|
Show legend Show as text |
Found 1 structure.
Displayed structure 1
| -8)-a-Neup5Ac-(2- | Show graphically |
|
Show legend Show as text |
Structure type: homopolymer
Trivial name: colominic acid sodium salt, colominic acid, type B polysaccharide, polysialic acid, PSA, oligosaccharide repeating unit, α-2,8-linked polysialic acid
Compound class: O-polysaccharide, K-antigen, O-antigen, CPS
Contained glycoepitopes: IEDB_136794,IEDB_141115,IEDB_142352,IEDB_146100,IEDB_149174,IEDB_150072,IEDB_150937,IEDB_153199,IEDB_226810,IEDB_558870,IEDB_983929,SB_170,SB_171,SB_172,SB_35,SB_42,SB_84
Colominic acid is a capsular homopolymer from Escherichia coli K1 composed of α(2-8)-linked N-acetyl-D-neuraminic acid (NeuAc) residues. Recently, we have described that NeuAc synthesis in this bacterium occurs through the action of NeuAc lyase (EC 4.1.3.3) [ Rodriguez-Aparicio, Ferrero and Reglero (1995) Biochem. J.308, 501-505]. In the present work we analysed and characterized this enzyme. E. coli K1 NeuAc lyase is detected from the early logarithmic phase of growth, is induced by NeuAc and is not repressed by glucose. The enzyme was purified to apparent homogeneity (312-fold) using two types of hydrophobic chromatographies (butyl-agarose and phenyl-Sepharose CL-4B), gel filtration on Sephacryl S-200, and anion-exchange chromatography on DEAE-FPLC. The pure enzyme, whose amino acid composition and N-terminal amino acid sequence are also established, has a native molecular mass, estimated by gel filtration, of 135 +/- 3 kDa, whereas its molecular mass in SDS/PAGE was 33 +/- 1 kDa. The enzyme was able to synthesize and cleave NeuAc in a reversible reaction. The maximal rate of catalysis was achieved in 125 mM Tris/HCl buffer, pH 7.8, at 37 degrees C. Under these conditions, the K(m) values calculated for N-acetyl-D-mannosamine and pyruvate (condensation direction), and NeuAc (hydrolysis direction) were 7.7, 8.3 and 4.8 mM respectively. NeuAc synthesis by the pure enzyme was activated by Ca2+ and inhibited by Mn2+ and NeuAc, whereas the enzyme cleavage direction was inhibited by Ca2+, Mn2+ and pyruvate. The reaction products, NeuAc and pyruvate, and Ca2+ are able to regulate the direction of this enzyme (synthesis or cleavage of sialic acid) and, accordingly, to modulate colominic acid biosynthesis.
biosynthesis, capsular, polysaccharide, Escherichia, Escherichia coli, acid, sialic acid, colominic acid, Escherichia coli K1, lyase
NCBI PubMed ID: 8694758No Abstract
colominic acid, chemical shift assignment, 2D NMR spectroscopy, polylactone
NCBI PubMed ID: 8839184Serum antibodies to Neisseria meningitidis group B (MenB) polysaccharide are reported not to elicit bacteriolysis in the presence of human complement. To reexamine this question, we evaluated the ability of two human IgM anti-MenB polysaccharide monoclonal antibodies (MAbs) and seven human MenB polysaccharide-reactive human IgM paraproteins to elicit bacteriolysis. In the presence of human complement, both MAbs and five of the seven paraproteins were bactericidal at antibody concentrations of 0.25-9.6 micrograms/mL (50% killing). Activity of the respective antibodies was enhanced 200- to > 10,000-fold when rabbit complement was used instead of human complement. With rabbit complement, the bactericidal activity of human IgM polyclonal antibody or MAb to Haemophilus influenzae type b (Hib) polysaccharide but not human IgG polyclonal antibody or MAb to Hib polysaccharide was similarly augmented. Thus, for both MenB and Hib, IgM antipolysaccharide antibodies elicit complement-mediated bactericidal activity in the presence of human complement, and the use of rabbit complement yields spuriously high activity.
Neisseria meningitidis, Neisseria, polysaccharide, capsular polysaccharide, antibody, serogroup B, bactericidal activity
NCBI PubMed ID: 7594665Lysophosphatidic acid (LPA) acyltransferases of Neisseria meningitidis and Neisseria gonorrhoeae were identified which share homology with other prokaryotic and eukaryotic LPA acyltransferases. In Escherichia coli, the conversion of LPA to phosphatidic acid, performed by the 1-acyl-sn-glycerol-3-phosphate acyltransferase PlsC, is a critical intermediate step in the biosynthesis of membrane glycerophospholipids. A Tn916-generated mutant of a serogroup B meningococcal strain was identified that exhibited increased amounts of capsular polysaccharide, as shown by colony immunoblots, and a threefold increase in the number of assembled pili. The single, truncated 3.8 kb Tn916 insertion in the meningococcal mutant was localized within a 771 bp open reading frame, The gonococcal equivalent of this gene was identified by transformation with the cloned meningococcal mutant gene. In N. gonorrhoeae, the mutation increased piliation fivefold. The insertions were found to be within a gene that was subsequently designated nlaA (neisserial LPA acyltransferase). The predicted neisserial LPA acyltransferases were homologous (>20% identity, >40% amino acid similarity) to the family of PlsC protein homologues. A cloned copy of the meningococcal nlaA gene complemented in trans a temperature-sensitive E. coli PlsCts- mutant. Tn916 and omega-cassette insertional inactivations of the neisserial nlaA genes altered the membrane glycerophospholipid compositions of both N. meningitidis and N. gonorrhoeae but were not lethal. Therefore, the pathogenic Neisseria spp. appear to be able to utilize alternative enzyme(s) to produce phosphatidic acid. This hypothesis is supported by the observation that, although the amounts of mature glycerophospholipids were altered in the meningococcal and the gonococcal nlaA mutants, glycerophospholipid synthesis was detectable at significant levels. In addition, acyltransferase enzymatic activity, while reduced in the gonococcal nlaA mutant, was increased in the meningococcal nlaA mutant. We postulate that the pathogenic Neisseria spp. are able to utilize alternate acyltransferases to produce glycerophospholipids in the absence of nlaA enzymatic activity. Implementation of these secondary enzymes results in alterations of glycerophospholipid composition that lead to pleiotropic effects on the cell surface components, including effects on capsule and piliation.
biosynthesis, Neisseria meningitidis, LOS, Neisseria, serogroup B, operon, polysialic acid, transcription, transport
NCBI PubMed ID: 8748025The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an α-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (∆neuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements ∆neuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.
biosynthesis, virulence, gene cluster, capsule, Escherichia coli K1
NCBI PubMed ID: 14729696The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with α(2-8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40 degrees C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP-Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43 degrees C (at least 25 times lower than those observed when the growth temperature was 37 degrees C). The synthesis of these enzymes increased rapidly when bacteria grown at 43 degrees C were transferred to 37 degrees C and decreased dramatically when cells grown at 37 degrees C were transferred to 43 degrees C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.
capsular polysaccharide, pyruvate, N-acetylneuraminic acid, polysialic acid biosynthesis, CTP, N-Acylneuraminate Cytidylyltransferase, CMP-Neu5Ac, Neu5Ac, Pasteurella haemolytica, polysialic acids
NCBI PubMed ID: 10094482To elucidate the role of secondary structure in the immune response against α(2→8)-linked polysialic acid, the capsular polysaccharide of Group B meningococci, we have investigated its solution dynamics by using specific models of molecular motion and hydrodynamic modeling to interpret experimental NMR data. (13)C-{(1)H} NMR relaxation times and steady-state NOE enhancements were measured for two aqueous solutions of α(2→8)-linked sialic acid polysaccharides. Each contained a unique distribution of polysaccharide chain lengths, with average lengths estimated at 40 or 400 residues. Models for rigid molecule tumbling, including two based on helical conformations proposed for the polysaccharide,(31) could not explain the NMR measurements. In general for these helices, the correlation times for their overall tumbling that best account for the NMR data correspond to polysaccharide chains between 9 and 18 residues in length, far short of the average lengths estimated for either solution. The effects of internal motions incorporated into these helices was modeled with an effective correlation time representing helix tumbling as well as internal motion. This modeling demonstrated that even with extreme amounts of internal motion, "flexible helices" of 25 residues or more still could not produce the NMR measurements. All data are consistent with internal and segmental motions dominating the nuclear magnetic relaxation of the polysaccharide and not molecular tumbling. Statistical distributions of correlation times have been found specifically for the pyranose rings, linkage groups, and methoxy groups that can account for the measured relaxation times and NOE enhancements. The distributions suggest that considerable flexibility attends the polysaccharide in solution, and the ranges of motional frequencies for the linkage groups and pyranose rings are comparable. We conclude that the Group B meningococcal polysaccharide is a random coil chain in solution, and therefore, does not have antigenic epitopes dependent upon a rigid, ordered conformation.
NMR, conformation, correlation, meningococcal, meningococci, capsular, polysaccharide, capsular polysaccharide, polysaccharides, group, molecular, molecule, conformational, dynamics, sialic acid, aqueous, solution, NOE, relaxation, polysialic acid, distribution, model, models, motion, modeling, enhancement, flexibility, helical, solutions
NCBI PubMed ID: 12617660We report esssentially complete 1H NMR assignments for the capsular polysaccharides from Neisseria meningitidis serotypes A, C, W-135, and Y. These polysaccharides are components of current polysaccharide vaccines against meningococcal infection and of the polysaccharide-protein conjugate vaccines under development. From these NMR data the pattern of O-acetylation was determined. O-Acetylation of the W-135 polysaccharide is reporded for the first time. We also show that, for the Types C and W-135 polysaccharides, a migration of O-acetyl groups occurs during storage in solution, and demonstrate that high field 1H NMR represents a simple and sensititve method to define the O-acetylation pattern of individual batches of these polysaccharides.
NMR, structure, Neisseria meningitidis, Neisseria, capsular, polysaccharide, capsular polysaccharide, capsular polysaccharides, polysaccharides, O-acetylation, production, vaccine, assignment, 1H NMR, pattern
Publication DOI: 10.1016/S0008-6215(96)00253-4In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. To study how outer membrane structure and biogenesis are affected by the absence of this normally major component, inner and outer membranes were separated and their composition analysed. The expression and assembly of integral outer membrane proteins appeared largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins was greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards phosphatidylethanolamine and phosphatidylglycerol species containing mostly shorter chain, saturated fatty acids, one of which was unique to the LPS-deficient outer membrane. The presence of the capsular polysaccharide turned out to be essential for viability without LPS, as demonstrated by using a strain in which LPS biosynthesis could be switched on or off through a tac promoter-controlled lpxA gene. Taken together, these results can help to explain why meningococci have the unique ability to survive without LPS
Lipopolysaccharide, biosynthesis, lipopolysaccharides, LPS, structure, Neisseria meningitidis, expression, gene, genetics, meningococci, metabolism, Neisseria, strain, capsular, polysaccharide, capsular polysaccharide, cell, chain, Research, acid, lipid, lipid A, protein, mutant, permeability, assembly, reduced, fatty acid, fatty acids, mutation, pathogen, composition, component, Base Sequence, vaccine, Cell Membrane, change, membrane, Membrane Lipids, Membrane Proteins, membranes, proteins, ability, species, outer membrane, phospholipid, acyltransferase, DNA Primers, inactivation, shift, absence, biogenesis, environment, iron, lipopolysaccharide-deficient, lipoprotein, lipoproteins, lpxA, outer membrane protein, outer membrane proteins, outer membranes, phosphatidylethanolamine, phospholipids, viable
NCBI PubMed ID: 11742971Polysialic acid (PSA) capsules are cell-associated homopolymers of α2,8-, α2,9-, or alternating α2,8/2,9-linked sialic acid residues that function as essential virulence factors in neuroinvasive diseases caused by certain strains of Escherichia coli and Neisseria meningitidis. PSA chains structurally identical to the bacterial α2,8-linked capsular polysaccharides are also synthesized by the mammalian central nervous system, where they regulate neuronal function in association with the neural cell adhesion molecule (NCAM). Despite the structural identity between bacterial and NCAM PSAs, the respective polysialyltransferases (polySTs) responsible for polymerizing sialyl residues from donor CMP-sialic acid are not homologous glycosyltransferases. To better define the mechanism of capsule biosynthesis, we established the functional interchangeability of bacterial polySTs by complementation of a polymerase-deficient E. coli K1 mutant with the polyST genes from groups B or C N. meningitidis and the control E. coli K92 polymerase gene. The biochemical and immunochemical results demonstrated that linkage specificity is dictated solely by the source of the polymerase structural gene. To determine the molecular basis for linkage specificity, we created chimeras of the K1 and K92 polySTs by overlap extension PCR. Exchanging the first 52 N-terminal amino acids of the K1 NeuS with the C terminus of the K92 homologue did not alter specificity of the resulting chimera, whereas exchanging the first 85 or reciprocally exchanging the first 100 residues did. These results demonstrated that linkage specificity is dependent on residues located between positions 53 and 85 from the N terminus. Site-directed mutagenesis of the K92 polyST N terminus indicated that no single residue alteration was sufficient to affect specificity, consistent with the proposed function of this domain in orienting the acceptor. The combined results provide the first evidence for residues critical to acceptor binding and elongation in polysialyltransferase.
Neisseria meningitidis, capsular polysaccharides, Escherichia coli, polysialic acid, sialyltransferases
NCBI PubMed ID: 12578835Neisseria meningitidis, Neisseria, capsular, group, Escherichia, Escherichia coli, evaluation, vaccines, vaccine, conjugate, conjugate vaccines, conjugate vaccine, polysaccharide-protein conjugate, polysaccharide-protein conjugate vaccine
Journal NLM ID: 0246127Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N-acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al., 1992). In this paper, we report the identification and characterization of cpsF, a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthase encoded by the Escherichia coli K1 gene, neuA. The enzymatic function of the protein encoded by cpsF was established by cloning the gene E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF, K1 antigen expression was restored. We concluded that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.
biosynthesis, Streptococcus, capsular polysaccharide, Escherichia coli, synthetase, polysaccharide biosynthesis
NCBI PubMed ID: 8830246The capsular polysaccharide of Neisseria meningitidis group B (MBPS) is a polymer of α (2→8) N-acetyl neuraminic acid. The polysaccharide is chemically identical to an autoantigen, polysialic acid (PSA), and is a poor immunogen, even when conjugated to protein carriers. Immunization of mice with MBPS-protein conjugate vaccines, in which N-acetyl groups have been replaced by propionyl groups (N-Pr MBPS), elicits serum bactericidal antibodies. A subpopulation of these antibodies do not cross-react with human PSA. The reasons for the increased immunogenicity of N-Pr MBPS and the antigenic targets of the bactericidal nonautoreactive antibodies are unknown. In this study, we investigated the antigenic targets of a protective murine monoclonal antibody (MAb) prepared against a N-Pr MBPS-tetanus toxoid conjugate vaccine. Binding of the MAb to N-Pr MBPS (as demonstrated by an enzyme-linked immunosorbent assay) and bactericidal activity were inhibited by de-N-acetylated MBPS and re-N-acetylated MBPS, which indicate that N-propionyl groups are not obligatory determinants for binding. The results of affinity selection from a preparation of N-Pr MBPS and matrix-assisted laser desorption ionization-time of flight mass spectroscopic analysis indicated that the minimal epitope recognized by the MAb is a MBPS disaccharide containing one de-N-acetylated residue. Thus, the bacterial capsular epitope recognized by this bactericidal, nonautoreactive, anti-group-B MAb likely contains de-N-acetyl residues.
Neisseria meningitidis, capsular polysaccharide, antibodies, epitope, monoclonal antibodies, monoclonal antibody, MAb, neuraminic acid, vaccines, conjugate, conjugate vaccine, toxoid, Epitope Mapping, Meningococcal Vaccines
NCBI PubMed ID: 15784554The capsule of N. meningitidis serogroup B, (α2→8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3' end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer
structure, Neisseria meningitidis, Bacterial Proteins, chemistry, disease, expression, gene, Bacterial, genetics, human, meningococcal, metabolism, Neisseria, role, strain, translocation, capsular, polysaccharide, analysis, capsular polysaccharide, polymer, Escherichia, Escherichia coli, acid, complex, level, protein, wild type, infectious disease, specific, spectrometry, mutant, mutants, serogroup, production, medicine, spectroscopy, surface, sequence, capsule, defined, Gram-negative, Infectious, mutation, intracellular, pathogen, pathogens, purified, mass fragmentography, relationship, location, CPS, serum, lipids, membrane, proteins, serogroup B, operon, killing, Polymers, human serum, polysialic acid, outer membrane, Bacterial Capsules, U.S.Gov't, control, phospholipid, Matrix-Assisted Laser Desorption-Ionization, glycerol, substitution, flow cytometry, Mass, sensitive, inactivation, P.H.S., accumulation, Blood Bactericidal Activity, Microscopy, capsules, electron microscopy, Humans, anchor, Research Support, Immunohistochemistry, Phosphatidic Acids
NCBI PubMed ID: 15731047Neisseria meningitidis is the only lipopolysaccharide (LPS)-producing gram-negative bacterial species shown to be viable also without LPS. It was thought that the presence of capsular polysaccharide is necessary for this unusual feature. However, we show now that no part of the capsule gene cluster is required for maintaining LPS deficiency in N. meningitidis.
Lipopolysaccharide, biosynthesis, Neisseria meningitidis, capsular polysaccharide, gene cluster, capsule, acyltransferases
NCBI PubMed ID: 16113348Polysialic acid (PSA) is a versatile biopolymer. PSA can be detected in a wide range of eukaryotes, including higher invertebrates and vertebrates, and also some prokaryotes, such as the neuroinvasive bacteria Neisseria meningitidis B. In mammals, PSA expression is developmentally regulated and plays significant roles in both neural development and in cancer. In bacteria, PSA is responsible for virulence. Spectroscopic methods, combined with immunological analysis, have played a significant role in understanding the relationship between PSA's structure and its biological functions. Chemical tools, including unnatural sialic acid analogs, have facilitated modulation of PSA's structure in cell culture and in vivo. The use of unnatural sialic acids has led to the development of vaccines for meningitis and cancer.
polysialic acid, glycoconjugate, neural cell adhesion molecule, conformational epitope, metabolic labelling
Publication DOI: 10.4052/tigg.16.305This chapter provides an overview of the molecular mechanisms involved in synthesis and expression of cell-surface polysaccharides in Gram-negative bacteria. Biosynthesis of many cell-surface components, including polysaccharides, involves enzymes and enzyme complexes found in the cytoplasmic membrane. The peptidoglycan layer is located immediately external to the cytoplasmic membrane and this layer is required for cell shape and rigidity. Gram-negative bacteria possess a periplasm that contains a variety of proteins and enzymes, including some involved in import and export of macromolecules. Biosynthesis of bacterial cell-surface polysaccharides involves a series of sequential processes: (1) biosynthesis of activated precursors in the cytoplasm, (2) formation of repeating units, (3) polymerization of repeating units, and (d) export of polysaccharides to the cell surface. The assembly of polysaccharide repeating units and subsequent polymerization reactions occur at the cytoplasmic membrane, using precursors synthesized in the cytoplasm. Genes for biosynthesis of cell-surface polysaccharides are chromosomal and are arranged in clusters of one or more transcriptional units. The synthesis of lipopolysaccharide (LPS) may be subject to complex regulation, but on-off switching is not possible due to the essential structural requirement for the lipid A-core LPS molecule. Most bacteria use extracellular polysaccharides (EPSs) for protection, and many regulatory strategies are directed to modulating EPS synthesis in response to appropriate environmental cues. Application of genetic and biochemical approaches has facilitated detailed analysis of complex, multicomponent systems, such as those involved in synthesis of cell-surface polysaccharides.
NCBI PubMed ID: 8310880disease, human, Salmon, polysialic acid, kidney, sialyltransferases, tumor, chicken, embryo, rat, heart, muscle, rainbow trout, egg, polysialoglycoprotein, polysialate, dolichol recognition sequences, E.coli K1 neural pathogenicity, neural cell adhesion molecules
Publication DOI: 10.1093/glycob/2.1.5We determined the heavy (H)- and light (L)-chain variable (V) region nucleotide and translated amino acid sequences of the human immunoglobulin M(kappa) monoclonal antibody (MAb) 5E1, which is specific for the polysaccharide capsule of Escherichia coli K1 and Neisseria meningitidis group B (poly[α(2→8)-N-acetylneuraminic acid]) and which is protective in animal models of infection. The 5E1 VH gene is a member of the VHIIIb family and is 97% homologous to the 9.1 germ line gene. The 5E1 VL gene is a member of the kappa I subgroup and is 98% homologous to the germ line gene, 15A, also known as KLO12. The VL and/or VH genes used by 5E1 are highly homologous to the V genes encoding antibodies to the Haemophilus influenzae type b polysaccharide and to antibodies reactive with self-antigens such as erythrocyte "i," DNA, and thyroid peroxidase. We also produced three murine anti-idiotype (Id) MAbs against 5E1. All three anti-Ids recognize a minor subset of antimeningococcal B polysaccharide antibodies present in serum from normal adults. Two of the anti-Ids define distinct Ids associated with antibodies having kappa I-15A V regions. These 15A-associated Ids are expressed by some heterologous human antimeningococcal B polysaccharide MAbs, and they also are independently expressed by two human MAbs that are specific for either the H. influenzae b polysaccharide or the i erythrocyte antigen and that utilize the kappa I-15A V region. Taken together, these data indicate that the 5E1 antibody uses V regions that recur in the human antibody repertoires to this polysaccharide and to structurally dissimilar polysaccharides and autoantigens. Thus, the poor immunogenicity of poly[α(2→8)-N-acetylneuraminic acid] cannot be explained by the unavailability of certain critical VH and VL genes required for generation of antibody response.
NCBI PubMed ID: 8168940The capsular polysaccharide of Neisseria meningitidis group B (MBPS) is a polymer of α(2→8)N-acetyl neuraminic acid, which is chemically identical to polysialic acid (PSA) expressed in human tissues. Antibodies from mice immunized with a MBPS-protein conjugate vaccine in which N-acetyl groups have been replaced by propionyl groups (N-Pr MBPS) can be bactericidal and show minimal or no cross-reactivity with human PSA. To investigate the molecular basis for antigen recognition, we cloned and sequenced the variable region (V) genes of five bactericidal anti-N-Pr MBPS murine mAbs and produced computer models of the combining sites. The results were compared to those reported in the literature for two autoreactive anti-MBPS. The V region genes of the anti-N-Pr MBPS mAbs and the anti-MBPS autoreactive mAbs are derived from a limited set of germline V, J, and D genes. However, the anti-N-Pr MBPS mAbs are more mutated than the anti-MBPS mAbs and the former use V-D-J editing that introduces arginine in H-CDR3. Models of the respective combining sites indicate that the anti-MBPS or anti-N-Pr MBPS mAbs that react with host PSA have relatively wide and shallow grooves with a high overall positive charge, consistent with recognition of extended helical polysaccharide structures recognized by the autoreactive mAbs. In contrast, anti-N-Pr MBPS mAbs that do not react with host PSA contain pockets and deep clefts that are consistent with recognition of discrete structural features of individual residues.
Autoantibodies, Autoantigens, Meningococcal vaccine, Structural models, V region genes
NCBI PubMed ID: 16140379Escherichia coli K1 is part of a reservoir of adherent, invasive facultative pathogens responsible for a wide range of human and animal disease including sepsis, meningitis, urinary tract infection and inflammatory bowel syndrome. A prominent virulence factor in these diseases is the polysialic acid capsular polysaccharide (K1 antigen), which is encoded by the kps/neu accretion domain inserted near pheV at 67 map units. Some E. coli K1 strains undergo form (phase) variation involving loss or gain of O-acetyl esters at carbon positions 7 or 9 of the individual sialic acid residues of the polysialic acid chains. Acetylation is catalysed by the receptor-modifying acetyl coenzyme-A-dependent O-acetyltransferase encoded by neuO, a phase variable locus mapping near the integrase gene of the K1-specific prophage, CUS-3, which is inserted in argW at 53.1 map units. As the first E. coli contingency locus shown to operate by a translational switch, further investigation of neuO should provide a better understanding of the invasive K1 pathotype. Minimal estimates of morbidity and economic costs associated with human infections caused by extraintestinal pathogenic E. coli strains such as K1 indicate at least 6.5 million cases with attendant medical costs exceeding $2.5 billion annually in the United States alone.
disease, gene, capsular polysaccharide, Escherichia coli K1, acetylation, polysialic acid, virulence factor, O-acetyl, meningitis
NCBI PubMed ID: 16677296The conformational properties of di- and trisaccharide fragments of the polysialic acid O-antigen capsular polysaccharide (CPS) of Neisseria meningitidis B (NmB) have been investigated by a combination of solution phase NMR spectroscopy and explicit-solvent molecular dynamics (MD) simulations. Simulations employing 100 ns of conventional MD, as well as 160 ns of replica exchange MD (REMD), with the GLYCAM06 force field were shown to be in agreement with experimental NMR scalar J-coupling and NOE values. The presence of conformational families has been determined by monitoring interglycosidic torsion angles, by comparing structural superimpositions, as well as via a Bayesian statistical analysis of the torsional data. Attempts to augment the immunogenicity of NmB CPS often involve chemical modifications of the N-acetyl moiety. Here the effects of these chemical group modifications on the conformational properties of the trisialoside have been probed via REMD simulations of the N-glycolyl, N-propionyl, N-propyl and N-butanoyl analogues. Although there were conformational families unique to each non-native analogue, the chemical modifications resulted in largely equivalent overall conformational phase-spaces compared to the native trisialoside. On the basis of the conformational distributions, these shared conformational properties suggest that a recurrent global conformational epitope may be present in both the native and chemically modified CPS fragments. Explanations are therefore provided for monoclonal antibody cross-reactivity, in terms of recognition of a shared global CPS conformation, as well as for lack of cross-reactivity, in terms of fine structural differences associated with the N-acyl groups, which may be dominant in highly matured antibody responses
NMR, conformation, Neisseria meningitidis, capsular polysaccharide, antibody response, vaccine
NCBI PubMed ID: 18954144The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis ofCMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis
biosynthesis, Escherichia coli, sialyltransferase, colominic acid, in vitro
NCBI PubMed ID: 2643517Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of α2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by α2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS286XGG), which is a hallmark of α/β-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to α/β-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an α/β-hydrolase fold topology, our data provide strong evidence that OatC belongs to the α/β-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.
biosynthesis, Neisseria meningitidis, Neisseria, capsular polysaccharide, capsule, polysialic acid, acyltransferase, meningitis
NCBI PubMed ID: 18986988Polysialic acids (PA) are protective capsular sialohomopolymers present in some bacteria which can invade the mammalian host and cause lethal bacteremia and meningitis. Biosynthesis and translocation of PA to the cell surface are equivalent in different species and bacterial strains which are produced. The diversity in PA structure is derived from the PA linkages and is a consequence of the specific sialyltransferase activities. The monomer acetylation and the polymer length could be important factors in the potential virulence. In vivo PA production is affected by different physical and chemical factors. The temperature of cellular growth strictly regulates PA genesis through a molecular complex and multifactorial mechanism that operate to transcription level.
Neisseria meningitidis, Escherichia coli, N-acetylneuraminic acid, polysialic acids
NCBI PubMed ID: 20349183The presence of cell-bound K1 capsule and K1 polysaccharide in culture supernatants was determined in a series of in-frame nonpolar core biosynthetic mutants from Escherichia coli KT1094 (K1, R1 core lipopolysaccharide [LPS] type) for which the major core oligosaccharide structures were determined. Cell-bound K1 capsule was absent from mutants devoid of phosphoryl modifications on L-glycero-D-manno-heptose residues (HepI and HepII) of the inner-core LPS and reduced in mutants devoid of phosphoryl modification on HepII or devoid of HepIII. In contrast, in all of the mutants, K1 polysaccharide was found in culture supernatants. These results were confirmed by using a mutant with a deletion spanning from the hldD to waaQ genes of the waa gene cluster to which individual genes were reintroduced. A nuclear magnetic resonance (NMR) analysis of core LPS from HepIII-deficient mutants showed an alteration in the pattern of phosphoryl modifications. A cell extract containing both K1 capsule polysaccharide and LPS obtained from an O-antigen-deficient mutant could be resolved into K1 polysaccharide and core LPS by column chromatography only when EDTA and deoxycholate (DOC) buffer were used. These results suggest that the K1 polysaccharide remains cell associated by ionically interacting with the phosphate-negative charges of the core LPS.
Lipopolysaccharide, biosynthesis, Escherichia coli
NCBI PubMed ID: 22522903Structural patterns of bacterial capsular antigens including capsular polysaccharides and exoglycans are given in this review. In addition, the immunological activity of capsular antigens and their role in type specificity of bacteria are discussed.
structure, capsular polysaccharides, bacterial capsular antigens, bacterial exoglycans, immunological activity, type specificity
NCBI PubMed ID: 17009947Monoclonal antibodies 13D9 and 6B9 are both specific for N-propionylated polysialic acid (NPrPSA); however, while 13D9 is protective against meningitis caused by group B meningococci and Escherichia coli capsular type K1 infection, 6B9 is not. The crystal structures of the Fabs from the two antibodies determined at 2.06 and 2.45 A resolutions, respectively, reveal markedly different combining sites, where only the surface of 13D9 is consistent with the recognition of extended helical epitopes known to exist in the capsular polysaccharides of etiological agents of meningitis. Interestingly, complementarity determining region H2 on 13D9 lies in a non-canonical conformation that docking studies show is a critical feature in the generation of negative free energy of binding. Finally, the model of extended NPrPSA decasaccharide bound to 13D9 derived from docking studies is consistent with saturation transfer difference nuclear magnetic resonance experiments. Together, these results provide further evidence that extended epitopes have the ability to break immune tolerance associated with the polysialic acid capsule of these pathogens.
antibody, crystal structure, polysialic acid, STD NMR
NCBI PubMed ID: 23704298Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.
biosynthesis, capsular polysaccharides, glycosyltransferases, gram negative bacteria, export, ABC transporters
NCBI PubMed ID: 23746650Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen-Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.
glycosyltransferases, protein structure, specificities, glycoprotein epitopes, glycan mimics
Publication DOI: 10.3389/fimmu.2014.00492Physicochemical techniques are a powerful tool for the structural characterization of carbohydrate-based vaccines. High-field Nuclear Magnetic Resonance (NMR) spectroscopy has been established as an extremely useful and robust method for tracking the industrial manufacturing process of these vaccines from polysaccharide bulk antigen through to the final formulation. Here, we describe the use of proton NMR for structural identity and conformity testing of carbohydrate-based vaccines.
carbohydrates, capsular polysaccharide, antigens, nuclear magnetic resonance spectroscopy, vaccines
NCBI PubMed ID: 26169742Neisseria meningitidis, a devastating pathogen exclusive to humans, expresses capsular polysaccharides that are the major meningococcal virulence determinants and the basis for successful meningococcal vaccines. With rare exceptions, the expression of capsule (serogroups A, B, C, W, X, Y) is required for systemic invasive meningococcal disease. Changes in capsule expression or structure (e.g. hypo- or hyper-encapsulation, capsule 'switching', acetylation) can influence immunologic diagnostic assays or lead to immune escape. The loss or down-regulation of capsule is also critical in meningococcal biology facilitating meningococcal attachment, microcolony formation and the carriage state at human mucosal surfaces. Encapsulated meningococci contain a cps locus with promoters located in an intergenic region between the biosynthesis and the conserved capsule transport operons. The cps intergenic region is transcriptionally regulated (and thus the amount of capsule expressed) by IS element insertion, by a two-component system, MisR/MisS and through sequence changes that result in post-transcriptional RNA thermoregulation. Reversible on-off phase variation of capsule expression is controlled by slipped strand mispairing of homo-polymeric tracts and by precise insertion and excision of IS elements (e.g. IS1301) in the biosynthesis operon. Capsule structure can be altered by phase-variable expression of capsular polymer modification enzymes or 'switched' through transformation and homologous recombination of different polymerases. Understanding the complex regulation of meningococcal capsule has important implications for meningococcal biology, pathogenesis, diagnostics, current and future vaccine development and vaccine strategies.
Neisseria meningitidis, capsular polysaccharide, Bacterial virulence regulation, capsule switching, meningococcal diseases
NCBI PubMed ID: 26089023The capsular polysaccharide is the main virulence factor in meningococcus. The capsular polysaccharides for meningococcal serogroups Y and W are almost identical polymers of hexose-sialic acid, suggesting the possibility of cross-protection between group Y and W vaccines. However, early studies indicated that they elicit different levels of cross-protection. Here we explore the conformations of the meningococcal Y and W polysaccharides with molecular dynamics simulations of three repeating unit oligosaccharide strands. We find differences in Y and W antigen conformation: the Y polysaccharide has a single dominant conformation, whereas W exhibits a family of conformations including the Y conformation. This result is supported by our NMR NOESY analysis, which indicates key close contacts for W that are not present in Y. These conformational differences provide an explanation for the different levels of cross-protection measured for the Y and W monovalent vaccines and the high group W responses observed in HibMenCY-TT vaccinees.
molecular modelling, Meningococcus, Neisseria meningitidis, capsular polysaccharide, carbohydrate antigens, cross-protection, molecular dynamics simulation
NCBI PubMed ID: 28501716Neisseria meningitidis is a major cause of bacterial meningitis worldwide especially in Africa. The capsular polysaccharide (CPS) is the main virulence factor and the target antigen for polysaccharide and conjugate vaccines. The high burden of serogroup A disease in the Meningitis Belt of sub-Saharan Africa led to the introduction of MenAfriVac®, which has successfully reduced the number of cases of group A disease. However, several outbreaks caused by other serogroups have been reported, including those due to serogroup X. The capsular polysaccharides of serogroups A and X are both homopolymers of amino sugars (?-D-ManNAc and ?-D-GlcNAc) containing phosphodiester linkages at C-6 and C-4, respectively. The similarity of the primary structures of the two polysaccharides suggests that serogroup A vaccination may provide cross-protection against serogroup X disease. Molecular dynamics simulations of a series of serogroup A and X oligosaccharides reveal that the MenA CPS behaves as a flexible random coil which becomes less conformationally defined as the length increases, whereas serogroup X forms a more stable regular helical structure. The presence of the MenX helix is supported by NMR analysis; it has four residues per turn and becomes more stable as the chain length increases. Licensed MenA vaccines are largely O-acetylated at C-3: simulations show that these O-acetyl groups are highly solvent exposed and their presence favors more extended conformations compared to the more compact conformations of MenA without O-acetylation. These findings may have implications for the design of optimal conjugate vaccines.
Neisseria meningitidis, capsular polysaccharide, molecular modeling, Antigen conformation
NCBI PubMed ID: 29940397Sialic acids are cytoprotectors, mainly localized on the surface of cell membranes with multiple and outstanding cell biological functions. The history of their structural analysis, occurrence, and functions is fascinating and described in this review. Reports from different researchers on apparently similar substances from a variety of biological materials led to the identification of a 9-carbon monosaccharide, which in 1957 was designated 'sialic acid.' The most frequently occurring member of the sialic acid family is N-acetylneuraminic acid, followed by N-glycolylneuraminic acid and O-acetylated derivatives, and up to now over about 80 neuraminic acid derivatives have been described. They appeared first in the animal kingdom, ranging from echinoderms up to higher animals, in many microorganisms, and are also expressed in insects, but are absent in higher plants. Sialic acids are masks and ligands and play as such dual roles in biology. Their involvement in immunology and tumor biology, as well as in hereditary diseases, cannot be underestimated. N-Glycolylneuraminic acid is very special, as this sugar cannot be expressed by humans, but is a xenoantigen with pathogenetic potential. Sialidases (neuraminidases), which liberate sialic acids from cellular compounds, had been known from very early on from studies with influenza viruses. Sialyltransferases, which are responsible for the sialylation of glycans and elongation of polysialic acids, are studied because of their significance in development and, for instance, in cancer. As more information about the functions in health and disease is acquired, the use of sialic acids in the treatment of diseases is also envisaged.
Biomolecular, sialic acids, History, Sialobiochemistry, Sialobiology, Sialochemistry
NCBI PubMed ID: 30509400Bacterial infections are the cause of different severe health conditions and new therapies to combat these pathogens have been widely investigated. Carbohydrates, being complex structures covering the surface of bacteria, are considered relevant targets for antibody and vaccine development. The biological activities in pathogenesis of bacterial capsular polysaccharides and lipopolisaccharides and their unique structures have boosted the study of the minimal antigenic binding epitopes and the structural details of antibody-carbohydrate recognition. This review describes the most recent advances on the field, examining the structure, conformation and dynamics of relevant bacterial carbohydrates and their complexes with antibodies. The understanding of key factors governing the recognition process is fundamental for the progress toward the development of specific and efficient bacterial therapeutics.
conformation, structure, capsular polysaccharides, epitope, recognition, dynamics, binding, vaccine, bacterial infection
NCBI PubMed ID: 33388123Bacterial extracellular polysaccharides are known as a cell-bound capsule, a sheath, or a slime, which is excreted into the environment. They play an important role in virulence of medical bacteria and plant-to-symbiont interaction and are used for serotyping of bacteria and production of vaccines. Some exopolysaccharides have commercial applications in industry, and claims of health benefits have been documented for an increasing number of them. Exopolysaccharides have diverse composition and structure, and some contain sugar and non-sugar components that are found in bacterial carbohydrates only. The present article provides an updated collection of the data on exopolysaccharides of various classes of gram-negative and gram-positive bacteria reported until the end of 2019. When known, biosynthesis pathways of exopolysaccharides are treated in a summary manner. References are made to structure and biosynthesis relatedness between exopolysaccharides of different bacterial taxa as well as between bacterial polysaccharides and mammalian glycosaminoglycans.
polysaccharide structure, Gram-negative bacteria, capsule, Biofilm, polysaccharide biosynthesis, gram-positive bacteria, Monosaccharide composition, Bacterial exopolysaccharide, non-sugar component
Publication DOI: 10.1016/B978-0-12-819475-1.00005-5Extracellular polysaccharides and glycoproteins of pathogenic bacteria assist in adherence, autoaggregation, biofilm formation, and host immune system evasion. As a result, considerable research in the field of glycobiology is dedicated to study the composition and function of glycans associated with virulence, as well as the enzymes involved in their biosynthesis with the aim to identify novel antibiotic targets. Especially, insights into the enzyme mechanism, substrate binding, and transition-state structures are valuable as a starting point for rational inhibitor design. An intriguing aspect of enzymes that generate or process polysaccharides and glycoproteins is the level of processivity. The existence of enzymatic processivity reflects the need for regulation of the final glycan/glycoprotein length and structure, depending on the role they perform. In this Review, we describe the currently reported examples of various processive enzymes involved in polymerization and transfer of sugar moieties, predominantly in bacterial pathogens, with a focus on the biochemical methods, to showcase the importance of studying processivity for understanding the mechanism.
processivity, Apparent processivity, Distributivity, Hyperglycosylation, Intrinsic processivity, Semiprocessive
NCBI PubMed ID: 31750644Vaccination is the most cost-effective way to control disease caused by encapsulated bacteria; the capsular polysaccharide (CPS) is the primary virulence factor and vaccine target. Neisseria meningitidis (Nm) serogroups B, C, Y and W all contain sialic acid, a common surface feature of human pathogens. Two protein-based vaccines against serogroup B infection are available for human use while four tetravalent conjugate vaccines including serogroups C, W and Y have been licensed. The tetravalent Menveo® conjugate vaccine is well-defined: a simple monomeric structure of oligosaccharides terminally conjugated to amino groups of the carrier protein CRM197. However, not only is there a surprisingly low limit for antigen chain attachment to CRM197, but different serogroup saccharides have consistently different CRM197 loading, the reasons for which are unclear. Understanding this phenomenon is important for the long-term goal of controlling conjugation to prepare conjugate vaccines of optimal immunogenicity. Here we use molecular modeling to explore whether antigen flexibility can explain the varying antigen loading of the conjugates. Because flexibility is difficult to separate from other structural factors, we focus on sialic-acid containing CPS present in current glycoconjugate vaccines: serogroups NmC, NmW and NmY. Our simulations reveal a correlation between Nm antigen flexibility (NmW > NmC > NmY) and the number of chains attached to CRM197, suggesting that increased flexibility enables accommodation of additional chains on the protein surface. Further, in silico models of the glycoconjugates confirm the relatively large hydrodynamic size of the saccharide chains and indicate steric constraints to further conjugation.
Neisseria meningitidis, capsular polysaccharide, molecular modeling, Antigen conformation, conjugation efficiency, glycoconjugate structure
NCBI PubMed ID: 33721150Glycosylation is a ubiquitous process that is universally conserved in nature. The various products of glycosylation, such as polysaccharides, glycoproteins, and glycolipids, perform a myriad of intra- and extracellular functions. The multitude of roles performed by these molecules is reflected in the significant diversity of glycan structures and linkages found in eukaryotes and prokaryotes. Importantly, glycosylation is highly relevant for the virulence of many bacterial pathogens. Various surface-associated glycoconjugates have been identified in bacteria that promote infectious behavior and survival in the host through motility, adhesion, molecular mimicry, and immune system manipulation. Interestingly, bacterial glycosylation systems that produce these virulence factors frequently feature rare monosaccharides and unusual glycosylation mechanisms. Owing to their marked difference from human glycosylation, bacterial glycosylation systems constitute promising antibacterial targets. With the rise of antibiotic resistance and depletion of the antibiotic pipeline, novel drug targets are urgently needed. Bacteria-specific glycosylation systems are especially promising for antivirulence therapies that do not eliminate a bacterial population, but rather alleviate its pathogenesis. In this review, we describe a selection of unique glycosylation systems in bacterial pathogens and their role in bacterial homeostasis and infection, with a focus on virulence factors. In addition, recent advances to inhibit the enzymes involved in these glycosylation systems and target the bacterial glycan structures directly will be highlighted. Together, this review provides an overview of the current status and promise for the future of using bacterial glycosylation to develop novel antibacterial strategies.
glycosylation, pathogenic bacteria, metabolic oligosaccharide engineering, antibacterial strategies, antivirulence
NCBI PubMed ID: 34630370Bacteria possess an extraordinary repertoire of cell envelope glycans that have critical physiological functions. Pathogenic bacteria have glycans that are essential for growth and virulence but are absent from humans, making them high-priority targets for antibiotic, vaccine, and diagnostic development. The advent of metabolic labeling with bioorthogonal chemical reporters and small-molecule fluorescent reporters has enabled the investigation and targeting of specific bacterial glycans in their native environments. These tools have opened the door to imaging glycan dynamics, assaying and inhibiting glycan biosynthesis, profiling glycoproteins and glycan-binding proteins, and targeting pathogens with diagnostic and therapeutic payload. These capabilities have been wielded in diverse commensal and pathogenic Gram-positive, Gram-negative, and mycobacterial species-including within live host organisms. Here, we review the development and applications of chemical reporters for bacterial glycans, including peptidoglycan, lipopolysaccharide, glycoproteins, teichoic acids, and capsular polysaccharides, as well as mycobacterial glycans, including trehalose glycolipids and arabinan-containing glycoconjugates. We cover in detail how bacteria-targeting chemical reporters are designed, synthesized, and evaluated, how they operate from a mechanistic standpoint, and how this information informs their judicious and innovative application. We also provide a perspective on the current state and future directions of the field, underscoring the need for interdisciplinary teams to create novel tools and extend existing tools to support fundamental and translational research on bacterial glycans.
biosynthesis, polysaccharides, glycoconjugates, glycan, Gram-negative bacteria, gram-positive bacteria
NCBI PubMed ID: 34905344Antimicrobial resistance (AMR) is emerging as the next potential pandemic. Different microorganisms, including the bacteria Acinetobacter baumannii, Clostridioides difficile, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, non-typhoidal Salmonella, and Staphylococcus aureus, and the fungus Candida auris, have been identified by the WHO and CDC as urgent or serious AMR threats. Others, such as group A and B Streptococci, are classified as concerning threats. Glycoconjugate vaccines have been demonstrated to be an efficacious and cost-effective measure to combat infections against Haemophilus influenzae, Neisseria meningitis, Streptococcus pneumoniae, and, more recently, Salmonella typhi. Recent times have seen enormous progress in methodologies for the assembly of complex glycans and glycoconjugates, with developments in synthetic, chemoenzymatic, and glycoengineering methodologies. This review analyzes the advancement of glycoconjugate vaccines based on synthetic carbohydrates to improve existing vaccines and identify novel candidates to combat AMR. Through this literature survey we built an overview of structure-immunogenicity relationships from available data and identify gaps and areas for further research to better exploit the peculiar role of carbohydrates as vaccine targets and create the next generation of synthetic carbohydrate-based vaccines.
carbohydrates, glycan, glycoconjugate vaccine
NCBI PubMed ID: 35608633Microbial polysaccharides are depolymerized within antigen-presenting cells by reactive nitrogen and reactive oxygen species with no obvious structure alterations. The exact chemical mechanisms of the in-cell processes vary with the structural features of the complex polymers. Free amine-containing carbohydrates are highly susceptible to action of reactive nitrogen species and acid sensitive linkages are prone to hydrolysis. The endosomal processing of glycoconjugates can modify the structure to a variable degree that correlates with ability to T-cell activation and immunogenicity of the antigen. The processed structures may direct innate and adaptive immune responses through modulation of polysaccharide-specific T cell response. During the in-cell processing of the glycoconjugate the immunodominant epitopes can be lost, leading to suboptimal antigenic properties. Consequently, the protective efficacy of the vaccine antigens can be altered. This may explain a differing effectiveness of vaccines, when the glycoconjugates are prepared from native or intact microbial glycans. The properties of both glycan and protein carrier should be considered in the design of optimal antigens, maximizing the glycopeptide specific T cell help and booster response. In this review, we highlight the importance of a prior knowledge of the carbohydrate structural features and present current understanding of the in-cell processing of glycoconjugates.
glycoconjugates, bacterial polysaccharides, vaccine, antigen processing, endosomes, reactive nitrogen species
NCBI PubMed ID: 37230635| New query | Export IDs | Home | Help |
Execution: 24 sec