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Evgeny Vinogradov - One of the best experts on this subject based on the ideXlab platform.

  • Tannerella forsythia strains display different cell-surface nonulosonic Acids: biosynthetic pathway characterization and first insight into biological implications
    Glycobiology, 2017
    Co-Authors: Valentin Friedrich, Matthias L. Braun, Marie-france Goneau, Zoe Anne Megson, Markus Windwarder, Bettina Janesch, Daniel Maresch, Ashu Sharma, Evgeny Vinogradov, Friedrich Altmann
    Abstract:

    Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium's cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic Acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic Acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of Legionaminic Acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes' functions. Using capillary electrophoresis (CE), CE-mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs.

  • Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics
    PLOS Pathogens, 2015
    Co-Authors: Sunita Gulati, Ian C. Schoenhofen, Jacek Stupak, Evgeny Vinogradov, Frank St. Michael, Dennis M. Whitfield, Bo Zheng, Jianjun Li, Makoto Ohnishi
    Abstract:

    Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5’-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic Acid (Neu5Ac), a sialic Acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation (“serum-resistance”). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, Legionaminic Acid (Leg5Ac7Ac) and pseudaminic Acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP-Leg5Ac7Ac-treated mice were sensitive to human complement ex vivo, simulating in vitro findings. These data reveal critical roles for the Sia exocyclic side-chain in gonococcal serum-resistance. Such CMP-NulO analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea.

  • characterization of the lipopolysaccharide o antigen of cronobacter turicensis hpb3287 as a polysaccharide containing a 5 7 diacetamido 3 5 7 9 tetradeoxy d glycero d galacto non 2 ulosonic Acid Legionaminic Acid residue
    Carbohydrate Research, 2011
    Co-Authors: Leann L Maclean, Evgeny Vinogradov, Franco Pagotto, Malcolm B Perry
    Abstract:

    Abstract Cronobacter turicensis , previously known as Enterobacter sakazakii , is a Gram-negative opportunistic food-borne pathogen that has been reported as a cause of life-threatening neonatal infections. From chemical and physical analyses involving composition analysis, methylation, two-dimensional high-resolution nuclear magnetic resonance, and mass spectrometry methods, the antigenic O-polysaccharide in the smooth-type lipopolysaccharide of C . turicensis (strain HPB 3287) was determined to be a high molecular mass polymer of a repeating pentasaccharide unit composed of d -galactose, d -glucose, 2-acetamido-2-deoxy- d -galactose, and 5,7-diacetamido-3,5,7,9-tetradeoxy- d - glycero - d - galacto -non-2-ulosonic Acid (Legionaminic Acid), in a molar ratio 2:1:1:1, and having the structure:

  • the cmp Legionaminic Acid pathway in campylobacter biosynthesis involving novel gdp linked precursors
    Glycobiology, 2009
    Co-Authors: Ian C. Schoenhofen, Jean-robert Brisson, Evgeny Vinogradov, Dennis M. Whitfield, Susan M Logan
    Abstract:

    The sialic Acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic Acid, or legion-aminic Acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an alpha2,4-linked homopolymer of Legionaminic Acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of Legionaminic Acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of Legionaminic Acid, and since Legionaminic Acid and sialic Acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic Acid mimic.

  • flagellar glycosylation in clostridium botulinum
    FEBS Journal, 2008
    Co-Authors: Susan M Twine, Jean-robert Brisson, David J. Mcnally, Evgeny Vinogradov, Catherine J Paul, James A Mullen, David R Mcmullin, Harold C Jarrell, John W Austin, John F Kelly
    Abstract:

    Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of Legionaminic Acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel Legionaminic Acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic Acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic Acid-like' nonulosonate sugar, Legionaminic Acid.

Ian C. Schoenhofen - One of the best experts on this subject based on the ideXlab platform.

  • biosynthesis of Legionaminic Acid and its incorporation into glycoconjugates
    Methods in Enzymology, 2017
    Co-Authors: Ian C. Schoenhofen, Martin N Young, Michel Gilbert
    Abstract:

    Legionaminic Acids are analogs of sialic Acid that occur in cell surface glycoconjugates of several bacteria. Because Legionaminic Acids share the same stereochemistry as sialic Acid but differ at C7 and C9, they are interesting analogs to probe the impact of varying exocyclic moieties (C7-C9) on biological activities such as susceptibilities to sialidases, interactions with Siglecs and immunogenicity. There are currently no reports on the bacterial enzymes that transfer Legionaminic Acids to these cell surface glycoconjugates, but some mammalian and bacterial sialyltransferases display donor promiscuity and can use CMP-Leg5,7Ac2 efficiently enough to transfer Leg5,7Ac2 to their natural acceptor glycans. When the natural activity with CMP-Leg5,7Ac2 is significant but relatively low, an alternate strategy has been to engineer versions with improved activity to transfer Leg5,7Ac2. Importantly, we have found that some bacterial sialyltransferases are very efficient for transferring Leg5,7Ac2 to small synthetic glycans with various aglycones. The two mammalian sialyltransferases that have been tested so far (porcine ST3Gal1 and human ST6Gal1) were found to be more efficient than the bacterial sialyltransferases for the modification of glycoproteins. We provide a review of the sialyltransferases selected to modify different types of glycoconjugates with Leg5,7Ac2, including small synthetic acceptors, glycolipids, and glycoproteins. In the first part, we also propose an optimized biosynthetic pathway for in vitro preparation of the donor CMP-Leg5,7Ac2, based on enzymes selected from two bacteria that naturally produce Legionaminic Acid.

  • total biosynthesis of Legionaminic Acid a bacterial sialic Acid analogue
    Angewandte Chemie, 2016
    Co-Authors: Mohamed I Hassan, Ian C. Schoenhofen, Dennis M. Whitfield, Benjamin R Lundgren, Michael Chaumun, Brady Clark, Christopher N Boddy
    Abstract:

    Legionaminic Acid, Leg5,7Ac2, a nonulosonic Acid like 5-acetamido neuraminic Acid (Neu5Ac, sialic Acid), is found in cell surface glycoconjugates of bacteria including the pathogens Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. The presence of Leg5,7Ac2 has been correlated with virulence in humans by mechanisms that likely involve subversion of the host's immune system or interactions with host cell surfaces due to its similarity to Neu5Ac. Investigation into its role in bacterial physiology and pathogenicity is limited as there are no effective sources of it. Herein, we construct a de novo Leg5,7Ac2 biosynthetic pathway by combining multiple metabolic modules from three different microbial sources (Saccharomyces cerevisiae, C. jejuni, and L. pneumophila). Over-expression of this de novo pathway in Escherichia coli that has been engineered to lack two native catabolic pathways, enables significant quantities of Leg5,7Ac2 (≈120 mg L−1 of culture broth) to be produced. Pure Leg5,7Ac2 could be isolated and converted into CMP-activated sugar for biochemical applications and a phenyl thioglycoside for chemical synthesis applications. This first total biosynthesis provides an essential source of Leg5,7Ac2 enabling study of its role in prokaryotic and eukaryotic glycobiology.

  • RESEARCH ARTICLE Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide- Mediated Complement Resistance and Design Novel Therapeutics
    2016
    Co-Authors: Sunita Gulati, Ian C. Schoenhofen, Jacek Stupak, Frank St. Michael, Dennis M. Whitfield, Bo Zheng, Andrew D. Cox, Evgeny V. Vinogradov, Makoto Ohnishi
    Abstract:

    Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neo-tetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyl-transferase, using host cytidine-5’-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic Acid (Neu5Ac), a sialic Acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting fac-tor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation (“serum-resistance”). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, Legionaminic Acid (Leg5Ac7Ac) and pseudaminic Acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac an

  • Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics
    PLOS Pathogens, 2015
    Co-Authors: Sunita Gulati, Ian C. Schoenhofen, Jacek Stupak, Evgeny Vinogradov, Frank St. Michael, Dennis M. Whitfield, Bo Zheng, Jianjun Li, Makoto Ohnishi
    Abstract:

    Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5’-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic Acid (Neu5Ac), a sialic Acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation (“serum-resistance”). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, Legionaminic Acid (Leg5Ac7Ac) and pseudaminic Acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP-Leg5Ac7Ac-treated mice were sensitive to human complement ex vivo, simulating in vitro findings. These data reveal critical roles for the Sia exocyclic side-chain in gonococcal serum-resistance. Such CMP-NulO analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea.

  • sialyltransferases with enhanced Legionaminic Acid transferase activity for the preparation of analogs of sialoglycoconjugates
    Glycobiology, 2015
    Co-Authors: David C Watson, Ian C. Schoenhofen, Warren W Wakarchuk, Sonia Leclerc, Martin N Young, Melissa J Schur, Michel Gilbert
    Abstract:

    Legionaminic Acids (Leg) are bacterial analogs of neuraminic Acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with Legionaminic Acid as a replacement for neuraminic Acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.

Dennis M. Whitfield - One of the best experts on this subject based on the ideXlab platform.

  • total biosynthesis of Legionaminic Acid a bacterial sialic Acid analogue
    Angewandte Chemie, 2016
    Co-Authors: Mohamed I Hassan, Ian C. Schoenhofen, Dennis M. Whitfield, Benjamin R Lundgren, Michael Chaumun, Brady Clark, Christopher N Boddy
    Abstract:

    Legionaminic Acid, Leg5,7Ac2, a nonulosonic Acid like 5-acetamido neuraminic Acid (Neu5Ac, sialic Acid), is found in cell surface glycoconjugates of bacteria including the pathogens Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. The presence of Leg5,7Ac2 has been correlated with virulence in humans by mechanisms that likely involve subversion of the host's immune system or interactions with host cell surfaces due to its similarity to Neu5Ac. Investigation into its role in bacterial physiology and pathogenicity is limited as there are no effective sources of it. Herein, we construct a de novo Leg5,7Ac2 biosynthetic pathway by combining multiple metabolic modules from three different microbial sources (Saccharomyces cerevisiae, C. jejuni, and L. pneumophila). Over-expression of this de novo pathway in Escherichia coli that has been engineered to lack two native catabolic pathways, enables significant quantities of Leg5,7Ac2 (≈120 mg L−1 of culture broth) to be produced. Pure Leg5,7Ac2 could be isolated and converted into CMP-activated sugar for biochemical applications and a phenyl thioglycoside for chemical synthesis applications. This first total biosynthesis provides an essential source of Leg5,7Ac2 enabling study of its role in prokaryotic and eukaryotic glycobiology.

  • RESEARCH ARTICLE Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide- Mediated Complement Resistance and Design Novel Therapeutics
    2016
    Co-Authors: Sunita Gulati, Ian C. Schoenhofen, Jacek Stupak, Frank St. Michael, Dennis M. Whitfield, Bo Zheng, Andrew D. Cox, Evgeny V. Vinogradov, Makoto Ohnishi
    Abstract:

    Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neo-tetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyl-transferase, using host cytidine-5’-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic Acid (Neu5Ac), a sialic Acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting fac-tor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation (“serum-resistance”). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, Legionaminic Acid (Leg5Ac7Ac) and pseudaminic Acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac an

  • Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics
    PLOS Pathogens, 2015
    Co-Authors: Sunita Gulati, Ian C. Schoenhofen, Jacek Stupak, Evgeny Vinogradov, Frank St. Michael, Dennis M. Whitfield, Bo Zheng, Jianjun Li, Makoto Ohnishi
    Abstract:

    Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5’-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic Acid (Neu5Ac), a sialic Acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation (“serum-resistance”). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, Legionaminic Acid (Leg5Ac7Ac) and pseudaminic Acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP-Leg5Ac7Ac-treated mice were sensitive to human complement ex vivo, simulating in vitro findings. These data reveal critical roles for the Sia exocyclic side-chain in gonococcal serum-resistance. Such CMP-NulO analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea.

  • the cmp Legionaminic Acid pathway in campylobacter biosynthesis involving novel gdp linked precursors
    Glycobiology, 2009
    Co-Authors: Ian C. Schoenhofen, Jean-robert Brisson, Evgeny Vinogradov, Dennis M. Whitfield, Susan M Logan
    Abstract:

    The sialic Acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic Acid, or legion-aminic Acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an alpha2,4-linked homopolymer of Legionaminic Acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of Legionaminic Acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of Legionaminic Acid, and since Legionaminic Acid and sialic Acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic Acid mimic.

  • Targeted metabolomics analysis of Campylobacter coli VC167 reveals Legionaminic Acid derivatives as novel flagellar glycans.
    Journal of Biological Chemistry, 2007
    Co-Authors: David J. Mcnally, Annie Aubry, Nam Huan Khieu, Cheryl P Ewing, Jean-robert Brisson, Patricia Guerry, Dennis M. Whitfield, Susan M Logan
    Abstract:

    Abstract Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic Acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic Acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of Legionaminic Acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as Legionaminic Acid biosynthetic enzymes. This is the first report of Legionaminic Acid in Campylobacter sp. and the first report of Legionaminic Acid derivatives as modifications on a protein.

Susan M Logan - One of the best experts on this subject based on the ideXlab platform.

  • the cmp Legionaminic Acid pathway in campylobacter biosynthesis involving novel gdp linked precursors
    Glycobiology, 2009
    Co-Authors: Ian C. Schoenhofen, Jean-robert Brisson, Evgeny Vinogradov, Dennis M. Whitfield, Susan M Logan
    Abstract:

    The sialic Acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic Acid, or legion-aminic Acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an alpha2,4-linked homopolymer of Legionaminic Acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of Legionaminic Acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of Legionaminic Acid, and since Legionaminic Acid and sialic Acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic Acid mimic.

  • Targeted metabolomics analysis of Campylobacter coli VC167 reveals Legionaminic Acid derivatives as novel flagellar glycans.
    Journal of Biological Chemistry, 2007
    Co-Authors: David J. Mcnally, Annie Aubry, Nam Huan Khieu, Cheryl P Ewing, Jean-robert Brisson, Patricia Guerry, Dennis M. Whitfield, Susan M Logan
    Abstract:

    Abstract Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic Acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic Acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of Legionaminic Acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as Legionaminic Acid biosynthetic enzymes. This is the first report of Legionaminic Acid in Campylobacter sp. and the first report of Legionaminic Acid derivatives as modifications on a protein.

R. M. Hall - One of the best experts on this subject based on the ideXlab platform.

  • 5,7-Di-N-acetyl-8-epiacinetaminic Acid: A new non-2-ulosonic Acid found in the K73 capsule produced by an Acinetobacter baumannii isolate from Singapore
    Scientific Reports, 2017
    Co-Authors: J. J. Kenyon, C. De Castro, Anna Notaro, R. M. Hall
    Abstract:

    Nonulosonic Acids are found in the surface polysaccharides of many bacterial species and are often implicated in pathogenesis. Here, the structure of a novel 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic Acid recovered from the capsular polysaccharide of a multiply antibiotic resistant Acinetobacter baumannii isolate was determined. The isolate carries a sugar synthesis module that differs by only a single gene from the module for the synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-L- glycero -L- altro -non-2-ulosonic Acid or 5,7-di- N -acetylacinetaminic Acid, recently discovered in the capsule of another A. baumannii isolate. The new monosaccharide is the C8-epimer of acinetaminic Acid (8eAci; 5,7-diacetamido-3,5,7,9-tetradeoxy-D- glycero -L- altro -non-2-ulosonic Acid) and the C7-epimer of Legionaminic Acid. This monosaccharide had not previously been detected in a biological sample but had been synthesized chemically.

  • 5,7-di-N-acetyl-acinetaminic Acid: A novel non-2-ulosonic Acid found in the capsule of an Acinetobacter baumannii isolate
    'Oxford University Press (OUP)', 2015
    Co-Authors: J. J. Kenyon, A. M. Marzaioli, C. De Castro, R. M. Hall
    Abstract:

    An Acinetobacter baumannii global clone 1 (GC1) isolate was found to carry a novel capsule biosynthesis gene cluster, designated KL12. KL12 contains genes predicted to be involved in the synthesis of simple sugars, as well as ones for N-acetyl-L-fucosamine (L-FucpNAc) and N-acetyl-D-fucosamine (D-FucpNAc). It also contains a module of 10 genes, 6 of which are required for 5,7-di-N-acetyl-Legionaminic Acid synthesis. Analysis of the composition of the capsule revealed the presence of N-acetyl-D-galactosamine, L-FucpNAc and D-FucpNAc, confirming the role of fnlABC and fnr/gdr genes in the synthesis of L-FucpNAc and D-FucpNAc, respectively. A non-2-ulosonic Acid, shown to be 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic Acid, was also detected. This sugar has not previously been recovered from biological source, and was designated 5,7-di-N-acetyl-acinetaminic Acid (Aci5Ac7Ac). Proteins encoded by novel genes, named aciABCD, were predicted to be involved in the conversion of 5,7-di-N-acetyl-Legionaminic Acid to Aci5Ac7Ac. A pathway for 5,7-di-N-acetyl-8-epiLegionaminic Acid biosynthesis was also proposed. In available A. baumannii genomes, genes for the synthesis of 5,7-di-N-acetyl-acinetaminic Acid were only detected in two closely related capsule gene clusters, KL12 and KL13, which differ only in the wzy gene. KL12 and KL13 are carried by isolates belonging to clinically important clonal groups, GC1, GC2 and ST25. Genes for the synthesis of N-acyl derivatives of Legionaminic Acid were also found in 10 further A. baumannii capsule gene clusters, and three carried additional genes for production of 5,7-di-N-acetyl- 8-epiLegionaminic Acid