jin Hyeok; Lee, Deukhee; Park, Sehyung

    2015-02-01

    Capsular laxity is a main contributing factor in recurrent shoulder instability and is suggested to be correlated with increased capsular volume. Arthroscopic capsular shift combined with Bankart repair can reduce the capsular volume and reinforce the redundant capsule; however, as the capsuloligamentous structure has viscoelastic properties, it is possible for the shifted and tensioned capsule of the glenohumeral joint to slowly stretch out again over time, resulting in an increase in capsular volume. To analyze changes in capsular volume of the glenohumeral joint over time after arthroscopic Bankart repair and capsular shift, the factors associated with these changes, and their relevance to outcomes. Case series; Level of evidence, 4. Included in this study were 105 patients (mean age, 25.8 ± 8.2 years) who underwent arthroscopic Bankart repair and capsular shift for anterior shoulder instability and computed tomography arthrography (CTA) at 3 months and 1 year postoperatively and whose various functional outcomes were evaluated preoperatively and at the last follow-up (>12 months). Among these patients, 27 also had preoperative CTA. These 27 patients were used to make comparisons between preoperative and 3-month postoperative CTA measurements, and all 105 patients were used for all other comparisons. Two raters measured the separate anterior and posterior capsular volume and cross-sectional area at the 5-o'clock position using 3-dimensional (3D) Slicer software. These measurements were subsequently adjusted for each glenoid size. The changes in capsular volume and cross-sectional area at the 5-o'clock position over time, the factors related to higher change in anterior capsular volume, and their correlation with outcomes were evaluated. Three months postoperatively, the total and anterior capsular volume and anterior cross-sectional area significantly decreased; however, these values increased again at 1 year postoperatively (all P < .01). The inter- and

  7. Structure of the polysaccharides from the lipopolysaccharide of Azospirillum brasilense Jm125A2.

    PubMed

    Sigida, Elena N; Fedonenko, Yuliya P; Shashkov, Alexander S; Zdorovenko, Evelina L; Konnova, Svetlana A; Ignatov, Vladimir V; Knirel, Yuriy A

    2015-10-30

    Two polysaccharides were obtained by mild acid degradation of the lipopolysaccharide of associative nitrogen-fixing bacteria Azospirillum brasilense Jm125A2 isolated from the rhizosphere of a pearl millet. The following structures of the polysaccharides were established by sugar and methylation analyses, Smith degradation, and (1)H and (13)C NMR spectroscopy: [Formula: see text] Structure 1 has been reported earlier for a polysaccharide from A. brasilense S17 (Fedonenko YP, Konnova ON, Zdorovenko EL, Konnova SA, Zatonsky GV, Shaskov AS, Ignatov VV, Knirel YA. Carbohydr Res 2008;343:810-6), whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Structural characterization of an acidic polysaccharide from Dalbergia sissoo Roxb. leaves.

    PubMed

    Rana, Vikas; Kumar, Vineet; Soni, P L

    2012-09-01

    The composition and structure of an acidic polysaccharide from the leaves of Dalbergia sissoo was studied using hydrolytic, methylation, (1)H/(13)C heteronuclear multiple quantum coherence (HMQC) and periodate oxidation experiments. The repeating unit of sissoo polysaccharide was found to be composed of α-L-rhamnose, β-D-glucuronic acid, β-D-galactose and β-D-glucose in the molar ratio of 1.00:1.00:2.00:2.33, respectively. The structure of polysaccharide was mainly composed of (1→2), (1→3), (1→4) linkages. Based on extensive laboratory experiments, the structure having the repeating units of the acidic polysaccharide from sissoo leaves, with unusual branching, was established.

  9. Capsule structure of Proteus mirabilis (ATCC 49565).

    PubMed Central

    Beynon, L M; Dumanski, A J; McLean, R J; MacLean, L L; Richards, J C; Perry, M B

    1992-01-01

    Proteus mirabilis 2573 (ATCC 49565) produces an acidic capsular polysaccharide which was shown from glycose analysis, carboxyl reduction, methylation, periodate oxidation, and the application of one dimensional and two-dimensional high-resolution nuclear magnetic resonance techniques to be a high-molecular-weight polymer of branched trisaccharide units composed of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine), 2-acetamido-2,6-dideoxy-L-galactose (N-acetyl-L-fucosamine), and D-glucuronic acid, having the structure: [formula: see text] P. mirabilis 2573 also produces an O:6 serotype lipopolysaccharide in which the O-chain component has the same structure as the homologous capsular polysaccharide. This is the first report of a defined capsular polysaccharide in this bacterial genus. PMID:1551839

  10. Structural identification and cytotoxic activity of a polysaccharide from the fruits of Lagenaria siceraria (Lau).

    PubMed

    Ghosh, Kaushik; Chandra, Krishnendu; Ojha, Arnab K; Sarkar, Siddik; Islam, Syed S

    2009-03-31

    A water-soluble polysaccharide, isolated from fruiting bodies of Lagenaria siceraria, is composed of methyl-alpha-d-galacturonate, 3-O-acetyl methyl-alpha-d-galacturonate, and beta-d-galactose in a ratio of nearly 1:1:1. Compositional analysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, 2D-COSY, TOCSY, NOESY, HMQC, and HMBC) revealed the presence of the following repeating unit in the polysaccharide: [carbohydrate structure: see text] This polysaccharide showed cytotoxic activity in vitro against human breast adenocarcinoma cell line (MCF-7).

  11. PolySac3DB: an annotated data base of 3 dimensional structures of polysaccharides

    PubMed Central

    2012-01-01

    Background Polysaccharides are ubiquitously present in the living world. Their structural versatility makes them important and interesting components in numerous biological and technological processes ranging from structural stabilization to a variety of immunologically important molecular recognition events. The knowledge of polysaccharide three-dimensional (3D) structure is important in studying carbohydrate-mediated host-pathogen interactions, interactions with other bio-macromolecules, drug design and vaccine development as well as material science applications or production of bio-ethanol. Description PolySac3DB is an annotated database that contains the 3D structural information of 157 polysaccharide entries that have been collected from an extensive screening of scientific literature. They have been systematically organized using standard names in the field of carbohydrate research into 18 categories representing polysaccharide families. Structure-related information includes the saccharides making up the repeat unit(s) and their glycosidic linkages, the expanded 3D representation of the repeat unit, unit cell dimensions and space group, helix type, diffraction diagram(s) (when applicable), experimental and/or simulation methods used for structure description, link to the abstract of the publication, reference and the atomic coordinate files for visualization and download. The database is accompanied by a user-friendly graphical user interface (GUI). It features interactive displays of polysaccharide structures and customized search options for beginners and experts, respectively. The site also serves as an information portal for polysaccharide structure determination techniques. The web-interface also references external links where other carbohydrate-related resources are available. Conclusion PolySac3DB is established to maintain information on the detailed 3D structures of polysaccharides. All the data and features are available via the web

  12. Structural studies of fungal cell-wall polysaccharides from two strains of Talaromyces flavus.

    PubMed

    Parra, E; Jiménez-Barbero, J; Bernabé, M; Leal, J A; Prieto, A; Gómez-Miranda, B

    1994-01-03

    The water-soluble cell-wall polysaccharides isolated from strains CBS 352.72 and 310.38 of Talaromyces flavus have been investigated by chemical analyses and NMR studies. Two different skeletons coexist, having the structures: [formula:see text]. The small differences between the polysaccharides isolated from both strains are probably due to slight diminution of branching in strain 352.72, as compared with strain 310.38.

  13. Giant amino acids designed on the polysaccharide scaffold and their protein-like structural interconversion.

    PubMed

    Tamaru, Shun-ichi; Tokunaga, Daisuke; Hori, Kaori; Matsuda, Sayaka; Shinkai, Seiji

    2014-02-07

    Amphoteric β-1,3-glucans possessing both amino groups and carboxylic acid groups on the C6 positions of glucose units were designed and synthesized from naturally produced curdlan. The amphoteric polysaccharides showed the isoelectric point and the pH responsive interconversion between the original triple helix and single-stranded random structures. Since the pH dependences are comparable to the typical properties of proteins, the polysaccharides can be considered as a new class of giant amino acids. Utilizing the pH responsiveness, pH-controlled catch-and-release has been realized for cationic peptides or anionic DNA. We believe that the amphoteric polysaccharide can act as a new potential polymer to construct stimuli-responsive smart materials on the basis of the polysaccharide scaffold.

  14. Extraction, Structural Characterization, and Potential Antioxidant Activity of the Polysaccharides from Four Seaweeds

    PubMed Central

    He, Jinzhe; Xu, Yaoyang; Chen, Hongbo; Sun, Peilong

    2016-01-01

    Four seaweed polysaccharides were extracted from Sarcodia ceylonensis, Ulva lactuca L., Gracilaria lemaneiformis, and Durvillaea antarctica, respectively, by microwave-assisted extraction. The effect of three significant variables (extraction time, extraction temperature, and the ratio of water to raw material) on the process for extracting polysaccharides was investigated, along with the optimization of the extraction using the response surface method (RSM) with a Box–Behnken design. The polysaccharide structure, monosaccharide composition, degree of sulfation, and molecular weight (MW) distribution were analyzed by infrared (IR) spectrometry, gas chromatography (GC), and high-performance gel permeation chromatography (HPGPC). IR spectrometry showed that Sarcodia ceylonensis polysaccharide (SCP), Ulva lactuca L. polysaccharide (ULLP), and Durvillaea antarctica polysaccharide (DAP) were all sulfated polysaccharides and, except Gracilaria lemaneiformis polysaccharide (GLP), all belong to β-pyranosidic polysaccharides. The average molecular weight (MW) of SCP, ULLP, GLP, and DAP was 466, 404, 591, and 482 kDa, respectively. The quantitative and comparative results with external standards indicated that the main monosaccharide in SCP and ULLP was mannose; and GLP and DAP were mainly composed of galactose and glucose, respectively. Then the in vitro antioxidant activity of all of the polysaccharides was evaluated using different assays—2,2–azino –bis (3-ethylbenzthiazoline-6- sulfonate) (ABTS), hydroxyl radical, nitrite scavenging capacity, and reducing power—and the relationship between their antioxidant activity and chemical characteristics were also examined. ULLP presented the highest ABTS radical scavenging activity; ULLP, SCP and DAP also showed a strong effect on the ABTS radical scavenging activity. SCP and ULLP exhibited excellent hydroxyl radical scavenging activities, about 83.33% ± 2.31% and 80.07% ± 2.17%, respectively, at 4 mg/mL. The

  15. Extraction, Structural Characterization, and Potential Antioxidant Activity of the Polysaccharides from Four Seaweeds.

    PubMed

    He, Jinzhe; Xu, Yaoyang; Chen, Hongbo; Sun, Peilong

    2016-11-28

    Four seaweed polysaccharides were extracted from Sarcodia ceylonensis, Ulva lactuca L., Gracilaria lemaneiformis, and Durvillaea antarctica, respectively, by microwave-assisted extraction. The effect of three significant variables (extraction time, extraction temperature, and the ratio of water to raw material) on the process for extracting polysaccharides was investigated, along with the optimization of the extraction using the response surface method (RSM) with a Box-Behnken design. The polysaccharide structure, monosaccharide composition, degree of sulfation, and molecular weight (MW) distribution were analyzed by infrared (IR) spectrometry, gas chromatography (GC), and high-performance gel permeation chromatography (HPGPC). IR spectrometry showed that Sarcodia ceylonensis polysaccharide (SCP), Ulva lactuca L. polysaccharide (ULLP), and Durvillaea antarctica polysaccharide (DAP) were all sulfated polysaccharides and, except Gracilaria lemaneiformis polysaccharide (GLP), all belong to β-pyranosidic polysaccharides. The average molecular weight (MW) of SCP, ULLP, GLP, and DAP was 466, 404, 591, and 482 kDa, respectively. The quantitative and comparative results with external standards indicated that the main monosaccharide in SCP and ULLP was mannose; and GLP and DAP were mainly composed of galactose and glucose, respectively. Then the in vitro antioxidant activity of all of the polysaccharides was evaluated using different assays-2,2-azino -bis (3-ethylbenzthiazoline-6- sulfonate) (ABTS), hydroxyl radical, nitrite scavenging capacity, and reducing power-and the relationship between their antioxidant activity and chemical characteristics were also examined. ULLP presented the highest ABTS radical scavenging activity; ULLP, SCP and DAP also showed a strong effect on the ABTS radical scavenging activity. SCP and ULLP exhibited excellent hydroxyl radical scavenging activities, about 83.33% ± 2.31% and 80.07% ± 2.17%, respectively, at 4 mg/mL. The reducing power of

  16. Antibody Binding to Cryptococcus neoformans Impairs Budding by Altering Capsular Mechanical Properties

    PubMed Central

    Cordero, Radames J. B.; Pontes, Bruno; Frases, Susana; Nakouzi, Antonio S.; Nimrichter, Leonardo; Rodrigues, Marcio L.; Viana, Nathan B.

    2013-01-01

    Abs to microbial capsules are critical for host defense against encapsulated pathogens, but very little is known about the effects of Ab binding on the capsule, apart from producing qualitative capsular reactions (“quellung” effects). A problem in studying Ab–capsule interactions is the lack of experimental methodology, given that capsules are fragile, highly hydrated structures. In this study, we pioneered the use of optical tweezers microscopy to study Ab–capsule interactions. Binding of protective mAbs to the capsule of the fungal pathogen Cryptococcus neoformans impaired yeast budding by trapping newly emerging buds inside the parental capsule. This effect is due to profound mAb-mediated changes in capsular mechanical properties, demonstrated by a concentration-dependent increase in capsule stiffness. This increase involved mAb-mediated cross-linking of capsular polysaccharide molecules. These results provide new insights into Ab-mediated immunity, while suggesting a new nonclassical mechanism of Ab function, which may apply to other encapsulated pathogens. Our findings add to the growing body of evidence that Abs have direct antimicrobial functions independent of other components of the immune system. PMID:23233725

  17. Cations modulate polysaccharide structure to determine FGF-FGFR signaling: a comparison of signaling and inhibitory polysaccharide interactions with FGF-1 in solution.

    PubMed

    Guimond, Scott E; Rudd, Timothy R; Skidmore, Mark A; Ori, Alessandro; Gaudesi, Davide; Cosentino, Cesare; Guerrini, Marco; Edge, Ruth; Collison, David; McInnes, Eric; Torri, Giangiacomo; Turnbull, Jeremy E; Fernig, David G; Yates, Edwin A

    2009-06-09

    For heparan sulfate (HS) to bind and regulate the activity of proteins, the polysaccharide must present an appropriate sequence and adopt a suitable conformation. The conformations of heparin derivatives, as models of HS, are altered via a change in the associated cations, and this can drastically modify their FGF signaling activities. Here, we report that changing the cations associated with an N-acetyl-enriched heparin polysaccharide, from sodium to copper(II), converted it from supporting signaling through the fibroblast growth factor receptor (FGF-1-FGFR1c) tyrosine kinase signaling system to being inhibitory in a cell-based BaF3 assay. Nuclear magnetic resonance and synchrotron radiation circular dichroism (SRCD) spectroscopy demonstrated that the polysaccharide conformation differed in the presence of sodium or copper(II) cations. Electron paramagnetic resonance confirmed the environment of the copper(II) ion on the N-acetyl-enriched polysaccharide was distinct from that previously observed with intact heparin, which supported signaling. Secondary structures in solution complexes of polysaccharides with FGF-1 (which either supported signaling through FGFR1c or were inhibitory) were determined by SRCD. This allowed direct comparison of the two FGF-1-polysaccharide complexes in solution, containing identical molecular components and differing only in their cation content. Subtle structural differences were revealed, including a reduction in the level of disordered structure in the inhibitory complex.

  18. Sulfated polysaccharides from brown seaweeds Saccharina japonica and Undaria pinnatifida: isolation, structural characteristics, and antitumor activity.

    PubMed

    Vishchuk, Olesya S; Ermakova, Svetlana P; Zvyagintseva, Tatyana N

    2011-12-13

    During the last decade brown seaweeds attracted much attention as a source of polysaccharides, namely laminarans, alginic acids, and sulfated polysaccharides-fucoidans, with various structures and biological activities. In this study, sulfated polysaccharides were isolated from brown seaweeds Saccharina japonica (formerly named Laminaria) and Undaria pinnatifida and their antitumor activity was tested against human breast cancer T-47D and melanoma SK-MEL-28 cell lines. The sulfated polysaccharide form S. japonica was highly branched partially acetylated sulfated galactofucan, built up of (1→3)-α-L-fucose residues. The sulfated polysaccharide from U. pinnatifida was partially acetylated highly sulfated galactofucan consisting of (1→3)- or (1→3);(1→4)-α-L-fucose residues. Fucoidans from S. japonica and U. pinnatifida distinctly inhibited proliferation and colony formation in both breast cancer and melanoma cell lines in a dose-dependent manner. These results indicated that the use of sulfated polysaccharides from brown seaweeds S. japonica and U. pinnatifida might be a potential approach for cancer treatment.

  19. New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids.

    PubMed

    Kondakova, A N; Toukach, F V; Senchenkova, S N; Arbatsky, N P; Shashkov, A S; Knirel, Y A; Bartodziejska, B; Zych, K; Rozalski, A; Sidorczyk, Z

    2003-04-01

    Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: [Figure: see text], where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N(epsilon)-[(R)-1-carboxyethyl]-L-lysine ("alaninolysine"), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.

  20. Structure characteristics of a water-soluble polysaccharide purified from dragon fruit (Hylocereus undatus) pulp.

    PubMed

    Xu, Lishan; Zhang, Yaojie; Wang, Lizhi

    2016-08-01

    Dragon fruit is a tropical fruit with good taste. It can bring health benefits to human body. As one of the major bioactive components in this fruit, the polysaccharides might contribute to the health benefits. However, the precise structure information remains unknown. A leading polysaccharide of dragon fruit pulp, DFPP, was purified and identified by NMR and GC-MS. →4-β-d-GlcpA-1→, →6-β-d-Galp-1→ and →4-α-l-Rhap-1→ constituted the backbone and α-l-Araf-1→5-α-l-Araf-1→ formed the branch chain. The precise structure was putatively identified as below. The molecular weight was 2.2×10(3)kDa. The structure information of polysaccharides will be helpful to understand this fruit.

  1. [Structural characterization of Astragalus polysaccharides using partial acid hydrolysis-hydrophilic interaction liquid chromatography-mass spectrometry].

    PubMed

    Liang, Tu; Fu, Qing; Xin, Huaxia; Li, Fangbing; Jin, Yu; Liang, Xinmiao

    2014-12-01

    Water-soluble polysaccharides from traditional Chinese medicine (TCM) have properties of broad-spectrum treatment and low toxicity, making them as important components in natural medicines and health products. In order to solve the problem of polysaccharides characterization caused by their complex structures, a "bottom-up" approach was developed to complete the characterization of polysaccharides from Astragalus. Firstly, Astragalus pieces were extracted with hot water and then were precipitated by ethanol to obtain Astragalus polysaccharides. Secondly, a partial acid hydrolysis method was carried out and the effects of time, acid concentration and temperature on hydrolysis were investigated. The degree of hydrolysis increased along with the increase of hydrolysis time and acid concentration. The temperature played a great role in the hydrolysis process. No hydrolysis of the polysaccharides occurred at low temperature, while the polysaccharides were almost hydrolyzed to monosaccharide at high temperature. Under the optimum hydrolysis conditions (4 h, 1.5 mol/L trifluoroacetic acid, and 80 °C), Astragalus polysaccharides were hydrolyzed to characteristic oligosaccharide fragments. At last, a hydrophilic liquid chromatography-mass spectrometry method was used for the separation and structural characterization of the polysaccharide hydrolysates. The results showed that the resulting polysaccharides were mainly 1--> 4 linear glucan, and gluco-oligosaccharides with the degrees of polymerization (DP) of 4 - 11 were obtained after partial acid hydrolysis. The significance of this study is that it is the guidance for the characterization of other TCM polysaccharides.

  2. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

    PubMed

    Lo Leggio, Leila; Simmons, Thomas J; Poulsen, Jens-Christian N; Frandsen, Kristian E H; Hemsworth, Glyn R; Stringer, Mary A; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S; De Maria, Leonardo; Harris, Paul V; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

    2015-01-22

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.

  3. Decoration of Chondroitin Polysaccharide with Threonine: Synthesis, Conformational Study, and Ice-Recrystallization Inhibition Activity.

    PubMed

    Laezza, Antonio; Casillo, Angela; Cosconati, Sandro; Biggs, Caroline I; Fabozzi, Antonio; Paduano, Luigi; Iadonisi, Alfonso; Novellino, Ettore; Gibson, Matthew I; Randazzo, Antonio; Corsaro, Maria M; Bedini, Emiliano

    2017-08-14

    Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

  4. Structure of the O-specific polysaccharide of the bacterium Proteus vulgaris O23.

    PubMed

    Perepelov, A V; Shashkov, A S; Babichka, D; Senchenkova, S N; Bartodziejska, B; Rozalski, A; Knirel, Y A

    2000-09-01

    An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Proteus vulgaris O23 (strain PrK 44/57) and found to contain 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, and D-galacturonic acid. Based on 1H- and 13C-NMR spectroscopic studies, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments, the following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: [figure], where the degree of O-acetylation of the terminal GalA residue at position 4 is about 80%. A structural similarity of the O-specific polysaccharides of P. vulgaris O23 and P. mirabilis O23 is discussed.

  5. Structure of the O-polysaccharide of Escherichia coli O132.

    PubMed

    Shashkov, Alexander S; Zhang, Wenwen; Perepelov, Andrei V; Weintraub, Andrej; Liu, Bin; Widmalm, Göran; Knirel, Yuriy A

    2016-06-02

    Mild acid degradation of the lipopolysaccharide of Escherichia coli O132 released its O-polysaccharide. Analysis by 1D and 2D (1)H and (13)C NMR spectroscopy prior and subsequent to O-deacetylation, in conjunction with sugar analysis, revealed a linear pentasaccharide repeating unit of the O-polysaccharide having the following structure: →2)-α-d-Galf-(1→3)-α-l-Rhap2Ac-(1→4)-α-d-Glcp-(1→2)-α-l-Rhap-(1→3)-β-d-GlcpNAc-(1→ Putative functions of genes in the O-antigen gene cluster of E. coli O132 are consistent with the O-polysaccharide structure.

  6. Quantitative Structure Activity Relationship Models for the Antioxidant Activity of Polysaccharides

    PubMed Central

    Nie, Kaiying; Wang, Zhaojing

    2016-01-01

    In this study, quantitative structure activity relationship (QSAR) models for the antioxidant activity of polysaccharides were developed with 50% effective concentration (EC50) as the dependent variable. To establish optimum QSAR models, multiple linear regressions (MLR), support vector machines (SVM) and artificial neural networks (ANN) were used, and 11 molecular descriptors were selected. The optimum QSAR model for predicting EC50 of DPPH-scavenging activity consisted of four major descriptors. MLR model gave EC50 = 0.033Ara-0.041GalA-0.03GlcA-0.025PC+0.484, and MLR fitted the training set with R = 0.807. ANN model gave the improvement of training set (R = 0.96, RMSE = 0.018) and test set (R = 0.933, RMSE = 0.055) which indicated that it was more accurately than SVM and MLR models for predicting the DPPH-scavenging activity of polysaccharides. 67 compounds were used for predicting EC50 of the hydroxyl radicals scavenging activity of polysaccharides. MLR model gave EC50 = 0.12PC+0.083Fuc+0.013Rha-0.02UA+0.372. A comparison of results from models indicated that ANN model (R = 0.944, RMSE = 0.119) was also the best one for predicting the hydroxyl radicals scavenging activity of polysaccharides. MLR and ANN models showed that Ara and GalA appeared critical in determining EC50 of DPPH-scavenging activity, and Fuc, Rha, uronic acid and protein content had a great effect on the hydroxyl radicals scavenging activity of polysaccharides. The antioxidant activity of polysaccharide usually was high in MW range of 4000–100000, and the antioxidant activity could be affected simultaneously by other polysaccharide properties, such as uronic acid and Ara. PMID:27685320

  7. Polysaccharides from Arctium lappa L.: Chemical structure and biological activity.

    PubMed

    Carlotto, Juliane; de Souza, Lauro M; Baggio, Cristiane H; Werner, Maria Fernanda de P; Maria-Ferreira, Daniele; Sassaki, Guilherme L; Iacomini, Marcello; Cipriani, Thales R

    2016-10-01

    The plant Arctium lappa L. is popularly used to relieve symptoms of inflammatory disorders. A crude polysaccharide fraction (SAA) resulting of aqueous extraction of A. lappa leaves showed a dose dependent anti-edematogenic activity on carrageenan-induced paw edema, which persisted for up to 48h. Sequential fractionation by ultrafiltration at 50kDa and 30kDa cut-off membranes yielded three fractions, namely RF50, RF30, and EF30. All these maintained the anti-edematogenic effect, but RF30 showed a more potent action, inhibiting 57% of the paw edema at a dose of 4.9mg/kg. The polysaccharide RF30 contained galacturonic acid, galactose, arabinose, rhamnose, glucose, and mannose in a 7:4:2:1:2:1 ratio and had a Mw of 91,000g/mol. Methylation analysis and NMR spectroscopy indicated that RF30 is mainly constituted by a type I rhamnogalacturonan branched by side chains of types I and II arabinogalactans, and arabinan. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Structures of three different neutral polysaccharides of Acinetobacter baumannii, NIPH190, NIPH201, and NIPH615, assigned to K30, K45, and K48 capsule types, respectively, based on capsule biosynthesis gene clusters.

    PubMed

    Shashkov, Alexander S; Kenyon, Johanna J; Arbatsky, Nikolay P; Shneider, Mikhail M; Popova, Anastasiya V; Miroshnikov, Konstantin A; Volozhantsev, Nikolay V; Knirel, Yuriy A

    2015-11-19

    Neutral capsular polysaccharides (CPSs) were isolated from Acinetobacter baumannii NIPH190, NIPH201, and NIPH615. The CPSs were found to contain common monosaccharides only and to be branched with a side-chain 1→3-linked β-d-glucopyranose residue. Structures of the oligosaccharide repeat units (K units) of the CPSs were elucidated by 1D and 2D (1)H and (13)C NMR spectroscopy. Novel CPS biosynthesis gene clusters, designated KL30, KL45, and KL48, were found at the K locus in the genome sequences of NIPH190, NIPH201, and NIPH615, respectively. The genetic content of each gene cluster correlated with the structure of the CPS unit established, and therefore, the capsular types of the strains studied were designated as K30, K45, and K48, respectively. The initiating sugar of each K unit was predicted, and glycosyltransferases encoded by each gene cluster were assigned to the formation of the linkages between sugars in the corresponding K unit.

  9. Polysaccharide components from the scape of Musa paradisiaca: main structural features of water-soluble polysaccharide component.

    PubMed

    Anjaneyalu, Y V; Jagadish, R L; Raju, T S

    1997-06-01

    Polysaccharide components present in the pseudo-stem (scape) of M. paradisiaca were purified from acetone powder of the scape by delignification followed by extraction with aqueous solvents into water soluble polysaccharide (WSP), EDTA-soluble polysaccharide (EDTA-SP), alkali-soluble polysaccharide (ASP) and alkali-insoluble polysaccharide (AISP) fractions. Sugar compositional analysis showed that WSP and EDTA-SP contained only D-Glc whereas ASP contained D-Glc, L-Ara and D-Xyl in approximately 1:1:10 ratio, respectively, and AISP contained D-Glc, L-Ara and D-Xyl in approximately 10:1:2 ratio, respectively. WSP was further purified by complexation with iso-amylalcohol and characterized by specific rotation, IR spectroscopy, Iodine affinity, ferricyanide number, blue value, hydrolysis with alpha-amylase and glucoamylase, and methylation linkage analysis, and shown to be a amylopectin type alpha-D-glucan.

  10. Bioactive potential and structural chracterization of sulfated polysaccharide from seaweed (Gracilaria corticata).

    PubMed

    Seedevi, Palaniappan; Moovendhan, Meivelu; Viramani, Shanmugam; Shanmugam, Annian

    2017-01-02

    The Sulfated polysaccharide was purified through anion-exchange and gel permeation column chromatography. The isolated sulfated polysaccharide from C. corticata contains 84% of carbohydrate, 0% of protein, 19.7% of ash and 29.4% of moisture was found. The carbon, hydrogen, nitrogen and sulfur content as 33.19%, 5.91%, 7.21% and 3.75%. The molecular weight of sulfated polysaccharide was found to be 43kDa. The sugar was composed of (90.11%), glucose (5.47%), xylose (2.30%) and mannose (2.12%). The structural feature of sulfated polysachharide was studied through FT-IR and (1)H NMR spectral analysis. Further the sulfated polysaccharide showed total antioxidant activity of 24.93%-75.21% at 50-250μg/ml, DPPH free radical scavenging activity of 23.12%-73.01% at 10-160μg/ml, ABTS scavenging activity of 15.8%-74.5% at 25-125μg/ml hydroxyl radical scavenging activity 12.87-69.19% at 25-125μg/ml and superoxide radical scavenging activity 28.10-78.11% at 50-250μg/ml respectively. The sulfated polysaccharide has shown good antibacterial activity against human pathogen.

  11. Structural, functional, and antioxidant properties of water-soluble polysaccharides from potatoes peels.

    PubMed

    Jeddou, Khawla Ben; Chaari, Fatma; Maktouf, Sameh; Nouri-Ellouz, Oumèma; Helbert, Claire Boisset; Ghorbel, Raoudha Ellouz

    2016-08-15

    Water-soluble polysaccharides were extracted from potato peel waste (PPW). The structure of the polysaccharides from PPW (PPPW) was examined by means of Fourier transform-infrared spectroscopy (FT-IR) analysis, X-ray diffractometry (XRD) and gas chromatography-mass spectrometry (GC-MS). The results suggest that the extracted polysaccharides form a semi-crystalline polymer constituted essentially of the functional groups CO, CH and OH. Acid hydrolysis of this polymer yielded glucose (76.25%) as the dominant sugar functional properties (water holding capacity: WHC, oil holding capacity: OHC, foaming, and emulsion properties) of this polymer were studied. The PPPW showed interesting water-holding and fat-binding capacities which were 4.097 ± 0.537 g/g and 4.398 ± 0.04 g/g, respectively. In addition, it presented good foaming and emulsion properties. The antioxidant activity of this polymer was also studied and revealed that the polysaccharides showed interesting 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity (IC50 PPPW=11.578 mg/mL), reducing power and β-carotene bleaching inhibition activities, and also a strong ABTS radical scavenging activity (IC50 PPPW=2mg/mL). Overall, the results suggest that the polysaccharide is a promising source of natural antioxidants and can be used as additive in food, pharmaceutical and cosmetic preparations.

  12. Structural Characterization and Antioxidant Activities of Polysaccharides Extracted from the Pulp of Elaeagnus angustifolia L.

    PubMed Central

    Chen, Qingqing; Chen, Juncheng; Du, Hongtao; Li, Qi; Chen, Jun; Zhang, Gechao; Liu, Hong; Wang, Junru

    2014-01-01

    In this study, two polysaccharides (Elaeagnus angustifolia L. polysaccharide-1 (PEA-1) and PEA-2) were prepared from Elaeagnus angustifolia L. Then, the preliminary structure and antioxidant activities of all the samples were investigated. The results showed that the average molecular weights for PEA-1 and PEA-2 were 9113 and 5020 Da, respectively. And, PEA-1 was mainly composed of rhamnose, xylose, mannose, glucose, and galactose, respectively. The components of PEA-2 were rhamnose, mannose, glucose, and galactose, respectively. Moreover, the Antioxidant assays demonstrated that PEA-1 possessed of strong free radicals scavenging activity and hydroxyl radicals scavenging activities, suggesting that PEA-1 could potentially be used as natural antioxidant. PMID:24972139

  13. Structure of the O-polysaccharide of Providencia stuartii O49.

    PubMed

    Bushmarinov, Ivan S; Ovchinnikova, Olga G; Kocharova, Nina A; Blaszczyk, Aleksandra; Toukach, Filipp V; Torzewska, Agnieszka; Shashkov, Alexander S; Knirel, Yuriy A; Rozalski, Antoni

    2004-06-01

    The O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Providencia stuartii O49 was studied using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. The polysaccharide was found to have the trisaccharide repeating unit with the following structure: -->6)-beta-D-Galp(1-->3)-beta-D-GalpNAc(1-->4)-alpha-D-Galp(1-->

  14. Structural characterization and antioxidant activities of polysaccharides extracted from the pulp of Elaeagnus angustifolia L.

    PubMed

    Chen, Qingqing; Chen, Juncheng; Du, Hongtao; Li, Qi; Chen, Jun; Zhang, Gechao; Liu, Hong; Wang, Junru

    2014-06-26

    In this study, two polysaccharides (Elaeagnus angustifolia L. polysaccharide-1 (PEA-1) and PEA-2) were prepared from Elaeagnus angustifolia L. Then, the preliminary structure and antioxidant activities of all the samples were investigated. The results showed that the average molecular weights for PEA-1 and PEA-2 were 9113 and 5020 Da, respectively. And, PEA-1 was mainly composed of rhamnose, xylose, mannose, glucose, and galactose, respectively. The components of PEA-2 were rhamnose, mannose, glucose, and galactose, respectively. Moreover, the Antioxidant assays demonstrated that PEA-1 possessed of strong free radicals scavenging activity and hydroxyl radicals scavenging activities, suggesting that PEA-1 could potentially be used as natural antioxidant.

  15. Structure of a glucosyl phosphate-containing O-polysaccharide of Proteus vulgaris O42.

    PubMed

    Perepelov, Andrei V; Bartodziejska, Beata; Shashkov, Alexander S; Wykrota, Marianna; Knirel, Yuriy A; Rozalski, Antoni

    2007-12-28

    An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O42 and studied by sugar and methylation analyses along with 1H, 13C and 31P NMR spectroscopy. The following structure of the polysaccharide having a linear pentasaccharide phosphate repeating unit was established: -->3)-alpha-L-FucpNAc4Ac-(1-->4)-alpha-D-Glcp-1-P-(O-->4)-alpha-D-GlcpNAc-(1-->3)-alpha-L-FucpNAc4Ac-(1-->3))-alpha-D-GlcpNAc6Ac-(1--> where the degree of O-acetylation is approximately 80% on GlcNAc and approximately 40% on each of the FucNAc residues. A weak serological cross-reaction of anti-P. vulgaris O42 serum with the lipopolysaccharide of P. vulgaris O39 was observed and accounted for by the sharing of a disaccharide fragment of the O-polysaccharides.

  16. Structural studies of the serotype-f polysaccharide antigen from Streptococcus mutans OMZ175.

    PubMed Central

    Linzer, R; Reddy, M S; Levine, M J

    1987-01-01

    The serotype f antigen of Streptococcus mutans has been described as a rhamnose-glucose polysaccharide associated with the bacterial cell wall. In this study, the structure of serotype f polysaccharide was examined by analyses of the methylated derivatives of the antigen and the periodate-oxidized antigen. Methylated derivatives were characterized with a gas chromatograph-mass spectrometer. The polysaccharide appeared to have a backbone of alternating 1,3- and 1,2,3-linked rhamnose units. Branching occurred at the 3-position of the 1,2,3-linked rhamnose. Side chains were composed of terminal alpha-linked glucose units. A small proportion of longer side chains containing 1,2- and 1,6-linked glucose units were noted in some preparations; however, these determinants were not reactive with serotype f antisera. PMID:2824381

  17. Structure of the O-specific polysaccharide of Proteus vulgaris O4 containing a new component of bacterial polysaccharides, 4,6-dideoxy-4.

    PubMed

    Perepelov, A V; Babicka, D; Senchenkova, S N; Shashkov, A S; Moll, H; Rozalski, A; Zähringer, U; Knirel, Y A

    2001-03-22

    A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O4 lipopolysaccharide followed by GPC. The polysaccharide was studied by chemical methods along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, and 1H,13C HMBC experiments. Solvolysis of the polysaccharide with trifluoromethanesulfonic (triflic) acid resulted in a GlcpA-(1 --> 3)-GlcNAc disaccharide and a novel amino sugar derivative, 4,6-dideoxy-4-[N-[(R)-3-hydroxybutyryl]-L-alanyl]amino-D-glucose [Qui4N(HbAla)]. On the basis of the data obtained, the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established: --> 4)-beta-D-GlcpA-(1 --> 3)-beta-D-GlcpNAc-(1 --> 2)-beta-D-Quip4N(HbAla)-(1 --> 3)-alpha-D-Galp-(1 -->. This structure is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied in a separate Proteus serogroup.

  18. Structural studies on the fucosamine-containing O-specific polysaccharide of Proteus vulgaris O19.

    PubMed

    Vinogradov, E V; Kaca, W; Knirel, Y A; Rózalski, A; Kochetkov, N K

    1989-03-01

    The polysaccharide chain of Proteus vulgaris O19 lipopolysaccharide contains D-galactose, N-acetyl-D-glucosamine N-acetyl-D-galactosamine and N-acetyl-L-fucosamine in the ratio 1:1:1:1. The structure of the polysaccharide was established by full acid hydrolysis and methylation analysis, as well as by non-destructive methods, i.e. the computer-assisted evaluation of the 13C-NMR spectrum and computer-assisted evaluation of the specific optical rotation by Klyne's rule. The polysaccharide is regular and built up of tetrasaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp-(1----3)-beta-D-GlcNAcp-(1----3)-alph a-D-Galp- (1----4)-alpha-D-GalNAcp-(1---- The O19-antiserum cross-reacts with lipopolysaccharide from P. vulgaris O42, the structure of which is still unknown. No cross-reactions were observed with O-polysaccharides Pseudomonas aeruginosa O7 and Salmonella arizonae O59 in spite of some structural similarities.

  19. Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects

    PubMed Central

    Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio

    2015-01-01

    Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection. PMID

  20. Structural identification and immunostimulating activity of a Laminaria japonica polysaccharide.

    PubMed

    Zha, Xue-Qiang; Lu, Chao-Qun; Cui, Shao-Hua; Pan, Li-Hua; Zhang, Hai-Lin; Wang, Jun-Hui; Luo, Jian-Ping

    2015-01-01

    In the present study, a new water-soluble polysaccharide (LJP-11) was obtained from Laminaria japonica by anion exchange DEAE-cellulose chromatography and Sephacryl S-500 chromatography. The average molecular weight of this polysaccharide was estimated to be about 2.89×10(6) Da by high performance liquid chromatography system. Gas chromatography showed that LJP-11 was composed of arabinose, mannose and glucose in a molar ratio of 1.0:1.16:6.33. LJP-11 contains a long backbone consisting of (1→4)-β-D-GlcpAc, (1→4)-α-D-Glcp, (1→6)-β-D-Glcp and (1→3,6)-α-D-Manp. The 1-linked β-L-Araf was linked to the C-6 of (1→3)-α-D-Manp and the sulfate group was attached to the C-4 of (1→6)-β-D-Glcp. Pharmacological tests displayed that LJP-11 can stimulate macrophages to release NO, IL-6, TNF-α and IL-10 as well as the up-regulation of their gene expressions, indicating LJP-11 has beneficial effects on immunostimulation. Moreover, LJP-11 exhibited positive effects on the translocation of NF-κB p65 from cytoplasm to nucleus and the phosphorylation of IκBα, ERK1/2, JNK1/2 and P38 in macrophages. These results suggested that the activation of MAPK and NF-κB signaling pathways is one of the mechanisms responsible for the immunostimulating activity of LJP-11. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Structural properties of polysaccharides from cultivated fruit bodies and mycelium of Cordyceps militaris.

    PubMed

    Liu, Xiao-Cui; Zhu, Zhen-Yuan; Tang, Ya-Li; Wang, Ming-fei; Wang, Zheng; Liu, An-Jun; Zhang, Yong-Min

    2016-05-20

    The structural properties of polysaccharides, respectively, obtained from the fermented mycelium and cultivated fruiting bodies of the Cordyceps militaris were investigated and compared in this paper. First, the crude polysaccharides were extracted from the mycelium and the fruiting bodies, respectively. The polysaccharides were successively purified by Sevag and chromatography on Sephadex G-100 column to produce two polysaccharides fractions termed CMPS-II and CBPS-II, respectively. The average molecular weights of CMPS-II and CBPS-II were 1.402×10(3) kDa and 1.273×10(3) kDa, respectively, and they were mainly composed of mannose, glucose and galactose in the mole ratios of 1:28.63:1.41 and 1:12.41:0.74, respectively, for CMPS-II and CBPS-II. Afterward, the structural features of CMPS-II and CBPS-II were investigated by a combination of chemical and instrumental analysis, such as FT-IR, periodate oxidation-Smith degradation, GC-MS, NMR and methylation analysis. The results indicated that structurally, both CMPS-II and CBPS-II were 1,3-branched-galactomannoglucan that had a linear backbone of (1→4)-linked α-D-glucopyranose (Glcp). Congo-red test revealed that CMPS-II and CBPS-II existed as triple-helical chains in 0.05-0.15 M NaOH solution.

  2. Structural diversity requires individual optimization of ethanol concentration in polysaccharide precipitation.

    PubMed

    Xu, Jun; Yue, Rui-Qi; Liu, Jing; Ho, Hing-Man; Yi, Tao; Chen, Hu-Biao; Han, Quan-Bin

    2014-06-01

    Ethanol precipitation is one of the most widely used methods for preparing natural polysaccharides, in which ethanol concentration significantly affects the precipitate yield, however, is usually set at 70-80%. Whether the standardization of ethanol concentration is appropriate has not been investigated. In the present study, the precipitation yields produced in varied ethanol concentrations (10-90%) were qualitatively and quantitatively evaluated by HPGPC (high-performance gel-permeation chromatography), using two series of standard glucans, namely dextrans and pullulans, as reference samples, and then eight natural samples. The results indicated that the response of a polysaccharide's chemical structure, with diversity in structural features and molecular sizes, to ethanol concentration is the decisive factor in precipitation of these glucans. Polysaccharides with different structural features, even though they have similar molecular weights, exhibit significantly different precipitation behaviors. For a specific glucan, the lower its molecular size, the higher the ethanol concentration needed for complete precipitation. The precipitate yield varied from 10% to 100% in 80% ethanol as the molecular size increased from 1kDa to 270kDa. This paper aims to draw scientists' attention to the fact that, in extracting natural polysaccharides by ethanol precipitation, the ethanol concentration must be individually optimized for each type of material.

  3. Polysaccharides of St. John's Wort Herb Stimulate NHDF Proliferation and NEHK Differentiation via Influence on Extracellular Structures and Signal Pathways

    PubMed Central

    Abakuks, S.; Deters, A. M.

    2012-01-01

    St. John's Wort herb extracts often contain undesirable or volitional polysaccharides. As polysaccharides exhibit structure-dependent biological functions in the present study water-soluble polysaccharides were extracted from herb material, fractionated by anion exchange chromatography into four main polysaccharide fractions (denominated as Hp1, Hp2, Hp3 and Hp4) and characterized by HPAEC-PAD, CE, IR and GC-MS. Biological activity on human skin keratinocytes and fibroblasts was assessed by investigation of their effect on proliferation, metabolism, cytotoxicity, apoptosis and differentiation. The underlying mechanisms were investigated in gene expression studies. Polysaccharide fraction Hp1 was mainly composed of β-D-glucose. Hp2, Hp3 and Hp4 contained pectic structures and arabinogalactan proteins varying in composition and quantity. Polysaccharides of Hp1 induced the keratinocyte differentiation by inhibiting the gene expression of the epidermal growth factor and insulin receptor. While the collagen secretion of fibroblasts was stimulated by each polysaccharide fraction only Hp1 stimulated the synthesis. The fibroblast proliferation was reduced by Hp1 and increased by Hp4. This effect was related to the influence on genes that referred to oxidative stress, metabolism, transcription processes and extracellular proteins. In conclusion polysaccharides have been shown as biologically active ingredients of aqueous St. John's Wort extracts with a relation between their structural characteristics and function. PMID:22848211

  4. Polysaccharides of St. John's Wort Herb Stimulate NHDF Proliferation and NEHK Differentiation via Influence on Extracellular Structures and Signal Pathways.

    PubMed

    Abakuks, S; Deters, A M

    2012-01-01

    St. John's Wort herb extracts often contain undesirable or volitional polysaccharides. As polysaccharides exhibit structure-dependent biological functions in the present study water-soluble polysaccharides were extracted from herb material, fractionated by anion exchange chromatography into four main polysaccharide fractions (denominated as Hp1, Hp2, Hp3 and Hp4) and characterized by HPAEC-PAD, CE, IR and GC-MS. Biological activity on human skin keratinocytes and fibroblasts was assessed by investigation of their effect on proliferation, metabolism, cytotoxicity, apoptosis and differentiation. The underlying mechanisms were investigated in gene expression studies. Polysaccharide fraction Hp1 was mainly composed of β-D-glucose. Hp2, Hp3 and Hp4 contained pectic structures and arabinogalactan proteins varying in composition and quantity. Polysaccharides of Hp1 induced the keratinocyte differentiation by inhibiting the gene expression of the epidermal growth factor and insulin receptor. While the collagen secretion of fibroblasts was stimulated by each polysaccharide fraction only Hp1 stimulated the synthesis. The fibroblast proliferation was reduced by Hp1 and increased by Hp4. This effect was related to the influence on genes that referred to oxidative stress, metabolism, transcription processes and extracellular proteins. In conclusion polysaccharides have been shown as biologically active ingredients of aqueous St. John's Wort extracts with a relation between their structural characteristics and function.

  5. Structural features of an immunostimulating and antioxidant acidic polysaccharide from the seeds of Cuscuta chinensis.

    PubMed

    Bao, Xingfeng; Wang, Zhan; Fang, Jinian; Li, Xiaoyu

    2002-03-01

    Three crude polysaccharide fractions, named CS-A, CS-B and CS-C, were prepared from the seeds of Cuscuta chinensis by hot water extraction and diluted alkali extraction subsequently, then EtOH precipitation, and tested for lymphocyte proliferation activity. CS-A showed a stimulating effect on concanavalin A or lipopolysaccharide induced mitogenic activity of lymphocytes. An acidic polysaccharide (CS-A-3beta) was purified from CS-A by anion exchange and gel filtration chromatography. The polysaccharide showed potent stimulating effects on lymphocyte proliferation and antibody production, but did not significantly influence the serum IgG level. Its structural features were elucidated by methylation analysis, partial acid hydrolysis, 1D and 2D NMR and ESI-mass spectroscopy. The data obtained indicated that CS-A-3beta had a backbone consisting of alpha-D-1,4-linked GalpA residues and beta-L-1,2-linked Rhap residues with branches at C-4 of Rhap residues and C-3 of GalpA residues, composed of arabinogalactan and glucobiose. This structure is typical for a pectic polysaccharide of the rhamnogalacturonan type. In addition, the effect of CS-A, CS-B, CS-C and CS-A-3beta on hydrogen peroxide induced cell lesion in rat pheochromocytoma line PC 12 was investigated. The results indicated that, besides its immunostimulating activity, CS-A-3beta had a protective effect against free radical-induced cell toxicity.

  6. Ultrasonic-assisted extraction, structure and antitumor activity of polysaccharide from Polygonum multiflorum.

    PubMed

    Zhu, Weili; Xue, Xiaoping; Zhang, Zhanjun

    2016-10-01

    Polygonum multiflorum is a popular Chinese herbal medicine with various pharmacological functions. In this study, the ultrasonic-assisted extraction condition, structural characterization and antitumor activity of a polysaccharide from roots of P. multiflorum were investigated. The ultrasonic-assisted extraction condition was optimized by single-factor experiments and response surface methodology. Results showed that the maximum extraction yield (5.49%) was obtained at ultrasonic power 158W, extraction temperature 62°C, extraction time 80min and ratio of water to material 20mL/g. The obtained crude polysaccharides were further purified to afford a neutral and an acidic fraction. The structure of the main neutral polysaccharide (named PPS with molecular weight of 3.26×10(5)Da) was characterized as a linear (1→6)-α-d-glucan by gas chromatography, Fourier transform-infrared spectroscopy, methylation analysis, 1D and 2D nuclear magnetic resonance. At the concentration of 400μg/mL, the inhibitory ratios of PPS on HepG-2 and BGC-823 cells were 53.35% and 38.58%, respectively. Results suggested this polysaccharide could be a potential natural antitumor agent. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Viscoelastic Properties and Nano-scale Structures of Composite Oligopeptide-Polysaccharide Hydrogels

    PubMed Central

    Hyland, Laura L.; Taraban, Marc B.; Feng, Yue; Hammouda, Boualem; Yu, Y. Bruce

    2012-01-01

    Biocompatible and biodegradable peptide hydrogels are drawing increasing attention as prospective materials for human soft tissue repair and replacement. To improve the rather unfavorable mechanical properties of our pure peptide hydrogels, in this work we examined the possibility of creating a double hydrogel network. This network was created by means of the co-assembly of mutually attractive but self-repulsive oligopeptides within an already existing fibrous network formed by the charged, biocompatible polysaccharides chitosan, alginate, and chondroitin. Using dynamic oscillatory rheology experiments, it was found that the co-assembly of the peptides within the existing polysaccharide network resulted in a less stiff material as compared to the pure peptide networks (the elastic modulus G′ decreased from 90 kPa to 10 kPa). However, these composite oligopeptide-polysaccharide hydrogels were characterized by a greater resistance to deformation (the yield strain γ grew from 4 % to 100 %). Small-angle neutron scattering (SANS) was used to study the 2D cross-sectional shapes of the fibers, their dimensional characteristics and the mesh sizes of the fibrous networks. Differences in material structures found with SANS experiments confirmed rheology data showing that incorporation of the peptides dramatically changed the morphology of the polysaccharide network. The resulting fibers were structurally very similar to those forming the pure peptide networks, but formedless stiff gels because of their markedly greater mesh sizes. Together, these findings suggest an approach for the development of highly deformation-resistant biomaterials. PMID:21994046

  8. Structural Characterisation of a Polysaccharide from Radix Ranunculus Ternati

    PubMed Central

    Huang, Xuefeng; Zhao, Yun; Jin, Xin

    2014-01-01

    A water soluble polysaccharide, HB-1, with a molecular weight of 23,930, was isolated from radix Ranunculi ternati. by hot water extraction, ethanol precipitation, deproteination,ultrafiltration and gel-filtration column chromatography. Its sugar composition was determined by GLC as Glc, Ara, and Gal in a molar ration of 16.071: 2.722: 1. And the absolute configuration of Glc was identified as D. Smith degradation and methylation reaction showed the proportion of —1Glc (A) was about 16%, —1Glc4— (B) about 62%, (C) about 14%, and —1Gal6— (D) about 8%. The repetitive unit was likely composed of 3 As, 3 Cs, 13 Bs and 1 D. Together with the average molecular weight, it was predictable that HB-1 consisted of about seven of the repetitive unit. The inhibition activity of HB-1 on human glioma cell line SF188 was also measured, only to find it inactive. PMID:25587330

  9. Study on molecular structural characteristics of tea polysaccharide.

    PubMed

    Guo, Li; Du, Xianfeng; Lan, Jing; Liang, Qin

    2010-08-01

    Tea polysaccharide (TPS) is attracting more attention gradually due to its particular biological properties. However, molecular characteristics of TPS are unclear since appropriate method is still absent. So, study on the molecular characteristics of TPS was carried out by high-performance size-exclusion chromatography (HPSEC), multi-angle laser light scattering (MALLS) and viscosimetry. The results showed that the molar masses of TPS ranged from 2.287 x 10(5) to 2.762 x 10(5)gmol(-1), the RMS radii distributed from 132.1 to 145.9 nm, and M(w)/M(n) is 1.028. The Mark-Houwink equation was established as [eta]=0.5423 M(w)(0.5379), and the intrinsic viscosity and molecular chain parameters were as follows: [eta]=1.007 dL g(-1), k(H)=0.845, k(K)=0.387, alpha=0.5379, K=0.5423. In addition, based on the slope of the RMS radius versus molar mass conformational plot being 0.24+/-0.00, we suggest that the molecular morphology of TPS is a homogeneous and spherical polymer with branch in solution.

  10. Two variants among Haemophilus influenzae serotype b strains with distinct bcs4, hcsA and hcsB genes display differences in expression of the polysaccharide capsule

    PubMed Central

    Schouls, Leo; van der Heide, Han; Witteveen, Sandra; Zomer, Bert; van der Ende, Arie; Burger, Marina; Schot, Corrie

    2008-01-01

    Background Despite nearly complete vaccine coverage, a small number of fully vaccinated children in the Netherlands have experienced invasive disease caused by Haemophilus influenzae serotype b (Hib). This increase started in 2002, nine years after the introduction of nationwide vaccination in the Netherlands. The capsular polysaccharide of Hib is used as a conjugate vaccine to protect against Hib disease. To evaluate the possible rise of escape variants, explaining the increased number of vaccine failures we analyzed the composition of the capsular genes and the expressed polysaccharide of Dutch Hib strains collected before and after the introduction of Hib vaccination. Results The DNA sequences of the complete capsular gene clusters of 9 Dutch Hib strains were assessed and two variants, designated type I and type II were found. The two variants displayed considerable sequence divergence in the hcsA and hcsB genes, involved in transport of capsular polysaccharide to the cell surface. Application of hcsA type specific PCRs on 670 Hib strains collected from Dutch patients with invasive Hib disease showed that 5% of the strains collected before 1996 were type II. No endogenous type II Hib strains were isolated after 1995 and all type II strains were isolated from 0–4 year old, non-vaccinated children only. Analysis of a worldwide collection of Hib strains from the pre-vaccination era revealed considerable geographic differences in the distribution of the type I and type II strains with up to 73% of type II strains in the USA. NMR analysis of type I and type II capsule polysaccharides did not reveal structural differences. However, type I strains were shown to produce twice as much surface bound capsular polysaccharide. Conclusion Type II strains were only isolated during the pre-vaccination era from young, non-vaccinated individuals and displayed a lower expression of capsular polysaccharide than type I strains. The higher polysaccharide expression may have

  11. Molecular modification of native coffee polysaccharide using subcritical water treatment: Structural characterization, antioxidant, and DNA protecting activities.

    PubMed

    Getachew, Adane Tilahun; Chun, Byung Soo

    2017-06-01

    Polysaccharides are an abundant resource in coffee beans and have proved to show numerous bioactivities. Despite their abundance, their activities are not always satisfactory mostly due to their structure and large molecular size. Molecular modifications of native polysaccharides can overcome this problem. In this study, we used a novel and green method to modify native coffee polysaccharides using subcritical water (SCW) treatment. The SCW treatment was used at the temperature of 180°C-220°C and pressure of 30-60bar. The molecular and structural modification of the polysaccharides was confirmed using several techniques such as FT-IR, UV spectroscopy, XRD, and TGA. The antioxidant activity of the modified polysaccharides was evaluated using several chemical and Saccharomyces cerevisiae-based high throughput assays. The modified polysaccharides showed high antioxidant activities in all tested assays. Moreover, the polysaccharides showed high DNA protection activities. Therefore, SCW could be employed as a green solvent for molecular modification of polysaccharides. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Anti-cancer effect and structural characterization of endo-polysaccharide from cultivated mycelia of Inonotus obliquus.

    PubMed

    Kim, Yong Ook; Park, Hae Woong; Kim, Jong Hoon; Lee, Jae Young; Moon, Seong Hoon; Shin, Chul Soo

    2006-05-30

    The endo-polysaccharide extracted from mycelia of Inonotus obliquus (Pers.:Fr.) Pil. (Hymenochaetaceae) is a specific activator of B cells and macrophages. However, the in vivo anti-cancer effects and the chemical structure of the endo-polysaccharide are unknown. We purified the endo-polysaccharide, investigated its anti-cancer effects via in vitro and in vivo assays, and performed a structural characterization. The endo-polysaccharide was extracted from I. obliquus mycelia cultivated in a 300-l pilot fermenter, followed by hot water extraction and ethanol precipitation. Purification was achieved by DEAE-cellulose ion-exchange chromatography and gel-permeation chromatography. Chemical analysis revealed that the purified endo-polysaccharide is an alpha-linked fucoglucomannan with a molecular weight of approximately 1,000 kDa. The anti-cancer activities of the endo-polysaccharide against various types of tumor cells were determined. No direct toxicity against either cancer or normal cells was observed. Intraperitoneal administration of the endo-polysaccharide significantly prolonged the survival rate of B16F10-implanted mice, resulting in a 4.07-fold increase in the survival rate at a dose of 30 mg/kg/day. After 60 days of feeding, approximately 67% of the initial number of mice survived with no tumor incidence based on macroscopic examination. These results indicate that the anti-cancer effect of endo-polysaccharide is not directly tumorcidal but rather is immuno-stimulating.

  13. Synthesis, Structural, and Adsorption Properties and Thermal Stability of Nanohydroxyapatite/Polysaccharide Composites

    NASA Astrophysics Data System (ADS)

    Skwarek, Ewa; Goncharuk, Olena; Sternik, Dariusz; Janusz, Wladyslaw; Gdula, Karolina; Gun'ko, Vladimir M.

    2017-02-01

    A series of composites based on nanohydroxyapatite (nHAp) and natural polysaccharides (PS) (nHAp/agar, nHAp/chitosan, nHAp/pectin FB300, nHAp/pectin APA103, nHAp/sodium alginate) was synthesized by liquid-phase two-step method and characterized using nitrogen adsorption-desorption, DSC, TG, FTIR spectroscopy, and SEM. The analysis of nitrogen adsorption-desorption data shows that composites with a nHAp: PS ratio of 4:1 exhibit a sufficiently high specific surface area from 49 to 82 m2/g. The incremental pore size distributions indicate mainly mesoporosity. The composites with the component ratio 1:1 preferably form a film-like structure, and the value of S BET varies from 0.3 to 43 m2/g depending on the nature of a polysaccharide. Adsorption of Sr(II) on the composites from the aqueous solutions has been studied. The thermal properties of polysaccharides alone and in nHAp/PS show the influence of nHAp, since there is a shift of characteristic DSC and DTG peaks. FTIR spectroscopy data confirm the presence of functional groups typical for nHAp as well as polysaccharides in composites. Structure and morphological characteristics of the composites are strongly dependent on the ratio of components, since nHAp/PS at 4:1 have relatively large S BET values and a good ability to adsorb metal ions. The comparison of the adsorption capacity with respect to Sr(II) of nHAp, polysaccharides, and composites shows that it of the latter is higher than that of nHAp (per 1 m2 of surface).

  14. Synthesis, Structural, and Adsorption Properties and Thermal Stability of Nanohydroxyapatite/Polysaccharide Composites.

    PubMed

    Skwarek, Ewa; Goncharuk, Olena; Sternik, Dariusz; Janusz, Wladyslaw; Gdula, Karolina; Gun'ko, Vladimir M

    2017-12-01

    A series of composites based on nanohydroxyapatite (nHAp) and natural polysaccharides (PS) (nHAp/agar, nHAp/chitosan, nHAp/pectin FB300, nHAp/pectin APA103, nHAp/sodium alginate) was synthesized by liquid-phase two-step method and characterized using nitrogen adsorption-desorption, DSC, TG, FTIR spectroscopy, and SEM. The analysis of nitrogen adsorption-desorption data shows that composites with a nHAp: PS ratio of 4:1 exhibit a sufficiently high specific surface area from 49 to 82 m(2)/g. The incremental pore size distributions indicate mainly mesoporosity. The composites with the component ratio 1:1 preferably form a film-like structure, and the value of S BET varies from 0.3 to 43 m(2)/g depending on the nature of a polysaccharide. Adsorption of Sr(II) on the composites from the aqueous solutions has been studied. The thermal properties of polysaccharides alone and in nHAp/PS show the influence of nHAp, since there is a shift of characteristic DSC and DTG peaks. FTIR spectroscopy data confirm the presence of functional groups typical for nHAp as well as polysaccharides in composites. Structure and morphological characteristics of the composites are strongly dependent on the ratio of components, since nHAp/PS at 4:1 have relatively large S BET values and a good ability to adsorb metal ions. The comparison of the adsorption capacity with respect to Sr(II) of nHAp, polysaccharides, and composites shows that it of the latter is higher than that of nHAp (per 1 m(2) of surface).

  15. Structural elucidation and antioxidant activity a novel Se-polysaccharide from Se-enriched Grifola frondosa.

    PubMed

    Li, Qian; Wang, Wei; Zhu, Yun; Chen, Yao; Zhang, Weijie; Yu, Ping; Mao, Guanghua; Zhao, Ting; Feng, Weiwei; Yang, Liuqing; Wu, Xiangyang

    2017-04-01

    Se-GFP-22, a heteropolysaccharide, with a weight-average Mw of 4.13×10(6)Da, was purified from the crude Se-polysaccharide (Se-GFP) isolated from fruit bodies of Se-enriched Grifola frondosa. Selenium was accumulated efficiently in Grifola frondosa during cultivation with Na2SeO3. The structure was investigated through FT-IR, GC, GC-MS, NMR, HPSEC-MALL-RI, particle size, Conge-red test, CD, AFM and SEM. Se-GFP-22 was deduced as a backbone chain of 1,4-α-d-Glcp units with a branched point at C6 of both 1,3,6-β-d-Manp and 1,4,6-α-d-Galp units. A typical absorption for selenium ester was existed in Se-GFP-22. Se-GFP-22 adopted as a spherical conformation with random coils. A novel Se-polysaccharide of different monosaccharide constituents, molecular weight, linkage types and high content of selenium has been isolated from G. frondosa. The antioxidant effect of Se-GFP-22 was more potent than that of G. frondosa polysaccharide (GFP-22), which may be influenced by the co-effect of polysaccharide and Se, molecular weight, degree of branching and configuration. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Structural and immunological feature of rhamnogalacturonan I-rich polysaccharide from Korean persimmon vinegar.

    PubMed

    Kim, Hoon; Hong, Hee-Do; Suh, Hyung-Joo; Shin, Kwang-Soon

    2016-08-01

    The crude polysaccharide (KPV-0) isolated from Korean persimmon vinegar was fractionated using gel filtration chromatography to enhance the immunostimulatory activity and to identify the structural features of active fraction. Among three fractions, KPV-I obtained in a void volume, demonstrated the potent production of macrophage-stimulating mediators, including tumor necrosis factor-α, interleukin (IL)-6, IL-12, and nitric oxide. KPV-I showed a combined single peak with high molecular weight of 55,000Da by high performance size exclusion chromatography. Component sugar analysis revealed that KPV-I contained mainly of arabinose, mannose, galactose, rhamnose and galacturonic acid. Single radial gel diffusion assay using β-glucosyl Yariv reagent showed that KPV-I contained arabinogalactan protein with 13.7%. Methylation analysis indicated that KPV-I contained 21 kinds of neutral glycosidic linkages, which seemed to be composed three kinds of polysaccharide; that is a rhamnogalacturonan-I (65-70%) derived from persimmon as a raw material, a mannan (20-25%) derived from fermentation-associated microorganisms, and a linear glucans (less than 10%). In conclusion, polysaccharide isolated from persimmon vinegar could augment the macrophage stimulation, and a large amounts of RG-I polysaccharide derived from persimmon is likely a crucial role in expression of the activity in persimmon vinegar. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Structure of the β-l-fucopyranosyl phosphate-containing O-specific polysaccharide of Escherichia coli O84.

    PubMed

    Knirel, Yuriy A; Qian, Chengqian; Senchenkova, Sofya N; Guo, Xi; Shashkov, Alexander S; Chizhov, Alexander O; Perepelov, Andrei V; Liu, Bin

    2016-07-01

    Fine structure of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) defines the serospecificity of bacterial cells, which is the basis for O-serotyping of medically and agriculturally important gram-negative bacteria including Escherichia coli. In order to obtain the O-polysaccharide for structural analysis, the lipopolysaccharide was isolated from cells of E. coli O84a by phenol/water extraction and degraded with mild acid. However, the O-polysaccharide was cleaved at a highly acid-labile β-l-fucopyranosyl phosphate (β-l-Fucp-1-P) linkage to give mainly a pentasaccharide that corresponded to the O-polysaccharide repeat. Therefore, the lipopolysaccharide and the pentasaccharide as well as their O-deacylated derivatives were studied using sugar analysis, NMR spectroscopy, and (for oligosaccharides) ESI HR MS, and the O84-polysaccharide structure was established. The O-polysaccharide is distinguished by the presence of β-l-Fucp-1-P and randomly di-O-acetylated 6-deoxy-d-talose, which are found for the first time in natural carbohydrates. The gene cluster for the O84-antigen biosynthesis was analysed and its content was found to be consistent with the O-polysaccharide structure.

  18. Evolution of the capsular operon of Streptococcus iniae in response to vaccination.

    PubMed

    Millard, Candice M; Baiano, Justice C F; Chan, Candy; Yuen, Benedict; Aviles, Fabian; Landos, Matt; Chong, Roger S M; Benedict, Suresh; Barnes, Andrew C

    2012-12-01

    Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.

  19. Evolution of the Capsular Operon of Streptococcus iniae in Response to Vaccination

    PubMed Central

    Millard, Candice M.; Baiano, Justice C. F.; Chan, Candy; Yuen, Benedict; Aviles, Fabian; Landos, Matt; Chong, Roger S. M.; Benedict, Suresh

    2012-01-01

    Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins. PMID:23001668

  20. Sulfation effect on levan polysaccharide chains structure with molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Coskunkan, Binnaz; Turgut, Deniz; Rende, Deniz; Malta, Seyda; Baysal, Nihat; Ozisik, Rahmi; Toksoy-Oner, Ebru

    Diversity in conformations and structural heterogeneity make polysaccharides the most challenging biopolymer type for experimental and theoretical characterization studies. Levan is a biopolymer chain that consists of fructose rings with β(2-6) linkages. It is a glycan that has great potential as a functional biopolymer in foods, feeds, cosmetics, pharmaceutical and chemical industries. Sulfated polysaccharides are group of macromolecules with sulfated groups in their hydroxyl parts with a range of important biological properties. Sulfate groups and their positions have a major effect on anticoagulant activity. It is reported that sulfate modified levan has anticoagulant activity such as heparin. In the current study, the effect of sulfation on the structure and dynamics of unmodified and sulfate modified levan are investigated via fully atomistic Molecular Dynamics simulations in aqueous media and varying salt concentrations at 310 K. This material is based upon work supported by the National Science Foundation under Grant No. CMMI-1538730.

  1. Effect of polysaccharide nanocrystals on structure, properties, and drug release kinetics of alginate-based microspheres.

    PubMed

    Lin, Ning; Huang, Jin; Chang, Peter R; Feng, Liangdong; Yu, Jiahui

    2011-07-01

    Polysaccharide nanocrystals, such as rod-like cellulose nanocrystals and chitin whiskers and platelet-like starch nanocrystals, were incorporated into alginate-based nanocomposite microspheres with the aim of enhancing mechanical strength and regulating drug release behavior. The structures and properties of the sols and the resultant nanocomposite microspheres were characterized by rheological testing, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and scanning electron microscopy (SEM). The presence of polysaccharide nanocrystals increased the stability of the crosslinked network structure, and the nanocomposite microspheres consequently exhibited prominent sustained release profiles, as demonstrated by inhibited diffusion of theophylline. Furthermore, based on the drug release results, the release kinetics and transport mechanisms were analyzed and discussed. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  2. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase

    PubMed Central

    Lo Leggio, Leila; Simmons, Thomas J.; Poulsen, Jens-Christian N.; Frandsen, Kristian E. H.; Hemsworth, Glyn R.; Stringer, Mary A.; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S.; De Maria, Leonardo; Harris, Paul V.; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J.; Walton, Paul H.

    2015-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes. PMID:25608804

  3. Chemical structure of the O-polysaccharide isolated from Pectobacterium atrosepticum SCRI 1039.

    PubMed

    Czerwicka, Małgorzata; Marszewska, Kinga; Bychowska, Anna; Dziadziuszko, Halina; Brzozowski, Krzysztof; Łojkowska, Ewa; Stepnowski, Piotr; Kaczyński, Zbigniew

    2011-12-27

    The lipopolysaccharide (LPS) of the bacterium Pectobacterium atrosepticum SCRI 1039 was hydrolyzed and the products were separated. A study of the obtained O-polysaccharide by means of chemical methods, GLC, GLC-MS, and NMR spectroscopy allowed us to identify a branched polymer with a pentasaccharide repeating unit of the structure shown below, in which the fucose residue was partially O-acetylated at C-2, C-3 or C-4.

  4. [The structure of O-specific polysaccharide chains of lipopolysaccharides from Citrobacter 032 and Salmonella arizonae 064 (Arizona 29)].

    PubMed

    Kocharova, N A; Vinogradov, E V; Knirel', Iu A; Shashkov, A S; Kochetkov, N K

    1988-05-01

    On the basis of acid hydrolysis, methylation, Smith degradation, selective cleavage with anhydrous hydrogen fluoride, and 13C NMR analysis, the repeating unit of the O-specific polysaccharide of Citrobacter O32 was concluded to have the following structure: (Formula: see text). The repeating unit of the Salmonella arizonae O64 O-specific polysaccharide has the same structure lacking the O-acetyl group.

  5. Structural and Immunological Activity Characterization of a Polysaccharide Isolated from Meretrix meretrix Linnaeus

    PubMed Central

    Li, Li; Li, Heng; Qian, Jianying; He, Yongfeng; Zheng, Jialin; Lu, Zhenming; Xu, Zhenghong; Shi, Jinsong

    2015-01-01

    Polysaccharides from marine clams perform various biological activities, whereas information on structure is scarce. Here, a water-soluble polysaccharide MMPX-B2 was isolated from Meretrix meretrix Linnaeus. The proposed structure was deduced through characterization and its immunological activity was investigated. MMPX-B2 consisted of d-glucose and d-galctose residues at a molar ratio of 3.51:1.00. The average molecular weight of MMPX-B2 was 510 kDa. This polysaccharide possessed a main chain of (1→4)-linked-α-d-glucopyranosyl residues, partially substituted at the C-6 position by a few terminal β-d-galactose residues or branched chains consisting of (1→3)-linked β-d-galactose residues. Preliminary immunological tests in vitro showed that MMPX-B2 could stimulate the murine macrophages to release various cytokines, and the structure-activity relationship was then established. The present study demonstrated the potential immunological activity of MMPX-B2, and provided references for studying the active ingredients in M. meretrix. PMID:26729136

  6. Structural characteristics of pineapple pulp polysaccharides and their antitumor cell proliferation activities.

    PubMed

    Wang, Ling; Tang, De-Qiang; Kuang, Yu; Lin, Feng-Jiao; Su, Yu

    2015-09-01

    Pineapple has a delicious taste and good health benefits. Bioactive polysaccharides are important components of pineapple that might contribute to its health benefits. Since little structural information on these polysaccharides is currently available, the aim of this study was to investigate their structural characteristics and bioactivities. The polysaccharides of pineapple pulp were fractionated into three fractions (PAPs 1-3) by anion exchange chromatography. Their structural characteristics were first identified, including molecular weights and glycosidic linkages. The monosaccharide compositions were revealed as PAP 1 (Ara, Xyl, Man, Glc and Gal), PAP 2 (Rha, Ara, Xyl, Man, Glc and Gal) and PAP 3 (Rha, Ara, Xyl, Man and Gal). Nuclear magnetic resonance (NMR) spectra suggested that PAP 2 had a backbone of → 4)-α-d-Manp-(1 → 2,4)-α-d-Manp-(1 → with branches attached to O-4 of Manp. The NMR data of α-l-Araf-(1→, →3)-α-l-Araf-(1→, →4)-β-d-Galp-(1 → and → 4)-α-d-GalpAMe-(1 → were assigned. PAPs 1 and 2 showed significant antitumor cell proliferation activities against breast carcinoma cell line and strong antioxidant activities. The above findings indicated that PAPs 1-3 contributed much to the health benefits of pineapple. They could be used as health-beneficial food additives in functional foods. © 2015 Society of Chemical Industry.

  7. Optimization of selenylation conditions for a pectic polysaccharide and its structural characteristic.

    PubMed

    Chen, Wenxia; Chen, Jiayu; Wu, Hongmei; Gou, Yuqiang; Hu, Fangdi; Liu, Linjie; Gao, Xia; Zhang, Pei

    2014-08-01

    CPP1b is a pectic polysaccharide isolated from Codonopsis pilosula, and it possesses potent antitumor activity. In this study, a HNO3-Na2SeO3 method was performed to synthesize selenium-CPP1b (sCPP1b). The effects of BaCl2 usage amount and HNO3 concentration on the yield and selenium content of sCPP1b were investigated by single-factor experiments. Reaction time, reaction temperature and the ratio of polysaccharide to Na2SeO3 were optimized by orthogonal experimental design at three levels of each of the three factors (L9(3)(4)) based on antitumor activity, selenium content and yield of sCPP1b. Our results showed that 5h of reaction time, 60°C of reaction temperature, and 2:2 ratio of polysaccharide to Na2SeO3 were the optimal selenylation modification conditions. The validation experiments completed under the optimal conditions gave the mean selenium content and yield of sCPP1b were 478.17μg/g (RSD=5.7%) and 595mg/g (RSD=1.6%), respectively. Selenylation modification can significantly increase the antitumor activity of CPP1b in vitro. The structural characterization of sCPP1b was further characterized by Fourier-transform infrared spectroscopy, thermogravimetric analysis, and high-performance gel permeation chromatography coupled with multi-angle laser light scattering. These structure analysis results indicated that sCPP1b has been successfully selenylation modified with similar structure to polysaccharide of CPP1b. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Identification of a capsular variant and characterization of capsular acetylation in Klebsiella pneumoniae PLA-associated type K57

    PubMed Central

    Hsu, Chun-Ru; Liao, Chun-Hsing; Lin, Tzu-Lung; Yang, Han-Ru; Yang, Feng-Ling; Hsieh, Pei-Fang; Wu, Shih-Hsiung; Wang, Jin-Town

    2016-01-01

    Klebsiella pneumoniae can cause community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) is important for its virulence. Among 79 capsular (K) types discovered thus far, K57 is often associated with PLA. Here, we report the identification of a K57 variant. Cps gene locus sequencing revealed differences between the K57 reference strain 4425/51 (Ref-K57) and a variant, the PLA isolate A1142. While Ref-K57 cps contained orf13 encoding a putative acetyltransferase, the insertion of a putative transposase-encoding gene at this position was detected in A1142. This variation was detected in other K57 clinical strains. Biochemical analyses indicated that A1142 was deficient in CPS acetylation. Genetic replacement and complementation verified that orf13 was responsible for CPS acetylation. Acetylation increased CPS immunoreactivity to antiserum and enhanced K. pneumoniae induction of pro-inflammatory cytokines through JNK and MAPK signaling. While acetylation diminished the serum resistance of bacteria, it promoted adhesion to intestinal epithelial cells possibly via increasing production of type I fimbriae. In conclusion, acetylation-mediated capsular variation in K57 was observed. Capsular acetylation contributed to the variety and antigenic diversity of CPS, influenced its biological activities, and was involved in K. pneumoniae-host interactions. These findings have implications for vaccine design and pathogenicity of K. pneumoniae. PMID:27550826

  9. The structure of the O-specific polysaccharide of Salmonella arizonae O62.

    PubMed

    Vinogradov, E V; Knirel, Y A; Kochetkov, N K; Schlecht, S; Mayer, H

    1994-02-03

    The O-specific polysaccharide was liberated by mild acid hydrolysis of the lipopolysaccharide (LPS) isolated from S. arizonae O62 by phenol-water extraction. The branched hexasaccharide repeating-unit of the O-specific chain of the O62 LPS contained L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galacturonic acid in molar ratios of 4:1:1. On the basis of methylation analysis, 1H and 13C NMR spectroscopy, including 2D shift-correlated (COSY) and 1D NOE spectroscopy, the following structure for the repeating unit of the O-specific polysaccharide was established: [formula: see text

  10. Structural analysis and antioxidant activities of polysaccharide isolated from Jinqian mushroom.

    PubMed

    Liu, Yong; Du, Yi-Qun; Wang, Jun-Hui; Zha, Xue-Qiang; Zhang, Jian-Bo

    2014-03-01

    Jinqian mushroom is a precious edible mushroom with delicious taste and high nutritional value. In this paper, a polysaccharide fraction JQPs was isolated and purified from the fruiting body of Jinqian mushroom. The chemical structure, chain conformation and antioxidant activities of JQPs were investigated. The results indicated that JQPs was mainly composed of glucose with trace amounts of xylose. The backbone of JQPs consisted of β-(1 → 3)-D-glucan with β-(1 → 6)-glucosyl side chain. The chain conformation analysis showed that JQPs was a triple helical polysaccharide. The antioxidant activity tests in vitro revealed that JQPs exhibited high DPPH radical and ABTS radical scavenging activities, moderate superoxide radical and hydroxyl radical scavenging activities, low reducing power and Fe(2+) chelating activities. The results suggested that JQPs could be used as a potential natural antioxidant. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Structural Characterization of the Extracellular Polysaccharide from Vibrio cholerae O1 El-Tor

    PubMed Central

    Yildiz, Fitnat; Fong, Jiunn; Sadovskaya, Irina; Grard, Thierry; Vinogradov, Evgeny

    2014-01-01

    The ability to form biofilms is important for environmental survival, transmission, and infectivity of Vibrio cholerae, the causative agent of cholera in humans. To form biofilms, V. cholerae produces an extracellular matrix composed of proteins, nucleic acids and a glycoconjugate, termed Vibrio exopolysaccharide (VPS). Here, we present the data on isolation and characterization of the polysaccharide part of the VPS (VPS-PS), which has the following structure: where α-D-Glc is partially (∼20%) replaced with α-D-GlcNAc. α-GulNAcAGly is an amide between 2-acetamido-2-deoxy-α-guluronic acid and glycine. Apparently, the polysaccharide is bound to a yet unidentified component, which gives it high viscosity and completely suppresses any NMR signals belonging to the sugar chains of the VPS. The only reliable method to remove this component at present is a treatment of the whole glycoconjugate with concentrated hydrochloric acid. PMID:24520310

  12. Structural elucidation of polysaccharide containing 3-O-methyl galactose from fruiting bodies of Pleurotus citrinopileatus.

    PubMed

    He, Pengfei; Zhang, Anqiang; Zhou, Saijing; Zhang, Fuming; Linhardt, Robert J; Sun, Peilong

    2016-11-03

    A water-soluble polysaccharide containing 3-O-methyl galactose (PCP60W) was isolated from fruiting bodies of Pleurotus citrinopileatus and purified by anion-exchange and gel column chromatography. This polysaccharide has an average molecular weight of 2.74 × 10(4) Da and its structure was elucidated using monosaccharide composition and methylation analysis combined with one- and two-dimensional (COSY, TOCSY, NOESY, HMQC and HMBC) NMR spectroscopy. PCP60W was shown to be a linear partially 3-O-methylated α-galactopyranan comprised of 6-linked galactose, 6-linked 3-O-methyl galactose and 4-linked glucose in a ratio of 3.0:1.0:0.6. This work provides additional evidence for the view that 3-O-methyl galactose is common to the genus Pleurotus.

  13. Structure analysis of water-soluble polysaccharide CPPS3 isolated from Codonopsis pilosula.

    PubMed

    Zhang, Ya-jun; Zhang, Li-xia; Yang, Jing-feng; Liang, Zhong-yan

    2010-04-01

    Codonopsis pilosula is a perennial species of flowering plants propagated in Northeast Asia. A water-soluble polysaccharide, CPPS(3), was extracted from the root of Codonopsis pilosula by boiled water extraction and ethanol precipitation. The molecular weight was estimated to be 7.4 x 10(4) Da determined by using Gel permeation chromatography. Monosaccharide composition and the structure of the polysaccharide were determined by gas spectroscopy, Fourier transform IR (FT-IR) spectroscopy, NMR spectroscopy and mass spectroscopy and some chemical method analysis was made. The components were galactose, arabinose and rhamnose in the molar ratio of 1.13:1.12:1. The main chain of CPPS(3) is illustrated to be (1-->3)-linked-beta-GalpNAc, (1-->3)-linked-alpha-Rhap and (1-->2,3)-beta-Galp.

  14. Characterization of the Kingella kingae Polysaccharide Capsule and Exopolysaccharide

    PubMed Central

    Starr, Kimberly F.; Porsch, Eric A.; Heiss, Christian; Black, Ian; Azadi, Parastoo; St. Geme, Joseph W.

    2013-01-01

    Recent evidence indicates that Kingella kingae produces a polysaccharide capsule. In an effort to determine the composition and structure of this polysaccharide capsule, in the current study we purified capsular material from the surface of K. kingae strain 269–492 variant KK01 using acidic conditions to release the capsule and a series of steps to remove DNA, RNA, and protein. Analysis of the resulting material by gas chromatography and mass spectrometry revealed N-acetyl galactosamine (GalNAc), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and galactose (Gal). Further analysis by NMR demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→. Disruption of the ctrA gene required for surface localization of the K. kingae polysaccharide capsule resulted in elimination of GalNAc and Kdo but had no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. Disruption of ctrA and deletion of pamABCDE resulted in a loss of all carbohydrates in surface extracts. These results establish that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→. The polysaccharide capsule and the exopolysaccharide require distinct genetic loci for surface localization. PMID:24098695

  15. Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides.

    PubMed

    Aboughe-Angone, Sophie; Nguema-Ona, Eric; Ghosh, Partha; Lerouge, Patrice; Ishii, Tadashi; Ray, Bimalendu; Driouich, Azeddine

    2008-01-14

    Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.

  16. Structure-function relationship of anticoagulant and antithrombotic well-defined sulfated polysaccharides from marine invertebrates.

    PubMed

    Pomin, Vitor H

    2012-01-01

    Marine sulfated polysaccharides (MSPs), such as sulfated fucans (SFs), sulfated galactans (SGs), and glycosaminoglycans (GAGs) isolated from invertebrate animals, are highly anionic polysaccharides capable of interacting with certain cationic proteins, such as (co)-factors of the coagulation cascade during clotting-inhibition process. Primarily, these molecular complexes between MSPs and coagulation-related proteins seem to be driven mostly by electrostatic interactions. However, through a systematic comparison using several novel well-defined sulfated polysaccharides composed of repetitive oligosaccharides with clear sulfation patterns, it was proved that those molecular interactions are essentially regulated by the stereochemistry of the glycans (which depends on a conjunction of anomeric configurations, sugar types, conformational preferences, glycosylation, and sulfation sites), rather than just a mere consequence of the electronegative density charges (mainly from number of sulfate groups). Here, we present an overview about the structure-function relationship of the invertebrate MSPs with regular structures as potential anticoagulant and antithrombotic agents, as pathologies related to the cardiovascular system are one of the major causes of mortality in the world. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. A sweet new wave: structures and mechanisms of enzymes that digest polysaccharides from marine algae.

    PubMed

    Hehemann, Jan-Hendrik; Boraston, Alisdair B; Czjzek, Mirjam

    2014-10-01

    Marine algae contribute approximately half of the global primary production. The large amounts of polysaccharides synthesized by these algae are degraded and consumed by microbes that utilize carbohydrate-active enzymes (CAZymes), thus creating one of the largest and most dynamic components of the Earth's carbon cycle. Over the last decade, structural and functional characterizations of marine CAZymes have revealed a diverse set of scaffolds and mechanisms that are used to degrade agars, carrageenan, alginate and ulvan-polysaccharides from red, brown and green seaweeds, respectively. The analysis of these CAZymes is not only expanding our understanding of their functions but is enabling the enhanced annotation of (meta)-genomic data sets, thus promoting an improved understanding of microbes that drive this marine component of the carbon cycle. Furthermore, this information is setting a foundation that will enable marine algae to be harnessed as a novel resource for biorefineries. In this review, we cover the most recent structural and functional analyses of marine CAZymes that are specialized in the digestion of macro-algal polysaccharides. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. The structure of the O-specific polysaccharide of Salmonella arizonae O21 (Arizona 22) containing N-acetylneuraminic acid.

    PubMed

    Vinogradov, E V; Knirel, Y A; Shashkov, A S; Paramonov, N A; Kochetkov, N K; Stanislavsky, E S; Kholodkova, E V

    1994-06-02

    The O-specific polysaccharide of S. arizonae O21 was found to contain 2-acetamido-2-deoxy-D-glucose, 2-acetamidino-2,6-dideoxy-L-galactose, N-acetylneuraminic acid, and O-acetyl groups. On the basis of 1H and 13C NMR studies of the intact and O-deacetylated polysaccharide and oligosaccharide fragments obtained by solvolysis with anhydrous hydrogen fluoride, partial methanolysis and partial hydrolysis, it was concluded that the O-specific polysaccharide has the following structure: [formula: see text

  19. Fungal polysaccharides.

    PubMed

    San-Blas, G; Suzuki, S; Hearn, V; Pinel, C; Kobayashi, H; Mendez, C; Niño, G; Nishikawa, A; San-Blas, F; Shibata, N

    1994-01-01

    Fungal polysaccharides are cell wall components which may act as antigens or as structural substrates. As antigens, the role of mannans in Saccharomyces cerevisiae and Candida albicans, and of glycoproteins in Aspergillus fumigatus are discussed. Analyses on beta-glucan synthetase in Paracoccidioides brasiliensis and the inhibitory effect of Hansenula mrakii killer toxin on beta-glucan biosynthesis are also considered.

  20. Structural characterization and immunomodulatory effect of a polysaccharide HCP-2 from Houttuynia cordata.

    PubMed

    Cheng, Bao-Hui; Chan, Judy Yuet-Wa; Chan, Ben Chung-Lap; Lin, Huang-Quan; Han, Xiao-Qiang; Zhou, Xuelin; Wan, David Chi-Cheong; Wang, Yi-Fen; Leung, Ping-Chung; Fung, Kwok-Pui; Lau, Clara Bik-San

    2014-03-15

    Immunomodulation of natural polysaccharides has been the hot topic of research in recent years. In order to explore the immunomodulatory effect of Houttuynia cordata Thunb., the water extract was studied and a polysaccharide HCP-2 with molecular weight of 60,000 Da was isolated by chromatography using DEAE Sepharose CL-6B and Sephacryl S-500 [corrected] HR columns. The structure characterization of HCP-2 was performed by Fourier transform infrared spectroscopy (FTIR), acidic hydrolysis, PMP derivation, HPLC analysis and nuclear magnetic resonance spectra (NMR). HCP-2 was elucidated as a pectic polysaccharide with a linear chain of 1,4-linked α-D-galacturonic acid residues in which part of the 6-carboxyl groups were methyl esterified and part of 2-hydroxyl groups were acetylated. The bioactivity assays showed that HCP-2 could increase the secretions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), macrophage inhibitory protein-1α (MIP-1α), macrophage inhibitory protein-1β (MIP-1β), and RANTES (regulated on activation, normal T cell expressed and secreted) in human peripheral blood mononuclear cells (PBMCs), which play critical roles in the innate immune system and shape the adaptive immunity. Our results implied that HCP-2 could be an immune enhancer.

  1. Extractability and structure of spent coffee ground polysaccharides by roasting pre-treatments.

    PubMed

    Simões, Joana; Nunes, Fernando M; Domingues, M Rosário; Coimbra, Manuel A

    2013-08-14

    The coffee residue left after the preparation of the brew (spent coffee grounds - SCG) is very rich in polysaccharides, namely galactomannans and arabinogalactans, which are polymers that can be used as dietary fibre and present immunostimulatory activity. Considering the huge amount of SCG produced all over the world, the reutilisation of this by-product by its application as food ingredients is very promising. However, the yields of extraction of these polysaccharides tend to be very low, namely the galactomannans. Based on the observation that the yield of galactomannans extracted from the ground coffee to the brew increase when the coffee is roasted, in this study, with the aim of increasing the yield of these polysaccharides, the SCG was roasted and then extracted with hot water and alkali solutions. The roasting at 160°C promoted an increment of 15% in the yield of galactomannan extractions and further improvement of the yield of extraction until 56% of all galactomannans was achieved by alkali extractions at 60 and 120°C. In these samples the galactomannans still kept their characteristic structure, including the acetylation and branching, determined by sugar linkage analysis and mass spectrometry. The yield of extraction of arabinogalactans under these conditions was 54%.

  2. Structure, chain conformation, and immunomodulatory activity of the polysaccharide purified from Bacillus Calmette Guerin formulation.

    PubMed

    Liu, Wei; Wang, Hong; Yu, Juping; Liu, Yameng; Lu, Weisheng; Chai, Yin; Liu, Chao; Pan, Chun; Yao, Wenbing; Gao, Xiangdong

    2016-10-05

    A polysaccharide, coded as BDP, purified from the injection powder of Bacillus Calmette Guerin (BCG) polysaccharide and nucleic acid, was composed mainly of α-D-(1→4)-linked glucan with (1→6)-linked branches and trace amounts of fucose and mannose from the results of FT-IR, HPAEC-PAD and NMR spectrum. The Mw, Mn, Mz, and [Formula: see text] were determined to be 1.320×10(5)g/mol, 1.012×10(5)g/mol, 2.139×10(5)g/mol, and 21.8±3.2%nm by using HPSEC-MALLS, respectively. The ν value from [Formula: see text] was calculated to be 0.52±0.01, which firstly clarified that BDP existed as random coils in 0.9% NaCl aqueous solution. AFM and SEM combined with Congo-red test also revealed that the polysaccharide was irregular globular like or curly structure. Furthermore, in vitro tests on RAW264.7 murine macrophages cells revealed that BDP exhibited significant immunomodulatory activity.

  3. Structural elucidation and immunostimulatory activity of polysaccharide isolated by subcritical water extraction from Cordyceps militaris.

    PubMed

    Luo, Xiaoping; Duan, Yuqing; Yang, Wenya; Zhang, Haihui; Li, Changzheng; Zhang, Jixian

    2017-02-10

    Water-soluble polysaccharides were obtained from Cordyceps militaris (C. militaris) (CMP) by subcritical water extraction (SWE). Two polysaccharides fractions, CMP-W1 and CMP-S1, were isolated from CMP using DEAE-52 cellulose and Sephadex G-150 column chromatography. The structural characteristics of CMP-W1 and CMP-S1 were investigated. The results showed that the molecular weight of CMP-W1 and CMP-S1 are 3.66×105Da and 4.60×105Da, respectively, and both of them were heteropolysaccharides composed of d-mannose, d-glucose, d-galactose with the molar ratios of 2.84:1:1.29 and 2.05:1:1.09, respectively. FT-IR spectra analysis suggested that CMP-W1 and CMP-S1 belonged to pyranose form sugar and protein free. For immunostimulatory activity assay in vitro, CMP-W1 and CMP-S1 significantly promoted lymphatic spleen cell proliferation of mice. Therefore, the polysaccharides obtained from C. militaris by SWE can be used as potential natural immunostimulant in functional foods or medicine.

  4. Structural characterization of polysaccharide obtained from red seaweed Gracilaria caudata (J Agardh).

    PubMed

    Barros, Francisco C N; da Silva, Draulio C; Sombra, Venicios G; Maciel, Jeanny S; Feitosa, Judith P A; Freitas, Ana L P; de Paula, Regina C M

    2013-01-30

    Seaweeds are considered an important source of bioactive molecules. In this work the marine red alga Gracilaria caudata was submitted to aqueous extraction of their polysaccharides for 2 h at 100 °C. The polysaccharide fraction (PGC) presented a recovery of 32.8%. The sulfate content of PGC, calculated by S%, is 1 ± 0.2% and the degree of sulfation accounts for 0.13 ± 0.2. High-Performance Size-Exclusion Chromatography demonstrated that PGC consists of a high molecular weight polysaccharide (2.5 × 10(5)gmol(-1)). Chemical analysis of PGC was performed by microanalysis, infrared (FT-IR) and nuclear magnetic resonance (NMR, 1 and 2D) spectroscopy. The structure of PGC is mainly constituted by the alternating residues 3-linked-β-D-galactopyranose and 4-linked-3,6-α-L-anhydrogalactose; however some hydroxyl groups were substituted by methyl groups and pyruvic acid acetal. The biological precursor of 3,6-α-L-anhydrogalactose (6-sulfate-α-l-galactose) was also detected.

  5. Structure and serology of the native polysaccharide antigen of type Ia group B Streptococcus

    PubMed Central

    Jennings, Harold J.; Rosell, Karl-Gunnar; Kasper, Dennis L.

    1980-01-01

    The native polysaccharide antigen isolated from type Ia group B Streptococcus by using pH-controlled growth conditions and extraction procedures contains D-galactose, D-glucose, 2-acetamido-2-deoxy-D-glucose, and sialic acid in the molar ratio of 2:1:1:1. The structure of the native type Ia antigen has been elucidated; it can be represented by the following repeating unit in which all the side-chain β-D-galactopyranose units are masked by sialic acid residues: [Formula: see text] Removal of all the sialic acid groups yields the incomplete type Ia polysacharide antigen with exposed terminal β-D-galactopyranose residues. Antisera to type Ia organisms produced in rabbits according to the Lancefield procedures contain antibodies specific for both the native and incomplete antigens. Although sialic acid is not itself a determinant in the formation of antibodies to the native polysaccharide, it is an essential part of a larger determinant. In order to maintain the high degree of immunologic specificity of the native antigen, this determinant must be at least a trisaccharide unit, because the native polysaccharide as isolated has terminal disaccharide units [α-D-NeuAcp-(2→3)-β-D-Galp] identical to those found in the human M and N blood group substances and fetuin. Formation of antibodies to the incomplete antigen is due to determinants terminating in β-D-galactopyranose residues. These determinants are probably generated by the removal of the masking sialic acid residues from the cell-associated native polysaccharide by degradative processes that occur in organisms grown without pH control. Images PMID:6156459

  6. Structure and Properties of Polysaccharide Based BioPolymer Gels

    NASA Astrophysics Data System (ADS)

    Prud'Homme, Robert K.

    2000-03-01

    Nature uses the pyranose ring as the basic building unit for a wideclass of biopolymers. Because of their biological origin these biopolymers naturally find application as food additives, rheology modifiers. These polymers range from being rigid skeletal material, such as cellulose that resist dissolution in water, to water soluble polymers, such as guar or carrageenan. The flexibility of the basic pyranose ring structure to provide materials with such a wide range of properties comes from the specific interactions that can be engineered by nature into the structure. We will present several examples of specific interactions for these systems: hydrogen bonding, hydrophobic interactions, and specific ion interactions. The relationship between molecular interations and rheology will be emphasized. Hydrogen bonding mediated by steric interference is used to control of solubility of starch and the rheology of guar gels. A more interesting example is the hydrogen bonding induced by chemical modification in konjac glucomannan that results in a gel that melts upon cooling. Hydrogen bonding interactions in xanthan lead to gel formation at very low polymer concentrations which is a result of the fine tuning of the polymer persistence length and total contour length. Given the function of xanthan in nature its molecular architecture has been optimized. Hydrophobic interactions in methylcellulose show a reverse temperature dependence arising from solution entropy. Carrageenan gelation upon the addition of specific cations will be addressed to show the interplay of polymer secondary structure on chemical reactivity. And finally the cis-hydroxyls on galactomannans permit crosslinking by a variety of metal ions some of which lead to "living gels" and some of which lead to permanently crosslinked networks.

  7. Automatic structure determination of regular polysaccharides based solely on NMR spectroscopy.

    PubMed

    Lundborg, Magnus; Fontana, Carolina; Widmalm, Göran

    2011-11-14

    The structural analysis of polysaccharides requires that the sugar components and their absolute configurations are determined. We here show that this can be performed based on NMR spectroscopy by utilizing butanolysis with (+)- and (-)-2-butanol that gives the corresponding 2-butyl glycosides with characteristic (1)H and (13)C NMR chemical shifts. The subsequent computer-assisted structural determination by CASPER can then be based solely on NMR data in a fully automatic way as shown and implemented herein. The method is additionally advantageous in that reference data only have to be prepared once and from a user's point of view only the unknown sample has to be derivatized for use in CASPER.

  8. A computer-assisted structural analysis of regular polysaccharides on the basis of 13C-n.m.r. data.

    PubMed

    Lipkind, G M; Shashkov, A S; Knirel, Y A; Vinogradov, E V; Kochetkov, N K

    1988-04-01

    A computerised approach to the structural analysis of unbranched regular polysaccharides is described, which is based on an evaluation of the 13C-n.m.r. spectra for all possible primary structures within the additive scheme starting from the chemical shifts of the 13C resonances of the constituent monosaccharides and the average values of the glycosylation effects. The analysis reveals a structure (or structures), the evaluated spectrum of which resembles most closely that observed. The approach has been verified by using a series of bacterial polysaccharides of known structure and, in combination with methylation analysis data, for the determination of the presently unknown structures of the O-specific polysaccharides from Salmonella arizonae O59 and O63, and Proteus hauseri O19.

  9. Capsular hook-assisted implantation of modified capsular tension ring.

    PubMed

    Khokhar, Sudarshan; Gupta, Shikha; Nayak, Bhagabat; Gogia, Varun

    2016-04-04

    A 16-year-old boy presented with decrease of vision over a period of 2 years. On examination, he was diagnosed to have microspherophakia with lenticular myopia with secondary glaucoma in both eyes. He was treated by lens aspiration and two-point capsular support using a modified capsular tension ring (M-CTR) and capsular tension segment (CTS) sutured to the sclera along with implantation of a foldable intraocular lens inside the bag. Lens aspiration was performed without artificial capsular hook support of the bag, as the lens was soft and vitreous was formed. However, M-CTR rotation into the bag was fraught with repeated adherence of the advancing end of the M-CTR into the loose bag causing simultaneous rotation of the bag with the rotation of the ring resulting in transient increase in bag subluxation. Capsular hooks provided appropriate countertraction to the unsupported bag, thus facilitating easy insertion and rotation of the ring into the bag. 2016 BMJ Publishing Group Ltd.

  10. Campylobacter jejuni capsular genotypes are related to Guillain-Barré syndrome.

    PubMed

    Heikema, A P; Islam, Z; Horst-Kreft, D; Huizinga, R; Jacobs, B C; Wagenaar, J A; Poly, F; Guerry, P; van Belkum, A; Parker, C T; Endtz, H P

    2015-09-01

    In about one in a thousand cases, a Campylobacter jejuni infection results in the severe polyneuropathy Guillain-Barré syndrome (GBS). It is established that sialylated lipo-oligosaccharides (LOS) of C. jejuni are a crucial virulence factor in GBS development. Frequent detection of C. jejuni with sialylated LOS in stools derived from patients with uncomplicated enteritis implies that additional bacterial factors should be involved. To assess whether the polysaccharide capsule is a marker for GBS, the capsular genotypes of two geographically distinct GBS-associated C. jejuni strain collections and an uncomplicated enteritis control collection were determined. Capsular genotyping of C. jejuni strains from the Netherlands revealed that three capsular genotypes, HS1/44c, HS2 and HS4c, were dominant in GBS-associated strains and capsular types HS1/44c and HS4c were significantly associated with GBS (p 0.05 and p 0.01, respectively) when compared with uncomplicated enteritis. In a GBS-associated strain collection from Bangladesh, capsular types HS23/36c, HS19 and HS41 were most prevalent and the capsular types HS19 and HS41 were associated with GBS (p 0.008 and p 0.02, respectively). Next, specific combinations of the LOS class and capsular genotypes were identified that were related to the occurrence of GBS. Multilocus sequence typing revealed restricted genetic diversity for strain populations with the capsular types HS2, HS19 and HS41. We conclude that capsular types HS1/44c, HS2, HS4c, HS19, HS23/36c and HS41 are markers for GBS. Besides a crucial role for sialylated LOS of C. jejuni in GBS pathogenesis, the identified capsules may contribute to GBS susceptibility. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

  11. Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds.

    PubMed

    Dourado, Fernando; Madureira, Pedro; Carvalho, Vera; Coelho, Ricardo; Coimbra, Manuel A; Vilanova, Manuel; Mota, Manuel; Gama, Francisco M

    2004-10-20

    The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.

  12. Structures of the O-polysaccharides and classification of Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups.

    PubMed

    Zych, Krystyna; Perepelov, Andrei V; Siwinska, Małgorzata; Knirel, Yuriy A; Sidorczyk, Zygmunt

    2005-11-01

    An acidic branched O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Proteus genomospecies 4 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H-detected 1H, 13C HSQC experiments. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established, which is unique among Proteus polysaccharide structures: [structure: see text] where Qui3NAc stands for 3-acetamido-3,6-dideoxyglucose. Based on the O-polysaccharide structure and serological data, we propose classifying Proteus genomospecies 4 into a new, separate Proteus serogroup, O56. A weak cross-reactivity of Proteus genomospecies 4 antiserum with LPS of Providencia stuartii O18 and Proteus vulgaris OX2 was observed and is discussed in view of a similarity of the O-polysaccharide structures. Structural and serological investigations showed that Proteus genomospecies 5 and 6 should be classified into the existing Proteus serogroups O8 and O69, respectively.

  13. X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid.

    PubMed

    Lizak, Christian; Worrall, Liam J; Baumann, Lars; Pfleiderer, Moritz M; Volkers, Gesa; Sun, Tianjun; Sim, Lyann; Wakarchuk, Warren; Withers, Stephen G; Strynadka, Natalie C J

    2017-07-19

    Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

  14. Salinity-Induced Anti-Angiogenesis Activities and Structural Changes of the Polysaccharides from Cultured Cordyceps Militaris

    PubMed Central

    Zeng, Yangyang; Han, Zhangrun; Qiu, Peiju; Zhou, Zijing; Tang, Yang; Zhao, Yue; Zheng, Sha; Xu, Chenchen; Zhang, Xiuli; Yin, Pinghe; Jiang, Xiaolu; Lu, Hong; Yu, Guangli; Zhang, Lijuan

    2014-01-01

    Cordyceps is a rare and exotic mushroom that grows out of the head of a mummified caterpillar. Many companies are cultivating Cordyceps to meet the increased demand for its medicinal applications. However, the structures and functions of polysaccharides, one of the pharmaceutical active ingredients in Cordyceps, are difficult to reproduce in vitro. We hypothesized that mimicking the salty environment inside caterpillar bodies might make the cultured fungus synthesize polysaccharides with similar structures and functions to that of wild Cordyceps. By adding either sodium sulfate or sodium chloride into growth media, we observed the salinity-induced anti-angiogenesis activities of the polysaccharides purified from the cultured C. Militaris. To correlate the activities with the polysaccharide structures, we performed the 13C-NMR analysis and observed profound structural changes including different proportions of α and β glycosidic bonds and appearances of uronic acid signals in the polysaccharides purified from the culture after the salts were added. By coupling the techniques of stable 34S-sulfate isotope labeling, aniline- and D5-aniline tagging, and stable isotope facilitated uronic acid-reduction with LC-MS analysis, our data revealed for the first time the existence of covalently linked sulfate and the presence of polygalacuronic acids in the polysaccharides purified from the salt added C. Militaris culture. Our data showed that culturing C. Militaris with added salts changed the biosynthetic scheme and resulted in novel polysaccharide structures and functions. These findings might be insightful in terms of how to make C. Militaris cultures to reach or to exceed the potency of wild Cordyceps in future. PMID:25203294

  15. Salinity-induced anti-angiogenesis activities and structural changes of the polysaccharides from cultured Cordyceps Militaris.

    PubMed

    Zeng, Yangyang; Han, Zhangrun; Qiu, Peiju; Zhou, Zijing; Tang, Yang; Zhao, Yue; Zheng, Sha; Xu, Chenchen; Zhang, Xiuli; Yin, Pinghe; Jiang, Xiaolu; Lu, Hong; Yu, Guangli; Zhang, Lijuan

    2014-01-01

    Cordyceps is a rare and exotic mushroom that grows out of the head of a mummified caterpillar. Many companies are cultivating Cordyceps to meet the increased demand for its medicinal applications. However, the structures and functions of polysaccharides, one of the pharmaceutical active ingredients in Cordyceps, are difficult to reproduce in vitro. We hypothesized that mimicking the salty environment inside caterpillar bodies might make the cultured fungus synthesize polysaccharides with similar structures and functions to that of wild Cordyceps. By adding either sodium sulfate or sodium chloride into growth media, we observed the salinity-induced anti-angiogenesis activities of the polysaccharides purified from the cultured C. Militaris. To correlate the activities with the polysaccharide structures, we performed the (13)C-NMR analysis and observed profound structural changes including different proportions of α and β glycosidic bonds and appearances of uronic acid signals in the polysaccharides purified from the culture after the salts were added. By coupling the techniques of stable (34)S-sulfate isotope labeling, aniline- and D5-aniline tagging, and stable isotope facilitated uronic acid-reduction with LC-MS analysis, our data revealed for the first time the existence of covalently linked sulfate and the presence of polygalacuronic acids in the polysaccharides purified from the salt added C. Militaris culture. Our data showed that culturing C. Militaris with added salts changed the biosynthetic scheme and resulted in novel polysaccharide structures and functions. These findings might be insightful in terms of how to make C. Militaris cultures to reach or to exceed the potency of wild Cordyceps in future.

  16. Diatom-Specific Oligosaccharide and Polysaccharide Structures Help to Unravel Biosynthetic Capabilities in Diatoms.

    PubMed

    Gügi, Bruno; Le Costaouec, Tinaïg; Burel, Carole; Lerouge, Patrice; Helbert, William; Bardor, Muriel

    2015-09-18

    Diatoms are marine organisms that represent one of the most important sources of biomass in the ocean, accounting for about 40% of marine primary production, and in the biosphere, contributing up to 20% of global CO₂ fixation. There has been a recent surge in developing the use of diatoms as a source of bioactive compounds in the food and cosmetic industries. In addition, the potential of diatoms such as Phaeodactylum tricornutum as cell factories for the production of biopharmaceuticals is currently under evaluation. These biotechnological applications require a comprehensive understanding of the sugar biosynthesis pathways that operate in diatoms. Here, we review diatom glycan and polysaccharide structures, thus revealing their sugar biosynthesis capabilities.

  17. Structural characterization and functional properties of antihypertensive Cymodocea nodosa sulfated polysaccharide.

    PubMed

    Kolsi, Rihab Ben Abdallah; Fakhfakh, Jawhar; Krichen, Fatma; Jribi, Imed; Chiarore, Antonia; Patti, Francesco Paolo; Blecker, Christophe; Allouche, Noureddine; Belghith, Hafedh; Belghith, Karima

    2016-10-20

    A sulfated polysaccharide was successfully isolated from Cymodocea nodosa (CNSP). This is the first report that indicates the chemical composition, structural characterization, functional and antihypertensive properties of this polysaccharide. The CNSP consisted mainly of sulfate (23.17%), total sugars (54.90%), galactose (44.89%), mannose (17.30%), arabinose (12.05%), xylose (9.18%), maltose (1.07%) and uronic acid (11.03%) with low water activity (0.49). CNSP had an XRD pattern that was typical for a semi-crystalline polymer with homogeneous structure. It also displayed an important anti-hypertensive activity (IC50=0.43mgml) with a dose-dependent manner using a synthetic substrate, N-hippuryl-His-Leu hydrate salt (HHL). Overall, the results indicate that CNSP have attractive chemical, functional and biological properties, with a preliminary structural may have a backbone of branched 6-O-sulfated (1→4) galactosidic linkages, which can be considered in the future as alternative additive in various foods, cosmetic and pharmaceutical preparations.

  18. Structural characterization, α-glucosidase inhibitory and DPPH scavenging activities of polysaccharides from guava.

    PubMed

    Zhang, Ziling; Kong, Fansheng; Ni, Hui; Mo, Zhixian; Wan, Jian-Bo; Hua, Dehong; Yan, Chunyan

    2016-06-25

    To explore the chemicals responsible for the health benefits of guava, water-soluble polysaccharides were extracted including GP90 and P90. They exhibited excellent α-glucosidase inhibition activity with an EC50 of 2.27μg/mL and 0.18mg/mL. This suggests that their activities were 1379- and 17-fold higher than the positive control. The DPPH scavenging activities of GP90 was even higher than Vc at some concentrations. Upon further isolation, a novel polysaccharide termed GB90-1B was obtained. Monosaccharide analysis, methylation analysis, and NMR were used to analyze the structural characterization of GB90-1B. Structural analysis revealed that its backbone consisted of (1→5)-linked-α-l-arabinose, (1→2,3,5)-linked-α-l-arabinose and (1→3)-linked-α-l-arabinose. Branch linkages included (1→6)-linked-α-d-glucose, (1→)-linked-α-d-glucose and (1→)-linked-α-l-arabinose. The structure of the repeating unit of GP90-1B was predicted. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Cereal non-cellulosic polysaccharides: structure and function relationship - an overview.

    PubMed

    Muralikrishna, G; Rao, M V S S T Subba

    2007-01-01

    The non-cellulosic polysaccharides present in cereals (2-8%) are mostly arabinoxylans, (1 --> 3),(1 --> 4)-beta -glucans, pectins and arabinogalactans. Of these, the arabinoxylans are known to absorb large amounts of water and influence significantly the water balance, rheological properties of dough, and the retrogradation of starch and bread quality. (1 --> 3),(1 --> 4)-beta -glucans are known as biological response modifiers (BMS) as they are believed to modulate the immune response. Cereal Pectins and arabinogalactans form a very small amount and do not contribute substantially to the functionality of noncellulosic polysaccharides. Detailed structural investigations on cereal hetero xylans using modern techniques were initiated in the 1990s and still pose a challenge to carbohydrate chemists because of their structural complexity. Nutritionally, they are classified under "unavailable carbohydrates" (dietary fiber) along with lignin and cellulose and are known to have beneficial effects in alleviating disease symptoms such as diabetes, atherosclerosis, and colon cancer. In this review isolation, purification, characterization, structural elucidation, functional, and nutritional attributes of cereal heteroxylans are covered with particular emphasis on recently characterized finger millet arabinoxylans.

  20. Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity*

    PubMed Central

    Borisova, Anna S.; Isaksen, Trine; Dimarogona, Maria; Kognole, Abhishek A.; Mathiesen, Geir; Várnai, Anikó; Røhr, Åsmund K.; Payne, Christina M.; Sørlie, Morten; Sandgren, Mats; Eijsink, Vincent G. H.

    2015-01-01

    The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation. PMID:26178376

  1. Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases

    PubMed Central

    Li, Xin; Beeson, William T.; Phillips, Christopher M.; Marletta, Michael A.; Cate, Jamie H. D.

    2013-01-01

    SUMMARY The use of cellulases remains a major cost in the production of renewable fuels and chemicals from lignocellulosic biomass. Fungi secrete copper-dependent polysaccharide monooxygenases (PMOs) that oxidatively cleave crystalline cellulose and improve the effectiveness of cellulases. However, the means by which PMOs recognize and cleave their substrates in the plant cell wall remain unclear. Here we present structures of Neurospora crassa PMO-2 and PMO-3 at 1.10 Å and 1.37 Å resolution, respectively. In the structures, dioxygen species are found in the active sites, consistent with the proposed cleavage mechanism. Structural and sequence comparisons between PMOs also reveal that the enzyme substrate-binding surfaces contain highly varied aromatic amino acid and glycosylation positions. The structures reported here provide evidence for a wide range of PMO substrate recognition patterns in the plant cell wall, including binding modes that traverse multiple glucan chains. PMID:22578542

  2. Microwave-Assisted Extraction, Chemical Structures, and Chain Conformation of Polysaccharides from a Novel Cordyceps Sinensis Fungus UM01.

    PubMed

    Cheong, Kit-Leong; Wang, Lan-Ying; Wu, Ding-Tao; Hu, De-Jun; Zhao, Jing; Li, Shao-Ping

    2016-09-01

    Cordyceps sinensis is a well-known tonic food with broad medicinal properties. The aim of the present study was to investigate the optimization of microwave-assisted extraction (MAE) and characterize chemical structures and chain conformation of polysaccharides from a novel C. sinensis fungus UM01. Ion-exchange and gel filtration chromatography were used to purify the polysaccharides. The chemical structure of purified polysaccharide was determined through gas chromatography-mass spectrometry. Moreover, high performance size exclusion chromatography combined with refractive index detector and multiangle laser light scattering were conducted to analyze the molecular weight (Mw ) and chain conformation of purified polysaccharide. Based on the orthogonal design L9 , optimal MAE conditions could be obtained through 1300 W of microwave power, with a 5-min irradiation time at a solid to water ratio of 1:60, generating the highest extraction yield of 6.20%. Subsequently, the polysaccharide UM01-S1 was purified. The UM01-S1 is a glucan-type polysaccharide with a (1→4)-β-d-glucosyl backbone and branching points located at O-3 of Glcp with a terminal-d-Glcp. The Mw , radius of gyration (Rg ) and hydrodynamic radius (Rh ) of UM01-S1 were determined as 5.442 × 10(6)  Da, 21.8 and 20.2 nm, respectively. Using the polymer solution theory, the exponent (ν) value of the power law function was calculated as 0.38, and the shape factor (ρ = Rg /Rh ) was 1.079, indicating that UM01-S1 has a sphere-like conformation with a branched structure in an aqueous solution. These results provide fundamental information for the future application of polysaccharides from cultured C. sinensis in health and functional food area. © 2016 Institute of Food Technologists®

  3. Structure of the O-specific polysaccharide of Proteus penneri 103 containing ribitol and 2-aminoethanol phosphates.

    PubMed

    Drzewiecka, Dominika; Toukach, Philip V; Arbatsky, Nikolay P; Zych, Krystyna; Shashkov, Alexander S; Knirel, Yuriy A; Sidorczyk, Zygmunt

    2002-09-27

    The O-specific polysaccharide of the lipopolysaccharide of Proteus penneri strain 103 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,(13)C HMQC, 1H, 31P HMQC, and HMBC experiments. It was found that the polysaccharide is built up of oligosaccharide-ribitol phosphate repeating units and thus resembles ribitol teichoic acids of Gram-positive bacteria. The following structure of the polysaccharide was established:where Etn and Rib-ol are ethanolamine and ribitol, respectively. This structure is unique among the known structures of Proteus O-antigens and, therefore, we propose classification of the strain studied into a new Proteus serogroup, O73. The molecular basis for cross-reactivity between O-antiserum against P. penneri 103 and O-antigens of P. mirabilis O33 and D52 is discussed.

  4. Capsule of Cryptococcus neoformans grows by enlargement of polysaccharide molecules.

    PubMed

    Frases, Susana; Pontes, Bruno; Nimrichter, Leonardo; Viana, Nathan B; Rodrigues, Marcio L; Casadevall, Arturo

    2009-01-27

    The human pathogenic fungus Cryptococcus neoformans has a distinctive polysaccharide (PS) capsule that enlarges during infection. The capsule is essential for virulence, but the mechanism for capsular growth is unknown. In the present study, we used dynamic light scattering (LS) analysis of capsular PS and optical tweezers (OT) to explore the architecture of the capsule. Analysis of capsular PS from cells with small and large capsules by dynamic LS revealed a linear correlation between PS effective diameter and microscopic capsular diameter. This result implied that capsule growth was achieved by the addition of molecules with larger effective diameter, such that some molecules can span the entire diameter of the capsule. Measurement of polystyrene bead penetration of C. neoformans capsules by using OT techniques revealed that the outer regions were penetrable, but not the inner regions. Our results provide a mechanism for capsular enlargement based on the axial lengthening of PS molecules and suggest a model for the architecture of a eukaryotic microbial capsule.

  5. PCR for capsular typing of Haemophilus influenzae.

    PubMed Central

    Falla, T J; Crook, D W; Brophy, L N; Maskell, D; Kroll, J S; Moxon, E R

    1994-01-01

    A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine. Images PMID:7814470

  6. Structure of the O-specific polysaccharide of Salmonella arizonae O45.

    PubMed

    Shashkov, A S; Vinogradov, E V; Knirel, Y A; Nifant'ev, N E; Kochetkov, N K; Dabrowski, J; Kholodkova, E V; Stanislavsky, E S

    1993-03-17

    The O-specific polysaccharide of Salmonella arizonae O45 (Arizona 11) is acidic and has a branched hexasaccharide repeating unit containing two residues of L-fucose, one residue each of D-galactose, D-ribose, D-glucuronic acid, and 2-acetamido-2-deoxy-D-glucose, and an O-acetyl group. It was studied with the help of 1H and 13C NMR spectroscopy, including 1D selective spin-decoupling and homonuclear Hartmann-Hahn spectroscopy, 2D homonuclear and 13C-1H heteronuclear shift-correlated (COSY) and NOE (ROESY) spectroscopy, as well as by methylation analysis, and selective cleavages with anhydrous HF (or dilute HCl) and lithium in ethylenediamine to yield two different tetrasaccharide fragments. As a result, the following structure of the polysaccharide was established: [formula: see text] Anomalous 13C chemical shifts were observed in the spectrum of the trisaccharide fragment alpha-L-Fucp-(1-->2)-beta-D-Gal p-(1-->3)-beta-D-Glc pNAc, structurally related to the Le(d) blood-group determinant, and rationalised by inter-residue proton-proton interactions.

  7. Structural characterization and antioxidant activity in vitro of polysaccharides from angelica and astragalus.

    PubMed

    Pu, Xiuying; Ma, Xiaolong; Liu, Lu; Ren, Jing; Li, Haibing; Li, Xiaoyue; Yu, Shuang; Zhang, Weijie; Fan, Wenbo

    2016-02-10

    In the present study, structural characterization and antioxidant activity of a fraction (AAP-2A) of polysaccharides from angelica and astragalus (AAP) were investigated. Characteriztion assay showed that AAP-2A had molecular weight (Mw), root-mean square (RMS) radius and polydispersity index (Mw/Mn) of 2.252 × 10(3)kDa, 28.4 nm and 1.038, respectively. There were infrared characteristic absorption peaks of polysaccharides in FT-IR spectroscopy. AAP-2A was composed of rhamnose (Rha), galactose (Gal), arabinose (Ara) and glucose (Glc) with a molar ratio of 1:2.13:3.22:6.18 in GC analysis. Methylation analysis combined with NMR spectroscopic analysis demonstrated that a preliminary structure of AAP-2A was proposed as follows: 1,3-linked Rhap, 1,3-linked Galp, 1,3-linked Araf, 1,5-linked Araf, 1,3,5-linked Araf, 1,4-linked Glcp and 1,4,6-linked Glcp interspersed with terminal Glcp. AAP-2A exhibited a surface with a sheet-like appearance in scanning electron microscope and stronger antioxidant capacity compared with AAP.

  8. Structural characterization of wall and lipidated polysaccharides from Clostridium perfringens ATCC 13124.

    PubMed

    Vinogradov, Evgeny; Aubry, Annie; Logan, Susan M

    2017-08-07

    Cell surface polysaccharides produced by C. perfringens ATCC 13124 were analyzed using NMR, chemical and immunological methods. Two distinct polymers were identified. The more abundant PS1 had a structure based on a polymer of β-mannosamine with a number of modifications, including varying levels of substitution at O-6 with PEtN, N-acetylation, and different linkages between monosaccharides. The shortest variant of PS1 represented a lipoteichoic acid. It contained only 1-4-linkages between ManNAc residues, minor branching α-Ribf, and glucosyl-glycerol at the reducing end, which was acylated with linear saturated fatty acids C16, C18, and C20 (dominant). Other non-lipidated variants of PS1 contained less PEtN, no α-Ribf, up to 50% 1-3-linkages, and up to 25% ManN with the free amino group. The minor polysaccharide PS2 had a linear regular structure with a -4-α-Rha-3-β-Gal-4-β-GalNAc3PCho- repeating unit, where PCho indicates phosphocholine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Structural characterization and inhibition on α-glucosidase activity of acidic polysaccharide from Annona squamosa.

    PubMed

    Ren, Yuan-Yuan; Zhu, Zhen-Yuan; Sun, Hui-Qing; Chen, Li-Jing

    2017-10-15

    The crude polysaccharide (TASP3) was extracted from the fruit pulp of Annona squamosa and then isolated and purified by the combination of grading-alcoholic precipitation and Sephadex G-200. The structure of purified polysaccharide (GASP3-3-I) was determined based on the physicochemical and instrumental analyses. The results indicated that GASP3-3-I was an acidic heteropolysaccharide and its average molecular weight was 2.28×10(6)Da. The monosaccharide composition was analysed by GC-MS and ion chromatography, respectively. It was revealed that GASP3-3-I was consisted of rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid with a molar ratio of 5.06:45.5:5.26:0.63:6.09:31.76:0.49:5.19. Moreover, periodate oxidation reaction, Smith degrading reaction, methylation, FT-IR and NMR were used to conduct the structural characterization of GASP3-3-I. The results indicated that glycosyl residues of GASP3-3-I were mainly composed of (1→) l-arabinose, (1→6), (1→3) and (1,3→6) d-galactose, (1→) d-xylose, (3→) and (3→6) d-glucose, (1→2) l-rhamnose. The α-glucosidase inhibitory activity assay showed that GASP3-3-I had a certain inhibition on α-glucosidase activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Differential regulation of polysaccharide-specific antibody responses to isolated polysaccharides, conjugate vaccines, and intact Gram-positive versus Gram-negative extracellular bacteria.

    PubMed

    Snapper, Clifford M

    2016-06-24

    Bacterial capsular polysaccharides are major virulence factors and are key targets in a number of licensed anti-bacterial vaccines. Their major characteristics are their large molecular weight and expression of repeating antigenic epitopes that mediate multivalent B cell receptor cross-linking. In addition, since the majority of these antigens cannot associate with MHC-II they fail to recruit CD4+ T cell help and are referred to as T cell-independent antigens. In this review I will discuss a series of studies from my laboratory that have underscored the importance of understanding polysaccharide-specific antibody responses within the context in which the PS is expressed (i.e. in isolation, as a component of conjugate vaccines, and expressed naturally by intact bacteria). We have shown that multivalent B cell receptor crosslinking, as mediated by polysaccharides, uniquely determines the qualitative response of the B cell to subsequent stimuli, but by itself is insufficient to induce antibody secretion or class switching. For these latter events to occur, second signals must act in concert with primary signals derived from the B cell receptor. The co-expression of polysaccharide and protein within intact bacteria promotes recruitment of CD4+ T cell help for the associated PS-specific IgG response, in contrast to isolated polysaccharides. Further, the particulate nature of extracellular bacteria confers properties to the polysaccharide-specific IgG response that makes it distinct immunologically from soluble conjugate vaccines. Finally, the underlying biochemical and/or structural differences that distinguish Gram-positive and Gram-negative bacteria appear to play critical roles in differentially regulating the associated polysaccharide-specific IgG responses to these groups of pathogens. These studies have a number of implications for the understanding and future design of polysaccharide-based vaccines. Published by Elsevier Ltd.

  11. Structure and optical properties of noble metal and oxide nanoparticles dispersed in various polysaccharide biopolymers

    NASA Astrophysics Data System (ADS)

    Djoković, V.; Božanic, D. K.; Vodnik, V. V.; Krsmanović, R. M.; Trandafilovic, L. V.; Dimitrijević-Branković, S.

    2011-10-01

    We present the results on the structure and the optical properties of noble metal (Ag, Au) and oxide (ZnO) nanoparticles synthesized by various methods in different polysaccharide matrices such as chitosan, glycogen, alginate and starch. The structure of the obtained nanoparticles was studied in detail with microscopic techniques (TEM, SEM), while the XPS spectroscopy was used to investigate the effects at the nanoparticle-biomolecule interfaces. The antimicrobial activity of the nanocomposite films with Ag nanoparticles was tested against the Staphylococcus aureus, Escherichia coli and Candida albicans pathogens. In addition, we will present the results on the structure and optical properties of the tryptophan amino acid functionalized silver nanoparticles dispersed in water soluble polymer matrices.

  12. 1H and 13C NMR characterization and secondary structure of the K2 polysaccharide of Klebsiella pneumoniae strain 52145.

    PubMed

    Corsaro, Maria Michela; De Castro, Cristina; Naldi, Teresa; Parrilli, Michelangelo; Tomás, Juan M; Regué, Miguel

    2005-09-26

    The complete (1)H and (13)C NMR characterization of the tetrasaccharide repeating unit from the K2 polysaccharide of Klebsiella pneumoniae strain 52145 is reported. [chemical structure] In addition a model for its secondary structure was suggested on the basis of dynamic and molecular calculations.

  13. Physicochemical Properties of Neisseria meningitidis Group X Polysaccharide Antigen

    PubMed Central

    Apicella, M. A.; Robinson, John A.

    1972-01-01

    Neisseria meningitidis group X occurs in human carrier populations and is rarely implicated in serious disease. This organism possesses a capsular group antigen which is an acidic polysaccharide. It is composed of the amino sugars, glucosamine, glucosamine-6-phosphate, galactosamine, and the simple hexose, glucose. The group X capsular antigen has an S20,w0 of 3.6, and the acidic nature of the polysaccharide is reflected in an isoelectric point of 3.65. The meningococcal A, B, C, and Y polysaccharide group antigens are also composed primarily of amino sugars. The chemical composition of the group X antigen most closely resembles the capsular antigen of N. meningitidis group Y, which is also predominately a carrier organism. Images PMID:4629206

  14. Chemical Modification of Polysaccharides

    PubMed Central

    Cumpstey, Ian

    2013-01-01

    This review covers methods for modifying the structures of polysaccharides. The introduction of hydrophobic, acidic, basic, or other functionality into polysaccharide structures can alter the properties of materials based on these substances. The development of chemical methods to achieve this aim is an ongoing area of research that is expected to become more important as the emphasis on using renewable starting materials and sustainable processes increases in the future. The methods covered in this review include ester and ether formation using saccharide oxygen nucleophiles, including enzymatic reactions and aspects of regioselectivity; the introduction of heteroatomic nucleophiles into polysaccharide chains; the oxidation of polysaccharides, including oxidative glycol cleavage, chemical oxidation of primary alcohols to carboxylic acids, and enzymatic oxidation of primary alcohols to aldehydes; reactions of uronic-acid-based polysaccharides; nucleophilic reactions of the amines of chitosan; and the formation of unsaturated polysaccharide derivatives. PMID:24151557

  15. Characterization of mucilage polysaccharides, arabinogalactanproteins and cell-wall hemicellulosic polysaccharides isolated from flax seed meal: A wealth of structural moieties.

    PubMed

    Ray, Sayani; Paynel, Florence; Morvan, Claudine; Lerouge, Patrice; Driouich, Azeddine; Ray, Bimalendu

    2013-04-02

    The present study aimed at analyzing the structural features of seed mucilage and cell-wall polysaccharides which accounted for 41% of the mass of flax meal (FM). A combination of high molar-mass mucilage-like polysaccharides (rhamnogalacturonan and arabinoxylan) was released from FM in water, together with arabinogalactan proteins and glucans. About half of FM homogalacturonans was extracted using a calcium chelator and boiling water. Hemicellulosic xyloglucans and xylans were further extracted with 1M KOH, in ∼13% FM-sugars yield. Structural characterization of the xyloglucan using specific enzyme hydrolysis, ion exchange chromatography (HPAEC) and matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectroscopy showed the presence of XXXG type xyloglucan, but also that of XXGG-structure, possibly characteristic of flax seeds. Hydrolysis of xylans with endo-(1→4)-β-D-xylanase, and analysis of the neutral and acidic oligosaccharides by MALDI-TOF-MS showed that xylan consisted of β-(1→4)-linked-D-xylopyranose backbone with some zones (DP 5-7) substituted with 4-O-MeGlcA\\GlcA\\Glc residues. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Structure of the O-polysaccharide leads to classification of Proteus penneri 31 in Proteus serogroup O19.

    PubMed

    Kondakova, Anna N; Zych, Krystyna; Senchenkova, Sof'ya N; Zabłotni, Agnieszka; Shashkov, Alexander S; Knirel, Yuriy A; Sidorczyk, Zygmunt

    2003-10-24

    O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide (LPS) of Proteus penneri strain 31. Sugar and methylation analyses along with NMR spectroscopic studies, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C and 1H,31P HMQC experiments, demonstrated the following structure of the polysaccharide: [carbohydrate structure: see text] where FucNAc is 2-acetamido-2,6-dideoxygalactose and EtnP is 2-aminoethyl phosphate. The polysaccharide studied has the same carbohydrate backbone as the O-polysaccharide of Proteus vulgaris O19. Based on this finding and close serological relatedness of the LPS of the two strains, it is proposed to classify P. penneri 31 in Proteus serogroup O19 as an additional subgroup. In contrast, D-GlcNAc6PEtn and alpha-L-FucNAc-(1-->3)-D-GlcNAc shared with a number of other Proteus O-polysaccharides could not provide any significant cross-reactivity of the corresponding LPS with rabbit polyclonal O-antiserum against P. penneri 31.

  17. Purification and Structural Characterization of Sulfated Polysaccharide from Sargassum myriocystum and its Efficacy in Scavenging Free Radicals.

    PubMed

    Badrinathan, S; Shiju, T M; Sharon Christa, A Suneeva; Arya, R; Pragasam, V

    2012-11-01

    Sulfated polysaccharides from marine algae are one of the commercially beneficial compounds with a range of pharmaceutical and biomedical applications. They are testified to be effective against free radicals and related health complications. This study aims to determine the antioxidant potential of the sulfated polysaccharide from Sargassum myriocystum, followed by its purification and structural characterization. Amount of extract obtained was 5% from 10 g of plant material. The carbohydrate and sulfate content were found to be 31 and 0.34 mg/10 g of plant material, respectively. Total sulfated polysaccharide extract showed a good radical scavenging activity at lower concentrations. The active principle from the total sulfated polysaccharide was fractionated in anion exchange and gel filtration columns followed by structural characterization using Fourier transform infrared and nuclear magnetic resonance spectroscopy. Fraction 12 closely matched with the Fourier transform infrared and nuclear magnetic resonance spectra of fucoidan. Based on the results obtained, we conclude that sulfated polysaccharide from Sargassum myriocystum is identified as Fucoidan with potential radical scavenging activity compared to butylated hydroxyl toluene.

  18. Structural Features of Alkaline Extracted Polysaccharide from the Seeds of Plantago asiatica L. and Its Rheological Properties.

    PubMed

    Yin, Jun-Yi; Chen, Hai-Hong; Lin, Hui-Xia; Xie, Ming-Yong; Nie, Shao-Ping

    2016-09-06

    Polysaccharide from the seeds of Plantago asiatica L. has many bioactivities, but few papers report on the structural and rheological characteristics of the alkaline extract. The alkaline extracted polysaccharide was prepared from seeds of P. asiatica L. and named herein as alkaline extracted polysaccharide from seeds of P. asiatica L. (PLAP). Its structural and rheological properties were characterized by monosaccharide composition, methylation, GC-MS and rheometry. PLAP, as an acidic arabinoxylan, was mainly composed of 1,2,4-linked Xylp and 1,3,4-linked Xylp residues. PLAP solution showed pseudoplastic behavior, and weak gelling properties at high concentration. Sodium and especially calcium ions played a significant role in increasing the apparent viscosity and gel strength.

  19. Capsular Weakness around Breast Implant: A Non-Recognized Complication

    PubMed Central

    Arquero, Pedro Salinero; Zanata, Fabiana Cristina; Ferreira, Lydia Masako; Nahas, Fabio Xerfan

    2015-01-01

    Capsular contraction is a frequent complication following breast augmentation. On the other hand, capsular weakness, a not widely recognized complication, may occur around the implant. A weak capsule allows the migration of the prosthesis to the lateral region of the thoracic region or inferiorly, towards the abdomen, due to gravitational forces. The cause of capsular weakness remains unresolved. Implant malposition, with lateral or downward displacement, breast asymmetry, improper contour, with implants moving in the pocket that compromise the aesthetic outcome of breast augmentation and require surgical correction may be different symptoms from the same clinical problem. Capsular weakness is a short or mid-term complication of breast augmentation. Most techniques aim to correct the malposition by making sutures to increase the resistance to the displacement of the implant, rearrange the structures using the capsule as flaps to remodel the envelope of the new pocket, obtaining a more stable and reliable result. In this article, four cases of displacement of breast prosthesis with capsular weakness are described and the surgical treatment that included a capsulotomy and capsulorraphy is described. PMID:26284187

  20. Structural characterization of the extracellular polysaccharide from Vibrio cholerae O1 El-Tor.

    PubMed

    Yildiz, Fitnat; Fong, Jiunn; Sadovskaya, Irina; Grard, Thierry; Vinogradov, Evgeny

    2014-01-01

    The ability to form biofilms is important for environmental survival, transmission, and infectivity of Vibrio cholerae, the causative agent of cholera in humans. To form biofilms, V. cholerae produces an extracellular matrix composed of proteins, nucleic acids and a glycoconjugate, termed Vibrio exopolysaccharide (VPS). Here, we present the data on isolation and characterization of the polysaccharide part of the VPS (VPS-PS), which has the following structure: -4)-α-GulpNAcAGly3OAc-(1-4)-β-D-Glcp-(1-4)-α-Glcp-(1-4)-α-D-Galp-(1- where α-D-Glc is partially (∼20%) replaced with α-D-GlcNAc. α-GulNAcAGly is an amide between 2-acetamido-2-deoxy-α-guluronic acid and glycine. Apparently, the polysaccharide is bound to a yet unidentified component, which gives it high viscosity and completely suppresses any NMR signals belonging to the sugar chains of the VPS. The only reliable method to remove this component at present is a treatment of the whole glycoconjugate with concentrated hydrochloric acid.

  1. Characterization of structural cell wall polysaccharides in cattail (Typha latifolia): Evaluation as potential biofuel feedstock.

    PubMed

    Rebaque, Diego; Martínez-Rubio, Romina; Fornalé, Silvia; García-Angulo, Penélope; Alonso-Simón, Ana; Álvarez, Jesús M; Caparros-Ruiz, David; Acebes, José L; Encina, Antonio

    2017-11-01

    Second generation bioethanol produced from lignocellulosic biomass is attracting attention as an alternative energy source. In this study, a detailed knowledge of the composition and structure of common cattail (Typha latifolia L.) cell wall polysaccharides, obtained from stem or leaves, has been conducted using a wide set of techniques to evaluate this species as a potential bioethanol feedstock. Our results showed that common cattail cellulose content was high for plants in the order Poales and was accompanied by a small amount of cross-linked polysaccharides. A high degree of arabinose-substitution in xylans, a high syringyl/guaiacyl ratio in lignin and a low level of cell wall crystallinity could yield a good performance for lignocellulose saccharification. These results identify common cattail as a promising plant for use as potential bioethanol feedstock. To the best of our knowledge, this is the first in-depth analysis to be conducted of lignocellulosic material from common cattail. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide.

    PubMed

    Liu, Jun; Meng, Chen-Guang; Yan, Ye-Hua; Shan, Ya-Na; Kan, Juan; Jin, Chang-Hai

    2016-01-01

    In this study, structural characterization, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide (catechin-g-TPS) were investigated. Crude polysaccharides were isolated from the fruit bodies of T. fuciformis and further purified on DEAE-52 and Sepharose CL-4B chromatography to afford a main purified fraction (named TPS). The molecular weight of TPS was determined as 5.82 × 10(5)Da by HPLC. Then, the free radical mediated grafting of catechin onto TPS was achieved by using a redox system. As compared with the unmodified TPS, catechin-g-TPS showed new bands within the range of 1300-1600 cm(-1) in FT-IR spectrum, and exhibited new signals at around δ 6.00 and 6.80 ppm in (1)H NMR spectrum. Thermogravimetric analysis indicated the thermal stability of catechin-g-TPS was higher than TPS. X-ray diffraction spectrum of catechin-g-TPS exhibited two sharp narrow diffraction peaks at 14.2 and 32.1°, corresponding to the crystalline peaks of catechin. Scanning electron microscopy observation revealed the surface of TPS was smooth, whereas the surface of catechin-g-TPS was much rough. These results all confirmed the successful grafting of catechin onto TPS. Moreover, catechin-g-TPS had higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power as compared to TPS.

  3. The Structure-Activity Relationship between Marine Algae Polysaccharides and Anti-Complement Activity

    PubMed Central

    Jin, Weihua; Zhang, Wenjing; Liang, Hongze; Zhang, Quanbin

    2015-01-01

    In this study, 33 different polysaccharides were prepared to investigate the structure-activity relationships between the polysaccharides, mainly from marine algae, and anti-complement activity in the classical pathway. Factors considered included extraction methods, fractionations, molecular weight, molar ratio of galactose to fucose, sulfate, uronic acid (UA) content, linkage, branching, and the type of monosaccharide. It was shown that the larger the molecular weights, the better the activities. The molar ratio of galactose (Gal) to fucose (Fuc) was a positive factor at a concentration lower than 10 µg/mL, while it had no effect at a concentration more than 10 µg/mL. In addition, sulfate was necessary; however, the sulfate content, the sulfate pattern, linkage and branching had no effect at a concentration of more than 10 µg/mL. Moreover, the type of monosaccharide had no effect. Laminaran and UA fractions had no activity; however, they could reduce the activity by decreasing the effective concentration of the active composition when they were mixed with the active compositions. The effect of the extraction methods could not be determined. Finally, it was observed that sulfated galactofucan showed good anti-complement activity after separation. PMID:26712768

  4. The Structure-Activity Relationship between Marine Algae Polysaccharides and Anti-Complement Activity.

    PubMed

    Jin, Weihua; Zhang, Wenjing; Liang, Hongze; Zhang, Quanbin

    2015-12-25

    In this study, 33 different polysaccharides were prepared to investigate the structure-activity relationships between the polysaccharides, mainly from marine algae, and anti-complement activity in the classical pathway. Factors considered included extraction methods, fractionations, molecular weight, molar ratio of galactose to fucose, sulfate, uronic acid (UA) content, linkage, branching, and the type of monosaccharide. It was shown that the larger the molecular weights, the better the activities. The molar ratio of galactose (Gal) to fucose (Fuc) was a positive factor at a concentration lower than 10 µg/mL, while it had no effect at a concentration more than 10 µg/mL. In addition, sulfate was necessary; however, the sulfate content, the sulfate pattern, linkage and branching had no effect at a concentration of more than 10 µg/mL. Moreover, the type of monosaccharide had no effect. Laminaran and UA fractions had no activity; however, they could reduce the activity by decreasing the effective concentration of the active composition when they were mixed with the active compositions. The effect of the extraction methods could not be determined. Finally, it was observed that sulfated galactofucan showed good anti-complement activity after separation.

  5. Structure-activity relationship of sulfated hetero/galactofucan polysaccharides on dopaminergic neuron.

    PubMed

    Wang, Jing; Liu, Huaide; Jin, Weihua; Zhang, Hong; Zhang, Quanbin

    2016-01-01

    Parkinson's disease (PD) is associated with progressive loss of dopaminergic neurons and more-widespread neuronal changes that cause complex symptoms. The aim of this study was to investigate the structure-activity relationship of sulfated hetero-polysaccharides (DF1) and sulfated galactofucan polysaccharides (DF2) on dopaminergic neuron in vivo and in vitro. Treatment with samples significantly ameliorated the depletion of both DA and TH-, Bcl-2- and Bax-positive neurons in MPTP-induced PD mice, DF1 showed the highest activity. The in vitro results found that DF1 and DF2 could reverse the decreased mitochondrial activity and the increased LDL release induced by MPP(+) (P<0.01 or P<0.001) which provides further evidence that DF1 and DF2 also exerts a direct protection against the neuronal injury caused by MPP(+). Furthermore, the administration of samples effectively decreased lipid peroxidation and increased the level/activities of GSH, GSH-PX, MDA and CAT in MPTP mice. Thus, the neuron protective effect may be mediated, in part, through antioxidant activity and the prevention of cell apoptosis. The chemical composition of DF1, DF2 and DF differed markedly, the DF1 fraction had the most complex chemical composition and showed the highest neuron protective activity. These results suggest that diverse monosaccharides and uronic acid might contribute to neuron protective activity.

  6. Capsules from Pathogenic and Non-Pathogenic Cryptococcus spp. Manifest Significant Differences in Structure and Ability to Protect against Phagocytic Cells

    PubMed Central

    Araujo, Glauber de S.; Fonseca, Fernanda L.; Pontes, Bruno; Torres, Andre; Cordero, Radames J. B.; Zancopé-Oliveira, Rosely M.; Casadevall, Arturo; Viana, Nathan B.; Nimrichter, Leonardo; Rodrigues, Marcio L.; Garcia, Eloi S.; de Souza, Wanderley; Frases, Susana

    2012-01-01

    Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular

  7. Study of macrophage activation and structural characteristics of purified polysaccharides from the fruiting body of Hericium erinaceus.

    PubMed

    Lee, Jong Seok; Min, Kyoung Min; Cho, Jae Youl; Hong, Eock Kee

    2009-09-01

    Most, if not all, Basidiomycetes mushrooms have biologically active polysaccharides showing potent antitumor activity with immunomodulating properties. These polysaccharides have various chemical compositions and belong primarily to the beta-glucan group. In this study, the crude water-soluble polysaccharide HEF-P, which was obtained from the fruiting body of Hericium erinaceus by hot water extraction and ethanol precipitation, was fractionated by DEAE-cellulose and Sepharose CL-6B column chromatographies. This process resulted in four polysaccharide fractions, named HEF-NP Fr I, HEF-NP Fr II, HEF-AP Fr I, and HEF-AP Fr II. Of these fractions, HEF-AP Fr II was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide and expression of cytokines (IL-1beta and TNF-beta). The molecular mass of HEF-AP Fr II was estimated by gel filtration to be 13 kDa. Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicate that HEF-AP Fr II is a low-molecular-mass polysaccharide with a laminarin-like triple helix conformation of a beta-1,3-branched-beta-1,6-glucan.

  8. Pectic polysaccharide from the green fruits of Momordica charantia (Karela): structural characterization and study of immunoenhancing and antioxidant properties.

    PubMed

    Panda, Bibhash C; Mondal, Soumitra; Devi, K Sanjana P; Maiti, Tapas K; Khatua, Somanjana; Acharya, Krishnendu; Islam, Syed S

    2015-01-12

    A water soluble pectic polysaccharide (PS) isolated from the aqueous extract of the green fruits of Momordica charantia contains D-galactose and D-methyl galacturonate in a molar ratio of nearly 1:4. It showed splenocyte, thymocyte as well as macrophage activations. Moreover, it exhibited potent antioxidant activities. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, and 1D and 2D NMR studies, the structure of the repeating unit of the pectic polysaccharide was established as: [Formula: see text]. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Antitumor polysaccharides from mushrooms: a review on the structural characteristics, antitumor mechanisms and immunomodulating activities.

    PubMed

    Meng, Xin; Liang, Hebin; Luo, Lixin

    2016-04-07

    Mushrooms are popular folk medicines that have attracted considerable attention because of their efficient antitumor activities. This review covers existing research achievements on the mechanisms of isolated mushroom polysaccharides, particularly (1→3)-β-D-glucans. Our review also describes the function in modulating the immune system and potential tumor-inhibitory effects of polysaccharides. The antitumor mechanisms of mushroom polysaccharides are mediated by stimulated T cells or other immune cells. These polysaccharides are able to trigger various cellular responses, such as the expression of cytokines and nitric oxide. Most polysaccharides could bind other conjugate molecules, such as polypeptides and proteins, whose conjugation always possess strong antitumor activities. The purpose of this review is to summarize available information, and to reflect the present situation of polysaccharide research filed with a view for future direction.

  10. [Somatic antigens of the Brucella genus. The structure of the O-specific polysaccharide chain of Brucella melitensis lipopolysaccharide].

    PubMed

    L'vov, V L; Malikov, V E; Shashkov, A S; Dranovskaia, E A; Dmitriev, B A

    1985-07-01

    The phenol-phase soluble antigenic lipopolysaccharide was isolated from Brucella melitensis, strain 565, by the routine phenol/water procedure followed by chromatography on Sepharose 4B. After mild acid hydrolysis and chromatography on Sephadex G-50, the lipopolysaccharide yielded a linear O-specific polysaccharide built up from 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The structure of the polysaccharide was deduced mainly from the nuclear magnetic resonance and methylation analyses. The phenol-soluble lipopolysaccharide, isolated from commercial vaccine strain B. abortus 19-BA, on mild hydrolysis afforded material, 13C and 1H-NMR spectra of which were identical to those of the O-specific polysaccharide from B. melitensis 565.

  11. Diatom-Specific Oligosaccharide and Polysaccharide Structures Help to Unravel Biosynthetic Capabilities in Diatoms

    PubMed Central

    Gügi, Bruno; Le Costaouec, Tinaïg; Burel, Carole; Lerouge, Patrice; Helbert, William; Bardor, Muriel

    2015-01-01

    Diatoms are marine organisms that represent one of the most important sources of biomass in the ocean, accounting for about 40% of marine primary production, and in the biosphere, contributing up to 20% of global CO2 fixation. There has been a recent surge in developing the use of diatoms as a source of bioactive compounds in the food and cosmetic industries. In addition, the potential of diatoms such as Phaeodactylum tricornutum as cell factories for the production of biopharmaceuticals is currently under evaluation. These biotechnological applications require a comprehensive understanding of the sugar biosynthesis pathways that operate in diatoms. Here, we review diatom glycan and polysaccharide structures, thus revealing their sugar biosynthesis capabilities. PMID:26393622

  12. Structural Investigation of Cell Wall Xylan Polysaccharides from the Leaves of Algerian Argania spinosa.

    PubMed

    Hachem, Kadda; Faugeron, Céline; Kaid-Harche, Meriem; Gloaguen, Vincent

    2016-11-21

    Xylan-type polysaccharides were isolated from the leaves of Argania spinosa (L.) Skeels collected in the Tindouf area (southwestern Algeria). Xylan fractions were obtained by sequential alkaline extractions and purified on Sepharose CL-4B. The xylan structure was investigated by enzymatic hydrolysis with an endo-β(1→4)-xylanase followed by chromatography of the resulting fragments on Biogel P2, characterization by sugar analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS ). The results show that the A. spinosa xylan is composed of a β-(1→4)-d-xylopyranose backbone substituted with 4-O-methyl-d-glucuronic acid and L-arabinose residues.

  13. Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines

    PubMed Central

    Lockyer, Kay; Gao, Fang; Derrick, Jeremy P.; Bolgiano, Barbara

    2015-01-01

    An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8 × 106 g/mol to larger than 20 × 106 g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines. PMID:25640334

  14. Structure and Antitumor and Immunomodulatory Activities of a Water-Soluble Polysaccharide from Dimocarpus longan Pulp

    PubMed Central

    Meng, Fa-Yan; Ning, Yuan-Ling; Qi, Jia; He, Zhou; Jie, Jiang; Lin, Juan-Juan; Huang, Yan-Jun; Li, Fu-Sen; Li, Xue-Hua

    2014-01-01

    A new water-soluble polysaccharide (longan polysaccharide 1 (LP1)) was extracted and successfully purified from Dimocarpus longan pulp via diethylaminoethyl (DEAE)-cellulose anion-exchange and Sephacryl S-300 HR gel chromatography. The chemical structure was determined using Infrared (IR), gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. The results indicated that the molecular weight of the sample was 1.1 × 105 Da. Monosaccharide composition analysis revealed that LP1 was composed of Glc, GalA, Ara and Gal in a molar ratio of 5.39:1.04:0.74:0.21. Structural analysis indicated that LP1 consisted of a backbone of →4)-α-d-Glcp-(1→4)-α-d-GalpA-(1→4)-α-d-Glcp-(1→4)-β-d-Glcp-(1→ units with poly saccharide side chains composed of →2)-β-d-Fruf-(1→2)-l-sorbose-(1→ attached to the O-6 position of the α-d-Glcp residues. In vitro experiments indicated that LP1 had significantly high antitumor activity against SKOV3 and HO8910 tumor cells, with inhibition percentages of 40% and 50%, respectively. In addition, LP1 significantly stimulated the production of the cytokine interferon-γ (IFN-γ), increased the activity of murine macrophages and enhanced B- and T-lymphocyte proliferation. The results of this study demonstrate that LP1 has potential applications as a natural antitumor agent with immunomodulatory activity. PMID:24663085

  15. Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines.

    PubMed

    Lockyer, Kay; Gao, Fang; Derrick, Jeremy P; Bolgiano, Barbara

    2015-03-10

    An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×10(6) g/mol to larger than 20×10(6) g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines.

  16. Quaternary structure of WzzB and WzzE polysaccharide copolymerases

    PubMed Central

    Kalynych, Sergei; Cherney, Maia; Bostina, Mihnea; Rouiller, Isabelle; Cygler, Miroslaw

    2015-01-01

    Bacteria have evolved cellular control mechanisms to ensure proper length specification for surface-bound polysaccharides. Members of the Polysaccharide Copolymerase (PCP) family are central to this process. PCP-1 family members are anchored to the inner membrane through two transmembrane helices and contain a large periplasm-exposed domain. PCPs are known to form homooligomers but their exact stoichiometry is controversial in view of conflicting structural and biochemical data. Several prior investigations addressing this question indicated a nonameric, hexameric, or tetrameric organization of several PCP-1 family members. In this work, we gathered additional evidence that E.coli WzzB and WzzE PCPs form octameric homo-oligomeric complexes. Detergent-solubilized PCPs were purified to homogeneity and subjected to blue native gel analysis, which indicated the presence of a predominant high-molecular product of over 500 kDa in mass. Molecular mass of WzzE and WzzB-detergent oligomers was estimated to be 550 kDA by size-exclusion coupled to multiangle laser light scattering (SEC-MALLS). Oligomeric organization of purified WzzB and WzzE was further investigated by negative stain electron microscopy and by X-ray crystallography, respectively. Analysis of EM-derived molecular envelope of WzzB indicated that the full-length protein is composed of eight protomers. Crystal structure of LDAO-solubilized WzzE was solved to 6 Å resolutions and revealed its octameric subunit stoichiometry. In summary, we identified a possible biological unit utilized for the glycan chain length determination by two PCP-1 family members. This provides an important step toward further unraveling of the mechanistic basis of chain length control of the O-antigen and the enterobacterial common antigen. PMID:25307743

  17. Noninvasive Shock Wave Treatment for Capsular Contractures After Breast Augmentation: A Rabbit Study.

    PubMed

    Chen, Po Chou; Kuo, Shyh Ming; Jao, Jo Chi; Yang, Shiou Wen; Hsu, Ching Wen; Wu, Yu Chiuan

    2016-06-01

    Capsular contracture is the most common complication of breast augmentation. Although numerous procedures are intended to prevent capsular contracture, their efficacy does not satisfy surgeons or patients. In the present study, we used shock waves to develop innovative protocols to treat capsular contracture in rabbits. We used shock waves to treat capsular contracture in a rabbit model. Six clinical parameters were evaluated to determine the treatment efficacy of shock waves on the pathological histology of capsular contracture. Dual-flip-angle T1-mapping magnetic resonance imaging was used to confirm the pathological findings. Among the parameters, myxoid change, vascular proliferation, and lymphoplasma cell infiltration around the capsule increased more after treatment than they did in a control group. Capsular thickness, inner thinner collagen layer, and capsule wall collagen deposition decreased after shock wave treatment; only the inner thinner collagen layer and capsule wall collagen deposition changed significantly. The MRI findings for both scar thickness and water content were consistent with pathological biology findings. This was the first pilot study and trial to treat capsular contractures using shock waves. We found that shock waves can cause changes in the structure or the composition of capsular contracture. We conclude that the treatment could decrease water content, loosen structure, decrease collagen deposition, and might alleviate scar formation from capsular contracture. We believe that the treatment could be a viable remedy for capsular contractures. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

  18. Structural, functional and pH sensitive release characteristics of water-soluble polysaccharide from the seeds of Albizia lebbeck L.

    PubMed

    Kumar Varma, Chekuri Ashok; Jayaram Kumar, K

    2017-11-01

    Plant polysaccharides, generally regarded as safe (GRAS), are gaining importance as excipients in drug delivery. Therefore, the current paper presents the studies on structural, functional and drug release study of water soluble polysaccharide (ALPS) from seeds of Albizia lebbeck L. High swelling, water holding capacity, foam stability and lower moisture content suggests its use as additive in food preparations. The apparent molecular weight of polysaccharide was found to be 1.98×10(2)kDa. Monosaccharide composition analysis indicated that ALPS consists of mannose (4.06%), rhamnose (22.79%), glucose (38.9%), galactose (17.84%) and xylose (16.42%). Micromeritic properties revealed that the polysaccharide possess potential for pharmaceutical applications. From the surface charge analysis, ALPS was found to be non-ionic polysaccharide. Morphological study reveals the polysaccharide with irregular particle shape and rough surface. Fourier transformed infrared spectroscopy (FTIR) study confirms the carbohydrate nature of polysaccharide. From the thermogravimetric analysis (TGA) data, the second mass loss (243-340°C) attributed to polysaccharide degradation. The drug release profile reveals the use of polysaccharide for the preparation of pH sensitive pharmaceutical dosage forms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria

    PubMed Central

    Cuthbertson, Leslie; Mainprize, Iain L.; Naismith, James H.; Whitfield, Chris

    2009-01-01

    Summary: Many bacteria export extracellular polysaccharides (EPS) and capsular polysaccharides (CPS). These polymers exhibit remarkably diverse structures and play important roles in the biology of free-living, commensal, and pathogenic bacteria. EPS and CPS production represents a major challenge because these high-molecular-weight hydrophilic polymers must be assembled and exported in a process spanning the envelope, without compromising the essential barrier properties of the envelope. Emerging evidence points to the existence of molecular scaffolds that perform these critical polymer-trafficking functions. Two major pathways with different polymer biosynthesis strategies are involved in the assembly of most EPS/CPS: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways. They converge in an outer membrane export step mediated by a member of the outer membrane auxiliary (OMA) protein family. OMA proteins form outer membrane efflux channels for the polymers, and here we propose the revised name outer membrane polysaccharide export (OPX) proteins. Proteins in the polysaccharide copolymerase (PCP) family have been implicated in several aspects of polymer biogenesis, but there is unequivocal evidence for some systems that PCP and OPX proteins interact to form a trans-envelope scaffold for polymer export. Understanding of the precise functions of the OPX and PCP proteins has been advanced by recent findings from biochemistry and structural biology approaches and by parallel studies of other macromolecular trafficking events. Phylogenetic analyses reported here also contribute important new insight into the distribution, structural relationships, and function of the OPX and PCP proteins. This review is intended as an update on progress in this important area of microbial cell biology. PMID:19258536

  20. Primary Frozen Shoulder Syndrome: Arthroscopic Capsular Release

    PubMed Central

    Arce, Guillermo

    2015-01-01

    Idiopathic adhesive capsulitis, or primary frozen shoulder syndrome, is a fairly common orthopaedic problem characterized by shoulder pain and loss of motion. In most cases, conservative treatment (6-month physical therapy program and intra-articular steroid injections) improves symptoms and restores shoulder motion. In refractory cases, arthroscopic capsular release is indicated. This surgical procedure carries several advantages over other treatment modalities. First, it provides precise and controlled release of the capsule and ligaments, reducing the risk of traumatic complications observed after forceful shoulder manipulation. Second, release of the capsule and the involved structures with a radiofrequency device delays healing, which prevents adhesion formation. Third, the technique is straightforward, and an oral postoperative steroid program decreases pain and allows for a pleasant early rehabilitation program. Fourth, the procedure is performed with the patient fully awake under an interscalene block, which boosts the patient's confidence and adherence to the physical therapy protocol. In patients with refractory primary frozen shoulder syndrome, arthroscopic capsular release emerges as a suitable option that leads to a faster and long-lasting recovery. PMID:26870652

  1. Structural Characterization and Antifatigue Effect In Vivo of Maca (Lepidium meyenii Walp) Polysaccharide.

    PubMed

    Tang, Weimin; Jin, Lu; Xie, Lianghua; Huang, Juqing; Wang, Nan; Chu, Bingquan; Dai, Xulin; Liu, Yu; Wang, Rui; Zhang, Ying

    2017-03-01

    Maca (Lepidium meyenii Walp) polysaccharides (MP) with purity of 99.2% were obtained to investigate their structural characteristics and antifatigue effect in vivo. The physicochemical properties of MP were analyzed through high-performance gel filtration chromatography, IR, monosaccharide composition, methylation, GC-MS, and NMR analyses. The antifatigue effect of MP was evaluated by using a mouse weight-loaded swimming model. MP is an acidic heteropolysaccharide with an average molecular weight (Mw ) of 793.5 kDa. It is composed of D-GalA: D-Glc: L-Ara: D-Man: D-Gal: L-Rha = 35.07:29.98:16.98:13.01:4.21:0.75 (mol, %). The findings revealed that MP contained β-1,3-Galp(A), β-1,3-Glcp, and α-1, 3-Manp linked alternatingly to form a backbone (5:4:1). MP (above mid-dosage 50 mg/kg bw/d) could effectively elongate swimming durations and accelerate average swimming speeds (within the 1st 5 min) of mice (P < 0.05) and improve the serous biochemical parameters of mice. Compared with the control model, high-dosage (100 mg/kg bw/d) MP treatment could significantly enhance glutathione peroxidase and creatine kinase activities (P < 0.05) and decreased lactate dehydrogenase activity (P < 0.01). High-dosage MP could significantly reduce the levels of blood urea nitrogen, lactic acid, and malondialdehyde (P < 0.05). MP is an acidic polysaccharide with a high D-GalA content, which could be responsible for the antifatigue effect of maca.

  2. Stability and structure of protein-polysaccharide coacervates in the presence of protein aggregates.

    PubMed

    Sanchez, Christian; Renard, Denis

    2002-08-21

    We have studied at pH 4.2 and three protein (Pr):polysaccharide (Pol) weight ratios (8:1, 2:1 and 1:1) the structure and stability of beta-lactoglobulin/acacia gum/water dispersions containing protein aggregates (BLG/AG/W) or free from aggregates (AF-BLG/AG/W). Phase diagrams were characteristic of complex coacervation. BLG/AG/W dispersions displayed a larger biphasic area than AF-BLG/AG/W dispersions, that moved towards the protein axis. It was concluded that protein aggregates affected complex coacervation both by entropic (size and molecular masses of aggregates) and enthalpic (surface properties of aggregates) effects. Laser light scattering measurements revealed that the particles diameter (d(43)) induced by demixing was controlled by protein aggregates in AF-BLG/AG/W dispersions. At 1 wt.% biopolymer concentration, particles were 15-20 times larger in AF-BLG/AG/W dispersions than in BLG/AG/W dispersions at (Pr:Pol) ratios of 2:1 or 1:1. Confocal scanning laser microscopy showed that AF-BLG/AG/W dispersions only contained spherical coacervates. BLG/AG/W dispersions contained both coacervates and aggregates coated with AG or/and BLG/AG coacervates. At a (Pr:Pol) ratio of 2:1 and 1:1, coacervates were vesicular or multivesicular. Coacervates were smaller in BLG/AG/W dispersions than in AF-BLG/AG/W dispersions. It was concluded that protein aggregates have the intrinsic ability to stabilize complex coacervates and could be used to design multifunctional delivery systems. This study showed that composite dispersions containing both protein aggregates embedded in protein-polysaccharide coacervates and free coacervates may be performed. In this respect, the design of protein aggregates with controlled size distribution and surface properties could open new possibilities both in the non-chemical control of coacervates stability and in the development of multifunctional delivery systems.

  3. Structure of a Bacterial ABC Transporter Involved in the Import of an Acidic Polysaccharide Alginate.

    PubMed

    Maruyama, Yukie; Itoh, Takafumi; Kaneko, Ai; Nishitani, Yu; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2015-09-01

    The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides.

  4. Analysis of E. coli K5 capsular polysaccharide heparosan

    PubMed Central

    Ly, Mellisa; Wang, Zhenyu; Laremore, Tatiana N.; Zhang, Fuming; Zhong, Weihong; Pu, Dennis; Zagorevski, Dmitri V.; Dordick, Jonathan S.; Linhardt, Robert J.

    2012-01-01

    Heparosan is the key precursor for the preparation of bioengineered heparin, a potential replacement for porcine intestinal heparin, an important anticoagulant drug. The molecular weight (MW) distribution of heparosan produced by the fermentation of E. coli K5 was investigated. Large-slab isocratic and mini-slab gradient polyacrylamide gel electrophoresis (PAGE) were used to analyze the MW and polydispersity of heparosan. A preparative method that allowed fractionation by continuous-elution PAGE was used to obtain heparosan MW standards. The MWs of the heparosan standards were determined by electrospray ionization Fourier-transform mass spectrometry (ESI-FT-MS). A ladder of the standards was then used to determine the MW properties of polydisperse heparosan samples. Unbleached and bleached heparosan produced by fermentation of E. coli K5 had similar number-averaged MWs (MN), weight-averaged MWs (MW), and MW ranges of 3,000 to 150,000 Da. PMID:20407891

  5. POLYS 2.0: An open source software package for building three-dimensional structures of polysaccharides.

    PubMed

    Engelsen, Søren B; Hansen, Peter I; Pérez, Serge

    2014-07-01

    This article describes an update of POLYS, the POLYSaccharide builder, for generating three-dimensional structures of polysaccharides and complex carbohydrates (Engelsen et al., Biopolymers 1996, 39, 417-433). POLYS is written in portable ANSI C and is now released under an open source license. Using this software, complex branched carbohydrate structures and polysaccharides can be constructed from their primary structure and the relevant monosaccharides stored in database containing information on optimized glycosidic linkage geometries. The constructed three-dimensional structures are described as Cartesian coordinate files which can be used as input to other molecular modeling software. The new version of POLYS includes a large database of monosaccharides and a helical generator to build and optimize regular single helix or double helix structures. To demonstrate the efficiency of POLYS to build carbohydrate structures, four examples of increasing complexity are presented in the manuscript, from simple alpha glucans over complex starch fragments and the double helical structure of amylopectin to the mega-oligosaccharide RhamnoGalacturonan II.

  6. Capsular Suspension Technique for Hip Arthroscopy

    PubMed Central

    Federer, Andrew E.; Karas, Vasili; Nho, Shane; Coleman, Struan H.; Mather, Richard C.

    2015-01-01

    Hip arthroscopy has recently become a common procedure to treat central and peripheral hip pathology. Capsulotomies are necessary in these procedures, and negotiating adequate visualization, as well as capsular preservation, is a challenge. We describe a capsular suspension technique that allows for adequate visualization of the central and peripheral compartments while facilitating preservation of the native hip capsule. This technique eliminates the need for additional personnel for retraction, potentially decreases iatrogenic hip injury, eliminates the need for excessive capsular debridement, and allows for capsular closure under minimal tension. PMID:26759769

  7. Agrobacterium rubi(T) DSM 6772 produces a lipophilic polysaccharide capsule whose degree of acetylation is growth modulated.

    PubMed

    De Castro, Cristina; Gargiulo, Valentina; Lanzetta, Rosa; Parrilli, Michelangelo

    2007-03-01

    The structure of the capsular polysaccharide produced from the type strain of Agrobacterium rubi DSM 6772 is demonstrated by means of chemical and spectroscopical methodologies. It is constituted from the quite rare monosaccharide 6-deoxy-L-talose, involved in alternating alpha-(1 --> 2) and alpha-(1 --> 3) linkages. This simple backbone is further complicated from the occurrence of O-acetyl substituents located always at O-2 of the O-3 substituted 6-deoxy-talose. This decoration is not stoichiometric and it depends on the growth stadium of the bacterium, leading to an almost regular acetylation pattern only at the stationary phase, where all the potential positions are substituted.

  8. Effects of extraction methods on the yield, chemical structure and anti-tumor activity of polysaccharides from Cordyceps gunnii mycelia.

    PubMed

    Zhu, Zhen-Yuan; Dong, Fengying; Liu, Xiaocui; Lv, Qian; YingYang; Liu, Fei; Chen, Ling; Wang, Tiantian; Wang, Zheng; Zhang, Yongmin

    2016-04-20

    This study was to investigate the effects of different extraction methods on the yield, chemical structure and antitumor activity of polysaccharides from Cordyceps gunnii (C. gunnii) mycelia. Five extraction methods were used to extract crude polysaccharides (CPS), which include room-temperature water extraction (RWE), hot-water extraction (HWE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE) and cellulase-assisted extraction (CAE). Then Sephadex G-100 was used for purification of CPS. As a result, the antitumor activities of CPS and PPS on S180 cells were evaluated. Five CPS and purified polysaccharides (PPS) were obtained. The yield of CPS by microwave-assisted extraction (CPSMAE) was the highest and its anti-tumor activity was the best and its macromolecular polysaccharide (3000-1000kDa) ratio was the largest. The PPS had the same monosaccharide composition, but their obvious difference was in the antitumor activity and the physicochemical characteristics, such as intrinsic viscosity, specific rotation, scanning electron microscopy and circular dichroism spectra.

  9. Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: A 600-MHz NMR study

    SciTech Connect

    Abeygunawardana, C.; Bush, C.A. ); Cisar, J.O. )

    1991-09-03

    Specific lectin-carbohydrate interactions between certain oral streptococci and actinomyces contribute to the microbial colonization of teeth. The receptor molecules of Streptococcus oralis, 34, ATCC 10557, and Streptococcus mitis J22 for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii are antigenically distinct polysaccharides, each formed by a different phosphodiester-linked oligosaccharide repeating unit. Receptor polysaccharide was isolated form S. oralis C104 cells and was shown to contain galactose, N-acetylgalactosamine, ribitol, and phosphate with molar ratios of 4:1:1:1. The {sup 1}H NMR spectrum of the polysaccharide shows that it contains a repeating structure. The individual sugars in the repeating unit were identified by {sup 1}H coupling constants observed in E-COSY and DQF-COSY spectra. NMR methods included complete resonance assignments ({sup 1}H and {sup 13}C) by various homonuclear and heteronuclear correlation experiments that utilize scalar couplings. Sequence and linkage assignments were obtained from the heteronuclear multiple-bond correlation (HMBC) spectrum. This analysis shows that the receptor polysaccharide of S. oralis C104 is a ribitol teichoic acid polymer composed of a linear hexasaccharide repeating unit containing two residues each of galactopyranose and galactofuranose and a residue each of GalNAc and ribitol joined end to end by phosphodiester linkages.

  10. An Interesting Class of Porous Polymer--Revisiting the Structure of Mesoporous α-D-Polysaccharide Gels.

    PubMed

    White, Robin J; Shuttleworth, Peter S; Budarin, Vitaliy L; De Bruyn, Mario; Fischer, Anna; Clark, James H

    2016-02-08

    The processes involved in the transformation of non-porous, native polysaccharides to their highly porous equivalents introduce significant molecular complexity and are not yet fully understood. In this paper, we propose that distinct changes in polysaccharide local short-range ordering promotes and directs the formation of meso- and micro-pores, which are investigated here using N2 sorption, FTIR, and solid-state (13)C NMR. It is found that an increase in the overall double helical amylose content, and their local association structures, are responsible for formation of the porous polysaccharide gel phase. An exciting consequence of this local ordering change is elegantly revealed using a (19)F NMR experiment, which identifies the stereochemistry-dependent diffusion of a fluorinated chiral probe molecule (1-phenyl-2,2,2-trifluoroethanol) from the meso- to the micro-pore region. This finding opens opportunities in the area of polysaccharide-based chiral stationary phases and asymmetric catalyst preparation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Molecular dimensions and structural features of neutral polysaccharides from the seed mucilage of Hyptis suaveolens L.

    PubMed

    Praznik, Werner; Čavarkapa, Andrea; Unger, Frank M; Loeppert, Renate; Holzer, Wolfgang; Viernstein, Helmut; Mueller, Monika

    2017-04-15

    The seed mucilage of Hyptis suaveolens L. includes acid - and neutral heteropolysaccharides in a ratio of about 1:1. The anionic charged fraction responsible for swelling and viscous behaviour possesses an average molar mass of Mw=350kg/mol, Mn=255kg/mol. The neutral polysaccharide fraction shows an average molar mass of Mw=47kg/mol and Mn=28kg/mol and is composed of d-Galp-, d-Glcp- and d-Manp residues in a molar ratio of about 3:2:1. The structural features present galactoglucan (30%) and galactoglucomannan (70%) with a high level of terminal β-linked d-Galp residues (18%). Structural details of galactoglucomannan are derived by combined enzymatic and chemical methods as well as NMR spectroscopy. Sequences of octa/nonasaccharide β-d-Glcp-(1→4)[β-d-Galp-(1→2)-α-d-Galp-(1→6)]-β-d-Manp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→4)[β-d-Galp-(1→2)-α-d-Galp-(1→6)]-β-d-Manp and lower mass tetrasaccharide repeating units β-d-Glcp-(1→4)[β-d-Galp-(1→2)-α-d-Galp-(1→6)]-β-d-Manp were found. The level of the prebiotic activity is related to the availability of β-linked d-Galp residues in the side chains of the molecules.

  12. Structural Characterization of a Water-Soluble Polysaccharide from the Fruiting Bodies of Agaricus bisporus

    PubMed Central

    He, Jinzhe; Zhang, Anqiang; Ru, Qiaomei; Dong, Dandan; Sun, Peilong

    2014-01-01

    An edible fungal polysaccharide termed as ABP was obtained by extraction with hot water, and followed successive chromatographic purification using DEAE-Sepharose Fast Flow column and Sephacryl S-300 High-Resolution column. A symmetrical peak was obtained on high-performance size-exclusion chromatography with an average molecular weight of 5.17 × 104 Da, which was named ABP, and its main components were d-glucose and d-mannose. Based on the study of methylation analysis, along with FT-IR, GC, GC-MS, 1D 1H and 13C NMR and 2D NMR (H-HCOSY, TOCSY, HMQC, and NOESY), its chemical structure was featured with a repeating unit (1→6) linking β-d-Glcp as the main backbone with (1→4)-linked α-d-Manp units. The structure of the mainly repeating units of ABP was established as: →6)-β-D-Glucp-(1→4)-α-D-Manp(1→6)-β-D-Glucp-(1→6)-β-D-Glucp-(1→ PMID:24406732

  13. Characterization of a bioactive polysaccharide from Ganoderma atrum: Re-elucidation of the fine structure.

    PubMed

    Zhang, Hui; Nie, Shaoping; Cui, Steve W; Xu, Ming; Ding, Huihuang; Xie, Mingyong

    2017-02-20

    The fine structure in terms of backbone and branch chain features of a bioactive polysaccharide from Ganoderma atrum (PSG-1) was re-elucidated systematically using high performance anion-exchange chromatography (HPAEC), methylation and GLC-MS analysis, and 1D & 2D NMR spectroscopy. Monosaccharide composition analysis revealed that PSG-1-F0.2 fraction mainly consisted of glucose (73.8%) and glucuronic acid (15.3%), with small amount of mannose (5.7%) and galactose (5.2%). Based on methylation, multistep partial acid hydrolysis and NMR study, were proposed to substitute at the O-6 position of β-(1→3)-glucan. The small amount of mannose and galactose residues were considered to be from the other fraction in PSG which was very difficult to be separated from PSG-1-F0.2. This revised structure as an acidic β-(1→3, 1→6)-glucan is considered to be more accurate than the previous proposal of PSG-1.

  14. Structural characterization and antioxidant activities of 2 water-soluble polysaccharide fractions purified from tea (Camellia sinensis) flower.

    PubMed

    Quan, Han; Qiong-Yao, Yu; Jiang, Shi; Chang-Yun, Xiong; Ze-Jie, Ling; Pu-Ming, He

    2011-04-01

    The water-soluble crude polysaccharide tea flower polysaccharide (TFP), obtained from tea (Camellia sinensis) flower by boiling-water extraction and ethanol precipitation, was fractionated by Sephadex G-100 column chromatography, giving 2 polysaccharide fractions termed TFP-1 and TFP-2. The structural features of TFP-1 and TFP-2 were investigated by high-performance liquid chromatography (HPLC), gel-permeation chromatography (GPC), rheometer, infrared (IR) spectra, nuclear magnetic resonance (NMR) spectroscopy, atomic force microscope (AFM), and scanning electron microscope (SEM). Results indicated that TFP-1 was composed of glucose: xylose: rhamnose: galactose=1.0:1.2:0.81:0.98 with a molecular weight of 167.5 KDa, while TFP-2 comprised glucose: xylose: rhamnose: arabinose=1.0:0.76:2.3:2.3 with a molecular weight of 10.1 KDa. The 1H NMR revealed that TFP-1 contained α-L-Rhap, α-D-Galp, α-D-GalpNAc, α-D-Xylp, α-D-Glcp, and β-D-Glcp residues, while TFP-2 was illustrated to have α-L-Rhap, α-L-Arap, α-D-Xylp, α-D-Glcp, and α-D-GlcpNAc residues. Antioxidant activities of these fractions were investigated using various in vitro assay systems compared with ascorbic acid. In conclusion, TFP-2 exhibited the higher antioxidant activities and could be explored as a novel potential antioxidant.   At present, commonly low-grade tea is preferred to extract the tea polysaccharide, to take full advantage of tea flower resource to extract polysaccharides can greatly reduce the cost of tea products. Thus, the search for plant-derived biomaterials from this study could generate natural value-added products from underutilized tea plant waste and used as a medicinal agent against chronic health problems, such as cancers, aging, and atherosclerosis caused by reactive free radicals that produced from oxidation.

  15. Structural features of a water soluble gum polysaccharide from Murraya paniculata fruits.

    PubMed

    Mondal, S K; Ray, B; Ghosal, P K; Teleman, A; Vuorinen, T

    2001-10-22

    A water soluble gum polysaccharide was isolated from Murraya paniculata fruits. Hydrolytic experiments, methylation analysis, periodate oxidation studies and NMR data revealed that the polysaccharide was extensively branched and it consisted of 1,3-, and 1,3,6-linked beta-D-galactopyranosyl units, terminal beta-D-galactopyranosyl units and terminal alpha-D-glucopyranosyl 1,4-beta-D-galactopyranosyl units. Small amounts of 4-O-methylglucuronic acid residues were also present.

  16. Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl

    PubMed Central

    Franklin, Michael J.; Nivens, David E.; Weadge, Joel T.; Howell, P. Lynne

    2011-01-01

    Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the genetic capacity to produce at least three secreted polysaccharides; alginate, Psl, and Pel. These polysaccharides differ in chemical structure and in their biosynthetic mechanisms. Since alginate is often associated with chronic pulmonary infections, its biosynthetic pathway is the best characterized. However, alginate is only produced by a subset of P. aeruginosa strains. Most environmental and other clinical isolates secrete either Pel or Psl. Little information is available on the biosynthesis of these polysaccharides. Here, we review the literature on the alginate biosynthetic pathway, with emphasis on recent findings describing the structure of alginate biosynthetic proteins. This information combined with the characterization of the domain architecture of proteins encoded on the Psl and Pel operons allowed us to make predictive models for the biosynthesis of these two polysaccharides. The results indicate that alginate and Pel share certain features, including some biosynthetic proteins with structurally or functionally similar properties. In contrast, Psl biosynthesis resembles the EPS/CPS capsular biosynthesis pathway of Escherichia coli, where the Psl pentameric subunits are assembled in association with an isoprenoid lipid carrier. These models and the environmental cues that cause the cells to produce predominantly one polysaccharide over the others are subjects of current investigation. PMID:21991261

  17. Structure of the O-antigen of Vibrio cholerae O155 that shares a putative D-galactose 4,6-cyclophosphate-associated epitope with V. cholerae O139 Bengal.

    PubMed

    Senchenkova, S N; Zatonsky, G V; Shashkov, A S; Knirel, Y A; Jansson, P E; Weintraub, A; Albert, M J

    1998-05-15

    The O-specific polysaccharide of Vibrio cholerae 0155 was studied by sugar and methylation analyses, dephosphorylation with 48% hydrofluoric acid, 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and heteronuclear single-quantum coherence (HSQC) experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established: carbohydrate sequence [see text]. An unusual component, D-galactose 4,6-cyclophosphate, has been reported previously as a component of the capsular polysaccharide and O-antigen of V. cholerae O139 Bengal and appears to be responsible for the known serological cross-reactivity between V. cholerae O139 and O155.

  18. Structural characteristics of water-soluble polysaccharides from Norway spruce (Picea abies).

    PubMed

    Shakhmatov, Evgeny G; Belyy, Vladimir A; Makarova, Elena N

    2017-11-01

    Arabinogalactan proteins and pectic polysaccharides were isolated from greenery of Picea abies by water extraction. Main elements of their structure were determined by ion-exchange chromatography, partial acid and enzymatic hydrolysis, and NMR spectroscopy. It was established that the backbone of pectin macromolecules of greenery of P. abies is represented by segments of partially methyl-esterified and acetylated 1,4-α-d-galactopyranosyluronan, and partially 2-O- and/or 3-O-acetylated RG-I with side chains consisting of highly-branched 1,5-α-l-arabinan segments. The carbohydrate part of AGP of greenery of P. abies consists of AG-II, the main chain of which is represented by 1,3-β-d-Galp and 1,3,6-β-d-Galp residues. The side chains of AG-II are formed of 1,6- and 1,3,6-β-d-Galp, 1,3- and 1,5-α-l-Araf, β-d-GlcpA and 1,4-β-d-GlcpA, T-α-l-Araf, T-α-l-Rhap and T-α-l-Fucp residues. The AGPs of P. abies are also characterized by the presence of an unusual 4-O-Me-α-l-Fucp monosaccharide, which, as far as we know, was not found in pectins or AGP earlier. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Structural characterisation and rheological properties of a polysaccharide from sesame leaves (Sesamum radiatum Schumach. & Thonn.).

    PubMed

    Nep, E I; Carnachan, S M; Ngwuluka, N C; Kontogiorgos, V; Morris, G A; Sims, I M; Smith, A M

    2016-11-05

    A polysaccharide from the leaves of Sesamum radiatum was extracted by maceration in deionized water followed by ethanol precipitation then chemically and physically characterised. Monosaccharide composition and linkages were determined by high performance anion exchange chromatography (HPAEC), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy respectively. Sesamum gum was composed of glucuronic acid, mannose, galactose, and xylose with trace quantities of glucose, rhamnose and arabinose. Proton and (13)C NMR spectroscopy, and linkage analysis revealed a glucuronomannan based structure comprising a backbone of →4)-β-d-GlcpA-(1→2)-α-d-Manp-(1→ with side-chains of galactose and xylose. Hydrated sesamum gum displayed temperature independent viscoelastic properties with no thermal hysteresis. Intrinsic viscosity was determined to be 3.31 and 4.40dLg(-1) in 0.1M NaCl and deionised water respectively, while the critical concentration was determined to be 0.1% w/v. The characterisation performed in this study will help direct potential applications of this material in foods and pharmaceuticals.

  20. Structural characterization and immunomodulatory activity of a water soluble polysaccharide isolated from Botrychium ternatum.

    PubMed

    Zhao, Xiaoyong; Li, Jinying; Liu, Yaqin; Wu, Datong; Cai, Pengfei; Pan, Yuanjiang

    2017-09-01

    As a folk medicine, Botrychium ternatum has been used for thousands of years in China. In the present work, a water soluble polysaccharide BTp1 was extracted and purified from B. ternatum. Based on the MALDI matrix 3-aminoquinoline-α-cyano-4-hydroxycinnamic acid, the molecular weight of BTp1 was determined to be 11638Da directly. Monosaccharide analysis showed that BTp1 was composed of arabinose (Ara). Combining enzymatic hydrolysis and subsequent MALDI-TOF analysis, a linear backbone of BTp1, consisted of (1→5)-linked α-l-Araf, was inferred quickly. Then according to NMR experiments, the whole structure of BTp1 was established. The repeating unit of BTp1 was deduced as a linear backbone with branches at O-2, O-3 and its neighboring O-2 positions terminated with (1→)-linked α-l-Araf, respectively. The immunomodulatory assay exhibited that BTp1 could significantly enhance the viability and promote the release of NO in RAW 264.7 cells, suggesting that BTp1 could be a potential immunomodulatory agent in pharmacological fields. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Unusual structures in the polysaccharides from the red seaweed Pterocladiella capillacea (Gelidiaceae, Gelidiales).

    PubMed

    Errea, María I; Matulewicz, María C

    2003-04-22

    Sequential extraction of tetrasporic Pterocladiella capillacea with water at room temperature and then at 50 degrees C led to the isolation of two products that were each fractionated with cetrimide to give a soluble fraction and a precipitate. The precipitates were then subjected to fractional solubilization in solutions of increasing sodium chloride concentration. The whole treatment yielded two major fractions in each case, one soluble in the cetrimide medium and the other soluble in 0.5 M NaCl, which were further fractionated by anion-exchange chromatography. Structural analysis, carried out by methylation, desulfation-methylation, 13C NMR spectroscopy and determination of the absolute configuration of the 2,6-di-O-methylgalactose units in the permethylated products, indicated the presence of xylogalactans, with low content of 3,6-anhydrogalactose and low molecular weight. These polysaccharides varied in the level of xylopyranosyl and sulfate substitution, primarily on the 6-position of the 3-linked beta-D-galactopyranosyl and on the 3-position of the 4-linked alpha-galactopyranosyl units. Moreover, herein we report, for the first time, the presence of 3-substituted, 4-linked D-galactopyranosyl residues in an alga belonging to the Gelidiales.

  2. Structure characterization and antitumor activity of a polysaccharide from the alkaline extract of king oyster mushroom.

    PubMed

    Liu, Xinkui; Wang, Lin; Zhang, Chunmei; Wang, Hongmin; Zhang, Xiaohong; Li, Yuexia

    2015-03-15

    A water-soluble polysaccharide, designated as KOMAP, was isolated and purified from the alkaline extract of king oyster mushroom, which was composed of glucose (Glc), mannose (Man) and arabinose (Ara) in a molar ratio of 6.2:2.1:2.0. It had an average molecular weight of 2.1×10(4)Da. GC-MS analysis revealed that KOMAP was a linear structure of the polymer with a backbone composed of β-1,4-linked glucopyranosyl and β-1,3,6-linked mannopyranosyl units, which was terminated with α-1-linked arabinofuranosyl unit at C-6 position of β-1,3,6-linked mannopyranosyl residue along the main chain in the ratio of 3.1:1. The results in the animal experiment showed that 50, 100 and 200mg/mL of KOMAP not only inhibited the tumor growth, but also increased relative thymus and spleen indices, LPS- or ConA-induced lymphocytes proliferation, and serum cytokine IL-2, TNF-α, and IFN-γ levels, as well as the activities of NK cells and CTLs in spleen of Renca tumor-bearing mice. In summary, our data indicate that the KOMAP exerts effective immunoregulatory and anti-tumor activities in vivo. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Computer-assisted analysis of the structure of regular branched polysaccharides containing 2,3-disubstituted rhamnopyranose and mannopyranose residues on the basis of 13C NMR data.

    PubMed

    Lipkind, G M; Shashkov, A S; Nifant'ev, N E; Kochetkov, N K

    1992-12-31

    A computer-assisted approach to the analysis of the structure of branched polysaccharides that contain 2,3-di-O-glycosylated alpha-rhamnopyranose and alpha-mannopyranose residues is based on evaluation of the 13C NMR spectra, using glycosylation effects and their deviations from additivity (delta delta values) at the branch points. This approach, in combination with monosaccharide and methylation analysis data, has been verified on a series of bacterial polysaccharides of known structure.

  4. Serogroup quantitation of multivalent polysaccharide and polysaccharide-conjugate meningococcal vaccines from China.

    PubMed

    Cook, Matthew C; Gibeault, Sabrina; Filippenko, Vasilisa; Ye, Qiang; Wang, Junzhi; Kunkel, Jeremy P

    2013-07-01

    The active components of most meningococcal vaccines are four antigenic serogroup capsular polysaccharides (A, C, Y, W135). The vaccines, monovalent or multivalent mixtures of either free polysaccharides or polysaccharides conjugated to antigenic carrier proteins, may be in liquid or lyophilised formulations, with or without excipients. Acid hydrolysis and chromatographic methods for serogroup quantitation, which were previously optimised and qualified using polysaccharide-based standards and a narrow range of real vaccines, are here challenged with multiple lots of a broad assortment of additional multivalent polysaccharide-based meningococcal vaccine products. Centrifugal filtration successfully removed all interfering lactose excipient without loss of polysaccharides to allow for the determination of Y and W135 serogroups. Replicate operations by three different analysts indicated high method reproducibility. Results indicated some lot-to-lot and product-to-product variations. However, all vaccines were within general specifications for each serogroup polysaccharide, with the exception of all lots of one polysaccharide vaccine - which by these methods were found to be deficient in the serogroup A component only. These robust techniques are very useful for the evaluation of antigen content and consistency of manufacture. The deformulation, hydrolysis and chromatographic methods may be adaptable for the evaluation of other types of polysaccharide-based vaccines.

  5. Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases

    PubMed Central

    Forsberg, Zarah; Mackenzie, Alasdair K.; Sørlie, Morten; Røhr, Åsmund K.; Helland, Ronny; Arvai, Andrew S.; Vaaje-Kolstad, Gustav; Eijsink, Vincent G. H.

    2014-01-01

    For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled cellulose-active LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu2+ (12–31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms. PMID:24912171

  6. Antibiotic modulation of capsular exopolysaccharide and virulence in Acinetobacter baumannii.

    PubMed

    Geisinger, Edward; Isberg, Ralph R

    2015-02-01

    Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low

  7. Antibiotic Modulation of Capsular Exopolysaccharide and Virulence in Acinetobacter baumannii

    PubMed Central

    Geisinger, Edward; Isberg, Ralph R.

    2015-01-01

    Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low

  8. Structure of the N-acetyl-L-rhamnosamine-containing O-polysaccharide of Proteus vulgaris TG 155 from a new Proteus serogroup, O55.

    PubMed

    Kondakova, Anna N; Kolodziejska, Katarzyna; Zych, Krystyna; Senchenkova, Sof'ya N; Shashkov, Alexander S; Knirel, Yuriy A; Sidorczyk, Zygmunt

    2003-09-10

    The O-polysaccharide of the lipopolysaccharide (LPS) of Proteus vulgaris TG 155 was found to contain 2-acetamido-2,6-dideoxy-L-mannose (N-acetyl-L-rhamnosamine, L-RhaNAc), a monosaccharide that occurs rarely in Nature. The following structure of the O-polysaccharide was established by NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H,13C HSQC experiments, along with chemical methods: [carbohydrate structure in text] Rabbit polyclonal O-antiserum against P. vulgaris TG 155 reacted with both core and O-polysaccharide moieties of the homologous LPS but showed no cross-reactivity with other LPS from the complete set of serologically different Proteus strains. Based on the unique O-polysaccharide structure and the serological data, we propose classifying P. vulgaris TG 155 into a new, separate Proteus O-serogroup, O55.

  9. The Role of Bacterial Protein Tyrosine Phosphatases in the Regulation of the Biosynthesis of Secreted Polysaccharides

    PubMed Central

    Morona, Renato

    2014-01-01

    Abstract Significance: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria. Recent Advances: Deletion and overexpression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases have suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. Critical Issues: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. Future Directions: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel antimicrobials. This is particularly the case for the polymerase and histidinol phosphatase family, which is predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism, and critical importance of these phosphatases in a wide range of bacteria. Antioxid. Redox Signal. 20, 2274–2289. PMID:24295407

  10. A novel polysaccharide isolated from mulberry fruits (Murus alba L.) and its selenide derivative: structural characterization and biological activities.

    PubMed

    Chen, Chun; Zhang, Bin; Fu, Xiong; Liu, Rui Hai

    2016-06-15

    A novel polysaccharide (MFP3P) was isolated from Murus alba L. through the hot water extraction method followed by chromatographic purification. The chemical structure of MFP3P was elucidated by acid hydrolysis, Smith degradation and methylation analysis, along with FT-IR, GC-MS, (1)H and (13)C NMR spectroscopy. Its morphological properties were further characterized by SEM and AFM. The selenide of the polysaccharide (MFP3P-Se) was obtained by the Na2SeO3/BaCl2 method. The antioxidant properties showed that MFP3P-Se exhibited higher peroxy radical-scavenging capacity than MFP3P in vitro. Moreover, MFP3P-Se had more significant hypoglycemic effects than MFP3P through promoting pancreatic cell proliferation and increasing glucose metabolism and insulin secretion.

  11. Structure of a glycerol teichoic acid-like O-specific polysaccharide of Proteus vulgaris O12.

    PubMed

    Perepelov, A V; Torzewska, A; Shashkov, A S; Senchenkova, S N; Rozalski, A; Knirel, Y A

    2000-02-01

    A phosphorylated O-specific polysaccharide (O-antigen) was obtained by mild acid degradation of Proteus vulgaris O12 lipopolysaccharide and studied by sugar and methylation analyses, 1H-, 13C- and 31P-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H, 13C and 1H, 31P heteronuclear multiple-quantum coherence experiments. It was found that the polysaccharide consists of pentasaccharide repeating units connected via a glycerol phosphate group, and has the following structure: where FucNAc is 2-acetamido-2,6-dideoxygalactose and the degree of O-acetylation at position 4 of GalNAc is approximately 25%. Immunochemical studies with P. vulgaris O12 O-antiserum suggested that the lipopolysaccharide studied shares common epitopes with the lipopolysaccharide core of P. vulgaris O8 and with the O-antigens of P. penneri strains 8 and 63.

  12. Structures, biological activities, and industrial applications of the polysaccharides from Hericium erinaceus (Lion's Mane) mushroom: A review.

    PubMed

    He, Xirui; Wang, Xiaoxiao; Fang, Jiacheng; Chang, Yu; Ning, Ning; Guo, Hao; Huang, Linhong; Huang, Xiaoqiang; Zhao, Zefeng

    2017-04-01

    Hericium erinaceus (Bull.) Pers., also known as Yamabushitake, Houtou and Lion's Mane, is capable of fortifying the spleen and nourishing the stomach, tranquilizing the mind, and fighting cancer. Over the past decade, it has been demonstrated that H. erinaceus polysaccharides possess various promising bioactivities, including antitumor and immunomodulation, anti-gastric ulcer, neuroprotection and neuroregeneration, anti-oxidation and hepatoprotection, anti-hyperlipidemia, anti-hyperglycemia, anti-fatigue and anti-aging. The purpose of the present review is to provide systematically reorganized information on extraction and purification, structure characteristics, biological activities, and industrial applications of H. erinaceus polysaccharides to support their therapeutic potentials and sanitarian functions. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Chemical structure and immunoinhibitory activity of a pectic polysaccharide containing glucuronic acid from the leaves of Diospyros kaki.

    PubMed

    Duan, Jinyou; Chen, Vincent L; Dong, Qun; Ding, Kan; Fang, Jinian

    2010-06-01

    Pectin is a family of complex polysaccharides characterized by the presence of 1,4-linked alpha-galacturonic acid residues in the backbone and is more recognized for its potential bioactive properties. Herein we reported the structural and biological features of a pectic fraction DL-4OAC-1 from the leaves of Diospyros kaki. Chemical and spectral analysis revealed this polysaccharide possessed a backbone composed of a repeating disaccharide [-->4)-alpha-GalAp-(1-->2)-alpha-Rhap-(1-->], substituted at some O-4 of Rhap residues and O-3 of GalAp residues by the side chains consisting of Araf, Galp, Xylp, and in particular terminal GlcAp residues. The immunological assay in vitro showed that this distinct pectic fraction could inhibit the LPS-induced B lymphocyte proliferation and had no effect on the ConA-induced T lymphocyte proliferation. Copyright 2010 Elsevier B.V. All rights reserved.

  14. Isolation, structure, and surfactant properties of polysaccharides from Ulva lactuca L. from South China Sea.

    PubMed

    Tian, Hua; Yin, Xueqiong; Zeng, Qinghuan; Zhu, Li; Chen, Junhua

    2015-08-01

    Two polysaccharides (ULP1 and ULP2) were isolated through ultrasonic-assisted extraction from green seaweed Ulva lactuca L. which was collected from the South China Sea. The highest yield of 17.57% was obtained under the conditions of 2% NaOH, 90 °C, material/water mass ratio 1:80, liquid extraction 5h and subsequent ultrasound-assisted extraction 1h. The structure of ULPs were characterized with periodate oxidation followed by Smith degradation, (1)H NMR, (13)C NMR spectroscopy, FTIR, and GPC. The molecular weights of ULP1 and ULP2 were 189 kDa and 230 kDa, respectively. The structural characteristics of ULP1 and ULP2 were quite similar. They were composed of rhamnose, xylose, glucose, and glucuronic acid. The content of rhamnose, xylose, glucose, glucuronic acid, sulfate was 51.2%, 12.3%, 20.1%, 16.4%, 12.0% for ULP1, respectively, and 60.8%, 14.2%, 8.2%, 16.8%, 26.8%, respectively, for ULP2. Both ULP1 and ULP2 showed good surface activity. 5 mg/mL ULP1 (2.62×10(-2) mmol/L) decreased the water surface tension to 51.63 mN/m. The critical micellar concentration of ULP1 and ULP2 was 1.01 mg/mL (5.3×10(-3) mmol/L) and 1.14 mg/mL (5.0×10(-3) mmol/L), respectively.

  15. Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides.

    PubMed

    Benincasa, Monica; Lagatolla, Cristina; Dolzani, Lucilla; Milan, Annalisa; Pacor, Sabrina; Liut, Gianfranco; Tossi, Alessandro; Cescutti, Paola; Rizzo, Roberto

    2016-08-10

    Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1-35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

  16. Polysaccharides from the green seaweed Codium decorticatum. Structure and cell wall distribution.

    PubMed

    Fernández, Paula Virginia; Raffo, María Paula; Alberghina, Josefina; Ciancia, Marina

    2015-03-06

    The cell wall polysaccharides from Codium decorticatum and their assembly were studied and these results were compared with those obtained previously for this genus. The water soluble polysaccharides are: (i) Pyruvylated and sulfated 3- and 6-linked β-D-galactans with sulfate mainly on C-4 and also on C-6. Pyruvate ketals are linked to O-3 and O-4 of terminal β-D-galactose or O-4 and O-6 of 3-linked β-D-galactose. (ii) Sulfated 3-linked β-L-arabinans substituted on C-2 or C-2 and C-4 predominantly with sulfate, but also with single stubs of arabinose, and (iii) 4-linked β-D-mannans with a low degree of sulfation on C-2. The whole polysaccharide system comprises 6.9% of sulfated polysaccharides and 32.9% of fibrillar polysaccharides, mostly insoluble mannans. By in situ localization it was possible to detect two similar fibrillar layers separated by a zone rich in charged polymers. Besides, arabinogalactan proteins co-localized with the fibrillar components. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Structure and anticoagulant property of a sulfated polysaccharide isolated from the green seaweed Monostroma angicava.

    PubMed

    Li, Na; Liu, Xue; He, Xiaoxi; Wang, Shuyao; Cao, Sujian; Xia, Zheng; Xian, Huali; Qin, Ling; Mao, Wenjun

    2017-03-01

    An anticoagulant-active polysaccharide PF2 was extracted with boiling water from the green seaweed Monostroma angicava, further purified by anion-exchange and size-exclusion chromatography. PF2 was a rhamnan-type sulfated polysaccharide with molecular weight of about 88.1kDa. Results of chemical and spectroscopic analyses demonstrated that PF2 consisted of→3)-α-l-Rhap-(1→ and →2)-α-l-Rhap-(1→residues, with partially branches at C-2 of→3)-α-l-Rhap-(1→residues. Sulfate groups were substituted at C-3 of →2)-α-l-Rhap-(1→ residues. The sulfated polysaccharide PF2 had a high anticoagulant action, and the mechanism of anticoagulant activity mediated by PF2 was mainly attributed to strong potentiation thrombin by heparin cofactor II. PF2 also exhibited weak effect on antithrombin-dependent thrombin or factor Xa inhibition. The fibrin(ogen)olytic activity and thrombolytic activity of PF2 were also evaluated. The investigation revealed that PF2 was a novel sulfated rhamnan differing from previously described sulfated polysaccharides from green seaweed and could be a potential anticoagulant polysaccharide.

  18. Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides

    PubMed Central

    Benincasa, Monica; Lagatolla, Cristina; Dolzani, Lucilla; Milan, Annalisa; Pacor, Sabrina; Liut, Gianfranco; Tossi, Alessandro; Cescutti, Paola; Rizzo, Roberto

    2016-01-01

    Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1–35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation. PMID:27681920

  19. Structure of the oligosaccharides isolated from Prosopis juliflora (Sw.) DC. seed polysaccharide.

    PubMed

    Bhatia, Himani; Gupta, P K; Soni, P L

    2014-01-30

    A water soluble polysaccharide isolated from Prosopis juliflora seed was purified and major homogenous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of d-galactose and d-mannose in the ratio 1:1.10, respectively. Partial hydrolysis of the polysaccharide furnished one hepta-(I), one octa-(II) and nona-(III) saccharides. Hydrolysis of oligosaccharide I, II and III followed by GLC analysis furnished d-galactose and d-mannose in the ratio 3:4, 3:5 and 5:4, respectively. Methylation analysis, periodate oxidation and (1)H NMR spectral studies of oligosaccharides indicated the presence of (1→4) mannose units linked to (1→6) galactose units.

  20. Uronic polysaccharide degrading enzymes.

    PubMed

    Garron, Marie-Line; Cygler, Miroslaw

    2014-10-01

    In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the β-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide.

  1. Structural elucidation of a cell wall fungal polysaccharide isolated from Ustilaginoidea virens, a pathogenic fungus of Oriza sativa and Zea mays.

    PubMed

    Leal, J A; Jiménez-Barbero, Jesús; Bernabé, Manuel; Prieto, Alicia

    2008-11-24

    The alkali-extractable water-soluble polysaccharides (F1SS) isolated from the outer cell wall of two strains of Ustilaginoidea virens have been studied by chemical and methylation analyses, and 1D and 2D (1)H and (13)C NMR spectroscopy. The structures of these polysaccharides are very similar, and can be described by the following idealized repeating unit: where n and m are approximately 1 and 2, respectively.

  2. Structure Elucidation of a Polysaccharide from Umbilicaria esculenta and Its Immunostimulatory Activity

    PubMed Central

    Zhang, Bi-Wei; Xu, Jin-Long; Zhang, Hua; Zhang, Qiang; Lu, Jie; Wang, Jun-Hui

    2016-01-01

    Umbilicaria esculenta has been used as a tonic food in China for several centuries owing to its pleasant flavor and health benefits. In this study, a water soluble polysaccharide, which we designated as UP2, with an average molecular weight of 3.33 × 105 Da, was isolated from U. esculenta cultivated in the Huangshan Mountain, by consecutive hot water extraction and anion-exchange chromatography. Gas chromatography analysis indicated that UP2 contained three kinds of monosaccharides, including mannose, glucose, and galactose at a molar ratio of 1.7:1.0:1.2. Linkage analysis of UP2 revealed the presence of (1 → 6)-linked glucosyl, (1 → 3,6)-linked glucosyl, t-linked galactosyl, (1 → 6)-linked galactosyl and (1 → 6)-linked mannosyl at a molar ratio of 0.7:4.6:4.1:2.2:9.1. Structural analysis determined that UP2 possessed a backbone consisting of (1 → 6)-linked β-D-glucopyranosyl and (1 → 6)-linked α-D-mannopyranosyl residues, which substituted at the O-3 position of (1 → 6)-linked β-D-glucopyranosyl residues by branches of (1 → 6)-linked α-D-galactopyranosyl and 1-linked β-D-galactopyranosyl residues. Immunostimulatory activity analysis showed that UP2 could stimulate the proliferation of RAW264.7 cells in a dose-dependent manner, and all the samples (20–500 μg/mL) were found to enhance nitric oxide production. The highest phagocytic activity of UP2 was observed at 200 μg/mL. Thus, UP2 may be a potential source of biological and pharmacological agents. PMID:27997616

  3. Structural, physicochemical, antioxidant and antitumor property of an acidic polysaccharide from Polygonum multiflorum.

    PubMed

    Zhu, Weili; Xue, Xiaoping; Zhang, Zhanjun

    2017-03-01

    In this study, the structural characterization, physicochemical property, antioxidant and antitumor activity of an acidic polysaccharide (APS) from Polygonum multiflorum were investigated. Monosaccharide composition analysis showed APS was composed of arabinose, rhamnose, galactose and galacturonic acid in the molar ratio of 1.23:1.32:1.48:1.00. The presence of uronic acid was also confirmed by the bands at 1740, 1645 and 1425cm(-1) on Fourier transform-infrared spectroscopy. Methylation and nuclear magnetic resonance analyses showed APS was mainly composed by the residues of →5)-α-l-Araf-(1→, →3)-β-d-Galp-(1→, →3,6)-β-d-Galp-(1→, →4)-α-d-GalAp-(1→ and →2)-α-l-Rhap-(1→ in the backbone. The non-reducing terminal α-l-Araf-(1→ was probably attached to the O-6 position of →3,6)-β-d-Galp-(1→ residues. Besides, APS exhibited rod-like and flaky shapes with rough surface. The initial decomposition of APS occurred at 172°C, and the rapidest weight loss rate of APS appeared at 320°C. Antioxidant activity assay showed the DPPH radical scavenging activity of APS was 67.5% at 1mg/mL. At the concentration of 400μg/mL, the antiproliferation activities of APS against HepG-2 and BGC-823 cells were 65.28% and 51.57%, respectively. Our results suggested APS could be a potential antioxidant and antitumor agent.

  4. Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots.

    PubMed Central

    Freshour, G.; Clay, R. P.; Fuller, M. S.; Albersheim, P.; Darvill, A. G.; Hahn, M. G.

    1996-01-01

    The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner. PMID:12226270

  5. Pseudomonas aeruginosa inactivation mechanism is affected by capsular extracellular polymeric substances reactivity with chlorine and monochloramine.

    PubMed

    Xue, Zheng; Hessler, Christopher M; Panmanee, Warunya; Hassett, Daniel J; Seo, Youngwoo

    2013-01-01

    The reactivity of capsular extracellular polymeric substances (EPS) to chlorine and monochloramine was assessed and compared in this study. The impact of capsular EPS on Gram-negative bacteria Pseudomonas aeruginosa inactivation mechanisms was investigated both qualitatively and quantitatively using a combination of batch experiments, viability tests with LIVE/DEAD staining, and Fourier transform infrared spectroscopy (FTIR). Both wild-type and isogenic mutant strains with different alginate EPS production capabilities were used to evaluate their susceptibility to chlorine and monochloramine. The mucA22 mutant strain, which overproduces the EPS composed largely of acidic polysaccharide alginate, exhibited high resistance and prolonged inactivation time to both chlorine and monochloramine relative to PAO1 (wild-type) and algT(U) mutant strains (alginate EPS deficient). Multiple analyses were combined to better understand the mechanistic role of EPS against chlorine-based disinfectants. The extracted EPS exhibited high reactivity with chlorine and very low reactivity with monochloramine, suggesting different mechanism of protection against disinfectants. Moreover, capsular EPS on cell membrane appeared to reduce membrane permeabilization by disinfectants as suggested by deformation of key functional groups in EPS and cell membrane (the C-O-C stretching of carbohydrate and the C=O stretching of ester group). The combined results supported that capsular EPS, acting either as a disinfectant consumer (for chlorine inactivation) or limiting access to reactive sites on cell membrane (for monochloramine inactivation), provide a protective role for bacterial cells against regulatory residual disinfectants by reducing membrane permeabilization.

  6. Arthroscopic Capsular Release of the Talocalcaneonavicular Joint.

    PubMed

    Lui, Tun Hing

    2016-12-01

    Arthrofibrosis of the talocalcaneonavicular joint can follow talar neck fracture especially if anterior approaches have been used for fracture fixation. Capsular release of the talocalcaneonavicular joint is indicated if the painful hindfoot stiffness cannot be controlled with conservative treatment. Open capsular release of the talocalcaneonavicular joint demands extensive soft tissue dissection and hinders early postoperative mobilization exercise of the joint. The purpose of this technical note is to describe a minimally invasive approach of arthroscopic capsular release of the talocalcaneonavicular joint that is composed of arthroscopic release of the talonavicular joint and the anterior subtalar joint. This allows immediate postoperative mobilization of the joint.

  7. Rheological Flow Behavior of Structural Polysaccharides from Edible Tender Cladodes of Wild, Semidomesticated and Cultivated 'Nopal' (Opuntia) of Mexican Highlands.

    PubMed

    López-Palacios, C; Peña-Valdivia, C B; Rodríguez-Hernández, A I; Reyes-Agüero, J A

    2016-12-01

    The aim of this study was to quantify the content of polysaccharides of edible tender cladodes (nopalitos) of three species of Opuntia and to evaluate the rheological flow behavior of isolated polysaccharides. A completely randomized experimental design was used to characterize a wild (O. streptacantha), a semidomesticated (O. megacantha) and a domesticated (O. ficus-indica) species. Mucilage content was higher (4.93 to 12.43 g 100 g(-1) dry matter), tightly bound hemicelluloses were lower (3.32 to 1.81 g 100 g(-1) dry matter) and pectins and loosely bound hemicelluloses were not different in wild than in domesticated species. Aqueous solution/suspensions of mucilage, pectins, hemicellulose and cellulose of all species showed non-Newtonian behavior under simple shear flow. The flow behavior of the structural polysaccharides was well described by the Ostwald de-Waele model. Pectins and mucilages exhibited the highest consistency indexes (K values ranged from 0.075 to 0.177 Pas(n)) with a moderated shear-thinning behavior (n values ranged from 0.53 to 0.67). Cellulose dispersions exhibited the most shear-thinning behavior (n values ranged from 0.17 to 0.41) and hemicelluloses showed a tendency to Newtonian flow (n values ranged from 0.82 to 0.97). The rheological flow properties of these polysaccharides may be useful to improve the textural and sensory qualities of some foods and pharmaceutical materials. Moreover, they can emerge as functional ingredients mainly due to the nutraceutical properties that have been attributed to nopalitos.

  8. Structure and serological specificity of a new acidic O-specific polysaccharide of Proteus vulgaris O45.

    PubMed

    Bartodziejska, B; Shashkov, A S; Torzewska, A; Grachev, A A; Ziolkowski, A; Paramonov, N A; Rozalski, A; Knirel, Y A

    1999-01-01

    The following structure of the O-specific polysaccharide (OPS) of Proteus vulgaris O45 lipopolysaccharide (LPS) was established using 1H- and 13C-NMR spectroscopy, including two-dimensional NOESY and H-detected 1H, 13C heteronuclear multiple-quantum coherence ( HMQC) experiments: [structure: see text text] Immunochemical studies, using rabbit polyclonal anti-P. vulgaris O45 serum and LPS, OPS and Smith-degraded OPS of P. vulgaris O45, showed the importance of beta-D-GlcA in manifesting the serological specificity of the O-antigen studied.

  9. Effects of light intensity on components and topographical structures of extracellular polysaccharides from the cyanobacteria Nostoc sp.

    PubMed

    Ge, Hongmei; Xia, Ling; Zhou, Xuping; Zhang, Delu; Hu, Chunxiang

    2014-02-01

    A study on the effects of light intensity (40 and 80 μE/m(2)/sec) on the components and topographical structures of extracellular polysaccharides (EPS) was carried out in cyanobacteria Nostoc sp.. EPS yield increased with light intensity. However, light intensity did not significantly affect the EPS fractions and monosaccharide composition. Higher light intensity generally resulted in higher protein content of EPS in similar fractions. The topographical structure of EPS, investigated by atomic force microscopy, appeared as spherical lumps, chains and networks. The long chains were observed at higher light intensity. Thus, light intensity affected the yield and nature of EPS.

  10. Polysaccharides: Occurrence, Significance, and Properties

    NASA Astrophysics Data System (ADS)

    Bemiller, James N.

    Polysaccharides are properties present significance in all living organisms where they carry out one or more of their diverse functions. While there is no specific category or definition of a complex polysaccharide, most are structurally complex. Polysaccharides contain 1-5 different monosaccharide (sugar) units. The different sugar units may have different anomeric configurations and/or be joined by different glycosidic linkages. Polysaccharides may be linear or branched. Branches may be short saccharide units on a linear backbone or the molecule may have a branch-on-branch structure; in either case, the branches may be isolated or clustered. Polysaccharides may contain non-carbohydrate groups. Esters or cyclic acetal groups, when present, can be removed by appropriate treatments. All polysaccharides are polydisperse, i. e., are present in a range of molecular weights rather than having a single molecular weight. Most are polymolecular, i. e., differ in fine structure from molecule to molecule. So most polysaccharides can be said to be structurally complex. They may be attached to protein molecules or to other polysaccharide molecules. They are solvated by water. Most dissolve in aqueous systems, especially if they are alkaline. Polysaccharides can be depolymerized by acids and heat, specific enzymes, and high pH systems following oxidation. Their hydroxyl groups can be esterified (acylated), etherified (alkylated), and oxidized. Amino groups can be acylated (and deacylated). Carboxyl groups can be converted into esters, amides, and amines. Structural modification makes the molecules even more complex and polymolecular and, perhaps, polydisperse.

  11. Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase GH61D from the Basidiomycota Fungus Phanerochaete chrysosporium*

    PubMed Central

    Wu, Miao; Beckham, Gregg T.; Larsson, Anna M.; Ishida, Takuya; Kim, Seonah; Payne, Christina M.; Himmel, Michael E.; Crowley, Michael F.; Horn, Svein J.; Westereng, Bjørge; Igarashi, Kiyohiko; Samejima, Masahiro; Ståhlberg, Jerry; Eijsink, Vincent G. H.; Sandgren, Mats

    2013-01-01

    Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain. PMID:23525113

  12. Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides.

    PubMed

    Pujar, Narahari S; Huang, Ngan Fong; Daniels, Christopher L; Dieter, Lance; Gayton, Marshall G; Lee, Ann L

    2004-09-01

    A comprehensive study of the base hydrolysis of all phosphodiester bond-containing capsular polysaccharides of the 23-valent pneumococcal vaccine is described here. Capsular polysaccharides from serotypes 6B, 10A, 17F, 19A, 19F, and 20 contain a phosphodiester bond that connects the repeating units in these polysaccharides (also referred to as backbone phosphodiester bonds), and polysaccharides from serotypes 11A, 15B, 18C, and 23F contain a phosphodiester bond that links a side chain to their repeating units. Molecular weight measurements of the polysaccharides, using high performance size exclusion chromatography with tandem multiangle laser light scattering and refractive index detection, was used to evaluate the kinetics of hydrolysis. The measurement of molecular weight provides a high degree of sensitivity in the case of small extents of reaction, thus allowing reliable measurements of the kinetics over short times. Pseudo-first-order rate constants for these polysaccharides were estimated using a simple model that accounts for the polydispersity of the starting sample. It was found that the relative order of backbone phosphodiester bond instability due to base hydrolysis was 19A > 10A > 19F > 6B > 17F, 20. Degradation of side-chain phosphodiester bonds was not observed, although the high degree of sensitivity in measurements is lost in this case, due to the low contribution of the side chains to the total polysaccharide molecular weight. In comparison with literature data on pneumococcal polysaccharide 6A, 19A was found to be the more labile, and hence appears to be the most labile pneumococcal polysaccharide studied to date. The rate of hydrolysis increased at higher pH and in the presence of divalent cation, but the extent was lower than expected based on similar data on RNA. Finally, the differences in the phosphodiester bond stabilities were analyzed by considering stereochemical factors in these polysaccharides. These results also provide a framework

  13. Multidimensional solid-state NMR studies of the structure and dynamics of pectic polysaccharides in uniformly 13C-labeled Arabidopsis primary cell walls

    SciTech Connect

    Dick-Perez, Marilu; Wang, Tuo; Salazar, Andre; Zabotina, Olga A.; Hong, Mei

    2012-07-08

    Plant cell wall (CW) polysaccharides are responsible for the mechanical strength and growth of plant cells; however, the high-resolution structure and dynamics of the CW polysaccharides are still poorly understood because of the insoluble nature of these molecules. Here, we use 2D and 3D magic-angle-spinning (MAS) solid-state NMR (SSNMR) to investigate the structural role of pectins in the plant CW. Intact and partially depectinated primary CWs of Arabidopsis thaliana were uniformly labeled with 13C and their NMR spectra were compared. Recent 13C resonance assignment of the major polysaccharides in Arabidopsis thaliana CWs allowed us to determine the effects of depectination on the intermolecular packing and dynamics of the remaining wall polysaccharides. 2D and 3D correlation spectra show the suppression of pectin signals, confirming partial pectin removal by chelating agents and sodium carbonate. Importantly, higher cross peaks are observed in 2D and 3D 13C spectra of the depectinated CW, suggesting higher rigidity and denser packing of the remaining wall polysaccharides compared with the intact CW. 13C spin–lattice relaxation times and 1H rotating-frame spin–lattice relaxation times indicate that the polysaccharides are more rigid on both the nanosecond and microsecond timescales in the depectinated CW. Taken together, these results indicate that pectic polysaccharides are highly dynamic and endow the polysaccharide network of the primary CW with mobility and flexibility, which may be important for pectin functions. This study demonstrates the capability of multidimensional SSNMR to determine the intermolecular interactions and dynamic structures of complex plant materials under near-native conditions. Copyright © 2012 John Wiley & Sons, Ltd.

  14. Campylobacter Polysaccharide Capsules: Virulence and Vaccines

    PubMed Central

    Guerry, Patricia; Poly, Frédéric; Riddle, Mark; Maue, Alexander C.; Chen, Yu-Han; Monteiro, Mario A.

    2012-01-01

    Campylobacter jejuni remains a major cause of bacterial diarrhea worldwide and is associated with numerous sequelae, including Guillain Barré Syndrome, inflammatory bowel disease, reactive arthritis, and irritable bowel syndrome. C. jejuni is unusual for an intestinal pathogen in its ability to coat its surface with a polysaccharide capsule (CPS). These capsular polysaccharides vary in sugar composition and linkage, especially those involving heptoses of unusual configuration and O-methyl phosphoramidate linkages. This structural diversity is consistent with CPS being the major serodeterminant of the Penner scheme, of which there are 47 C. jejuni serotypes. Both CPS expression and expression of modifications are subject to phase variation by slip strand mismatch repair. Although capsules are virulence factors for other pathogens, the role of CPS in C. jejuni disease has not been well defined beyond descriptive studies demonstrating a role in serum resistance and for diarrhea in a ferret model of disease. However, perhaps the most compelling evidence for a role in pathogenesis are data that CPS conjugate vaccines protect against diarrheal disease in non-human primates. A CPS conjugate vaccine approach against this pathogen is intriguing, but several questions need to be addressed, including the valency of CPS types required for an effective vaccine. There have been numerous studies of prevalence of CPS serotypes in the developed world, but few studies from developing countries where the disease incidence is higher. The complexity and cost of Penner serotyping has limited its usefulness, and a recently developed multiplex PCR method for determination of capsule type offers the potential of a more rapid and affordable method. Comparative studies have shown a strong correlation of the two methods and studies are beginning to ascertain CPS-type distribution worldwide, as well as examination of correlation of severity of illness with specific CPS types. PMID:22919599

  15. Characterization of structures and antiviral effects of polysaccharides from Portulaca oleracea L.

    PubMed

    Dong, Cai-Xia; Hayashi, Kyoko; Lee, Jung-Bum; Hayashi, Toshimitsu

    2010-04-01

    Three polysaccharides including a neutral polysaccharide (RN), an acidic polysaccharide (RA) and a pectic polysaccharide (RP) were isolated from aerial part of Portulaca oleracea L. and evaluated for their anti-herpes simplex virus type 2 (HSV-2) and anti-influenza A virus (IFV-A). RN was found to consist of glucose (Glc), mannose (Man) and arabinose (Ara) with small amounts of galactose (Gal), and identified to be an arabinoglucomannan. RA was mainly composed of Gal and Ara with a small proportion of glucuronic acid (GlcA). It was characterized as a type II arabinogalactan (AGII), which consisted of a 1,3-, 1,6- and 1,3,6-linked galactopyranosyl (Galp) and non-reducing terminal and 1,5-linked arabinofuranosyl (Araf) residues. RP was deduced to be a pectin, which consisted of a predominant amount of galacturonic acid (GalA) with small amounts of Gal, rhamnose (Rha) and Ara. The GalA residues were found to be highly methyl-esterified and partially acetylated. Results of antiviral tests showed that only RP had anti-HSV-2 activity. Furthermore, its anti-HSV-2 target was elucidated to be the step of virus penetration into host cells. No marked virucidal activity of RP was observed.

  16. An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity.

    PubMed

    Norheim, Gunnstein; Sanders, Holly; Mellesdal, Jardar W; Sundfør, Idunn; Chan, Hannah; Brehony, Carina; Vipond, Caroline; Dold, Chris; Care, Rory; Saleem, Muhammad; Maiden, Martin C J; Derrick, Jeremy P; Feavers, Ian; Pollard, Andrew J

    2015-01-01

    Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial.

  17. An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity

    PubMed Central

    Norheim, Gunnstein; Sanders, Holly; Mellesdal, Jardar W.; Sundfør, Idunn; Chan, Hannah; Brehony, Carina; Vipond, Caroline; Dold, Chris; Care, Rory; Saleem, Muhammad; Maiden, Martin C. J.; Derrick, Jeremy P.; Feavers, Ian; Pollard, Andrew J.

    2015-01-01

    Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial. PMID:26390123

  18. Streptococcus iniae cpsG alters capsular carbohydrate composition and is a cause of serotype switching in vaccinated fish.

    PubMed

    Heath, Candice; Gillen, Christine M; Chrysanthopoulos, Panagiotis; Walker, Mark J; Barnes, Andrew C

    2016-09-25

    Streptococcus iniae causes septicaemia and meningitis in marine and freshwater fish wherever they are farmed in warm-temperate and tropical regions. Although serotype specific, vaccination with bacterins (killed bacterial cultures) is largely successful and vaccine failure occurs only occasionally through emergence of new capsular serotypes. Previously we showed that mutations in vaccine escapes are restricted to a limited repertoire of genes within the 20-gene capsular polysaccharide (cps) operon. cpsG, a putative UDP-galactose 4-epimerase, has three sequence types based on the insertion or deletion of the three amino acids leucine, serine and lysine in the substrate binding site of the protein. To elucidate the role of cpsG in capsular polysaccharide (CPS) biosynthesis and capsular composition, we first prepared isogenic knockout and complemented mutants of cpsG by allelic exchange mutagenesis. Deletion of cpsG resulted in changes to colony morphology and cell buoyant density, and also significantly decreased galactose content relative to glucose in the capsular polysaccharide as determined by GC-MS, consistent with epimerase activity of CpsG. There was also a metabolic penalty of cpsG knockout revealed by slower growth in complex media, and reduced proliferation in whole fish blood. Moreover, whilst antibodies raised in fish against the wild type cross-reacted in whole cell and cps ELISA, they did not cross-opsonise the mutant in a peripheral blood neutrophil opsonisation assay, consistent with reported vaccine escape. We have shown here that mutation in cpsG results in altered CPS composition and this in turn results in poor cross-opsonisation that explains some of the historic vaccination failure on fish farms in Australia.

  19. Structural characterization and anti-aging activity of a novel extracellular polysaccharide from fungus Phellinus sp. in a mammalian system.

    PubMed

    Ma, Xiao-Kui; Guo, Dan Dan; Peterson, Eric Charles; Dun, Ying; Li, Dan Yang

    2016-08-10

    Little is known about the chemical structure of purified extracellular polysaccharides from Phellinus sp., a fungal species with known medicinal properties. A combination of IR spectroscopy, methylation analysis and NMR were performed for the structural analysis of a purified extracellular polysaccharide derived from Phellinus sp. culture, denoted as SHP-1, along with an evaluation of the anti-aging effect in vivo of the polysaccharide supplementation. The structure of SHP-1 was established, with a backbone composed of →2,4)-α-d-glucopyranose-(1→ and →2)-β-d-mannopyranose-(1→ and two terminal glucopyranose branches. Biochemical analysis from mammalian animal experiments demonstrated that SHP-1 possesses the ability to enhance antioxidant enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD) activities, Trolox equivalent antioxidant capacity (TEAC) in serum of d-galactose-aged mice, while reducing lipofuscin levels, another indicator of cell aging, indicating a potential association with anti-aging activities in a dose dependent manner. This compound had a favourable influence on immune organ indices, and a marked amelioration ability of histopathological hepatic lesions such as necrosis, karyolysis and reduced inflammation and apoptosis in mouse hepatocytes. These results suggest that SHP-1 has strong antioxidant activities and a significant protective effect against oxidative stress or hepatotoxicity induced by d-galactose in mice and it could be developed as a food ingredient or a pharmaceutical to prevent many age-associated diseases such as major depressive disorder and hepatotoxicity. To our knowledge, this is the first report on the antioxidant effects of a novel purified exopolysaccharide derived from Phellinus sp.

  20. Following fungal melanin biosynthesis with solid-state NMR: biopolymer molecular structures and possible connections to cell-wall polysaccharides.

    PubMed

    Zhong, Junyan; Frases, Susana; Wang, Hsin; Casadevall, Arturo; Stark, Ruth E

    2008-04-22

    Melanins serve a variety of protective functions in plants and animals, but in fungi such as Cryptococcus neoformans they are also associated with virulence. A recently developed solid-state nuclear magnetic resonance (NMR) strategy, based on the incorporation of site-specific (13)C-enriched precursors into melanin, followed by spectroscopy of both powdered and solvent-swelled melanin ghosts, was used to provide new molecular-level insights into fungal melanin biosynthesis. The side chain of an l-dopa precursor was shown to cyclize and form a proposed indole structure in C. neoformans melanin, and modification of the aromatic rings revealed possible patterns of polymer chain elongation and cross-linking within the biopolymer. Mannose supplied in the growth medium was retained as a beta-pyranose moiety in the melanin ghosts even after exhaustive degradative and dialysis treatments, suggesting the possibility of tight binding or covalent incorporation of the pigment into the polysaccharide fungal cell walls. In contrast, glucose was scrambled metabolically and incorporated into both polysaccharide cell walls and aliphatic chains present in the melanin ghosts, consistent with metabolic use as a cellular nutrient as well as covalent attachment to the pigment. The prominent aliphatic groups reported previously in several fungal melanins were identified as triglyceride structures that may have one or more sites of chain unsaturation. These results establish that fungal melanin contains chemical components derived from sources other than l-dopa polymerization and suggest that covalent linkages between l-dopa-derived products and polysaccharide components may serve to attach this pigment to cell wall structures.

  1. New Ulvan-Degrading Polysaccharide Lyase Family: Structure and Catalytic Mechanism Suggests Convergent Evolution of Active Site Architecture.

    PubMed

    Ulaganathan, ThirumalaiSelvi; Boniecki, Michal T; Foran, Elizabeth; Buravenkov, Vitaliy; Mizrachi, Naama; Banin, Ehud; Helbert, William; Cygler, Miroslaw

    2017-03-23

    Ulvan is a complex sulfated polysaccharide biosynthesized by green seaweed and contains predominantly rhamnose, xylose, and uronic acid sugars. Ulvan-degrading enzymes have only recently been identified and added to the CAZy ( www.cazy.org ) database as family PL24, but neither their structure nor catalytic mechanism(s) are yet known. Several homologous, new ulvan lyases, have been discovered in Pseudoalteromonas sp. strain PLSV, Alteromonas LOR, and Nonlabens ulvanivorans, defining a new family PL25, with the lyase encoded by the gene PLSV_3936 being one of them. This enzyme cleaves the glycosidic bond between 3-sulfated rhamnose (R3S) and glucuronic acid (GlcA) or iduronic acid (IdoA) via a β-elimination mechanism. We report the crystal structure of PLSV_3936 and its complex with a tetrasaccharide substrate. PLSV_3936 folds into a seven-bladed β-propeller, with each blade consisting of four antiparallel β-strands. Sequence conservation analysis identified a highly conserved region lining at one end of a deep crevice on the protein surface. The putative active site was identified by mutagenesis and activity measurements. Crystal structure of the enzyme with a bound tetrasaccharide substrate confirmed the identity of base and acid residues and allowed determination of the catalytic mechanism and also the identification of residues neutralizing the uronic acid carboxylic group. The PLSV_3936 structure provides an example of a convergent evolution among polysaccharide lyases toward a common active site architecture embedded in distinct folds.

  2. Immunomodulating polysaccharides from Lessertia frutescens leaves: isolation, characterization and structure activity relationship.

    PubMed

    Zhang, Bingzhao; Leung, Wei Kee; Zou, Yuanfeng; Mabusela, Wilfred; Johnson, Quinton; Michaelsen, Terje E; Paulsen, Berit Smestad

    2014-03-14

    Sutherlandia frutescens (syn. Lessertia frutescens) is an indigenous plant in Southern Africa and has been extensively studied from the ethnobotanical point of view. Amongst the various traditional uses, several illnesses involving the immune system have been reported. Due to some of the therapeutic effects observed, in relation to the traditional uses reported by the "khoi san" and "nama" people on cancer related illnesses, the plant has been given the local name kankerbos (cancerbush). Recently the plant has also been used amongst HIV/AIDS patients to stimulate the immune system. Leaves of Sutherlandia frutescens were extracted sequentially with ethanol, 50% ethanol/water, and water at 50 and 100°C. The polysaccharides were extracted with water and fractionated by ion exchange chromatography and gel filtration to obtain enriched polysaccharide fractions. The bioactivities of the fractions were tested in the complement assay. Some of the fractions were treated with the enzyme pectinase, and the fragments thus produced were separated by gel filtration and their activities tested. Monosaccharide compositions and linkage analyses were determined for the relevant fractions. The leaves of Sutherlandia frutescens contain polysaccharides of the pectin type. Fractions from both the water extracts of 50 and 100°C were bioactive. Fractions chosen for further studies showed that the fragment with the highest M(W) after the pectinase treatment had a substantially higher biological effect than the parent molecules. Based on a comparison of the different fractions it was concluded that galactose-rich regions were important for the bioactivity, these being of the AGII and AGI type, with the latter probably being more important than the former. Fragments rich in xylose also gave higher activity than those without it. Our theory that the polysaccharides present in the leaves of Sutherlandia frutescens could be of importance as immunomodulating agents was confirmed. It was also

  3. Polysaccharides of the red algae.

    PubMed

    Usov, Anatolii I

    2011-01-01

    Red algae (Rhodophyta) are known as the source of unique sulfated galactans, such as agar, agarose, and carrageenans. The wide practical uses of these polysaccharides are based on their ability to form strong gels in aqueous solutions. Gelling polysaccharides usually have molecules built up of repeating disaccharide units with a regular distribution of sulfate groups, but most of the red algal species contain more complex galactans devoid of gelling ability because of various deviations from the regular