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PubMed1987

Serological relationships between Escherichia coli and Salmonella smooth- and rough-mutant lipopolysaccharides as revealed by enzyme-linked immunosorbent assay for human immunoglobulin G antiendotoxin antibodies.

Reactions of sera from healthy blood donors to smooth and rough mutant lipopolysaccharides (LPS) from Escherichia coli O111:B4 and Salmonella minnesota which had been complexed with polymyxin B were determined by enzyme-linked immunosorbent assay. Relative enzyme-linked immunosorbent assay reactivities were examined for prima facie evidence of cross-reactivity by analyses of correlation and relative scatter. Patterns of reactivity were interpreted in relation to published structures. Evidence was obtained for two dominant epitopes associated with the lipid A-2-keto-3-deoxyoctulosonic acid and heptose regions of the LPS core. Sera demonstrated apparent exclusive antibody homogeneity in that given sera showed only one of the two possible specificities, which occurred with equal frequency. Cross-reactivity was found in the lipid A-2-keto-3-deoxyoctulosonic acid region for all LPS. Cross-reactivity was found in the heptose region for larger rough mutant S. minnesota LPS and smooth S. minnesota LPS and for E. coli O111:B4 LPS but not for E. coli rough mutant J5 LPS, whose heptose region appears to be different from those of the other organisms.

Antibodies, Bacterial↗

PubMed2002

The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.

All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep(3)-phosphorylated 3-deoxy-D-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, D-glycero-D-manno-heptose (DD-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked beta1-->4, to the DD-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DD-Hepalpha1-->6Glcbeta1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.

Acylation↗

PubMed2005

Identification of a D-glycero-D-manno-heptosyltransferase gene from Helicobacter pylori.

We have identified a Helicobacter pylori d-glycero-d-manno-heptosyltransferase gene, HP0479, which is involved in the biosynthesis of the outer core region of H. pylori lipopolysaccharide (LPS). Insertional inactivation of HP0479 resulted in formation of a truncated LPS molecule lacking an alpha-1,6-glucan-, dd-heptose-containing outer core region and O-chain polysaccharide. Detailed structural analysis of purified LPS from HP0479 mutants of strains SS1, 26695, O:3, and PJ1 by a combination of chemical and mass spectrometric methods showed that HP0479 likely encodes alpha-1,2-d-glycero-d-manno-heptosyltransferase, which adds a d-glycero-d-manno-heptose residue (DDHepII) to a distal dd-heptose of the core oligosaccharide backbone of H. pylori LPS. When the wild-type HP0479 gene was reintegrated into the chromosome of strain 26695 by using an "antibiotic cassette swapping" method, the complete LPS structure was restored. Introduction of the HP0479 mutation into the H. pylori mouse-colonizing Sydney (SS1) strain and the clinical isolate PJ1, which expresses dd-heptoglycan, resulted in the loss of colonization in a mouse model. This indicates that H. pylori expressing a deeply truncated LPS is unable to successfully colonize the murine stomach and provides evidence for a critical role of the outer core region of H. pylori LPS in colonization.

Animals↗

PubMed2003

A Salmonella typhimurium rfaE mutant recovers invasiveness for human epithelial cells when complemented by wild type rfaE (controlling biosynthesis of ADP-L-glycero-D-mannoheptose-containing lipopolysaccharide).

Invasion of host cells is essential for the pathogenicity of Salmonella. The author's group has recently reported the cloning of the rfaE gene of Salmonella typhimurium, previously implicated in biosynthesis of the lipopolysaccharide (LPS)-inner core [Jin et al. (2001); Kim (2002)]. The product of the rfaE gene is involved in ADP-L-glycero-D-manno-heptose biosynthesis. rfaE mutants synthesize heptose-deficient LPS (Re-LPS) consisting only of lipid A and 3-deoxy-D-manno-octulosonic acid (KDO). Mutants that make incomplete LPS are rough mutants and "deep-rough" mutants affected in the heptose region of the inner core have reduced growth rate and increased sensitivity to high temperature. Complementation of S. typhimurium rfaE mutant strain SL1102 (rfaE543) with rfaE demonstrated conclusively that this gene restored the smooth phenotype, and the LPS produced by the complemented strain was indistinguishable from that of wild type smooth strains. In vitro infection experiments showed that complementation with rfaE permitted invasion of human Chang epithelial cells, larynx epidermal carcinoma HEp-2 cells and intestinal epithelial Henle-407 cells. These data imply that the structure of the LPS that is synthesized is critical for Salmonella invasiveness.

Bacterial Proteins↗

PubMed1984

Lipopolysaccharides of the Heddleston serotypes of Pasteurella multocida.

Lipopolysaccharides (LPS) were extracted from 13 of the 16 Heddleston serotypes of Pasteurella multocida by phenol-chloroform-petroleum ether (PCP). Serotypes 3, 9, and 13 were extracted only by phenol-water (PW). After extraction of LPS of serotype 9 by PW, an additional LPS was isolated by PCP. All LPS contained glucose, 2-keto-3-deoxyoctonate, and heptose. Two isomers of heptose, D-glycero-D-mannoheptose and L-glycero-D-mannoheptose, were found in serotypes 2 and 5. Antisera made against purified LPS of serotypes 2 and 5 reacted with both heat-stable antigens and LPS from serotypes 2 and 5 in the gel-diffusion precipitin test. Antisera against serotype 2 LPS protected turkeys against challenge with capsulated serotype 5, indicating that a structural relationship exists between LPS of strains that cause hemorrhagic septicemia and fowl cholera. Rhamnose was a component of serotype 9 LPS, and galactose was found in all LPS, except for serotype 11. The LPS of serotype 13 contained an isomer of heptose that has not been identified. The LPS had buoyant densities in CsCl of 1.40 +/- 0.0148 g/ml, and all hemagglutinated chicken and turkey, but not sheep or horse, RBC.

Animals↗

PubMed1994

The structure of lipooligosaccharide produced by Neisseria gonorrhoeae, strain 15253, isolated from a patient with disseminated infection. Evidence for a new glycosylation pathway of the gonococcal lipooligosaccharide.

We studied the structure of the lipooligosaccharide (LOS) that is produced by Neisseria gonorrhoeae, strain 15253. This strain, recovered from a patient with disseminated infection, produces predominantly a single LOS component, and its oligosaccharide (OS) structure is different from those of previously studied LOSs. Definition of this OS structure provides additional information on the LOS biosynthesis. We determined that the 15253 OS has an unusual structure: 2 lactosyl residues at its nonreducing ends shown below, [formula: see text] where KDO is 2-keto-3-deoxy-mannooctulosonic acid and Hep is heptose. Comparison of this OS structure with those determined previously indicates the presence of a new glycosylation pathway for gonococcal OS biosynthesis: elongation of a GlcNAc-linked heptose, in contrast to elongation of the other heptose by sequential addition of glycoses which results in the antigenic similarity with human glycolipids. The current study provides not only additional structural information on LOS expressed during different clinical states of infection but also evidence for the diversity of gonococcal LOS biosynthesis. This evidence may be helpful in understanding the pathogenesis involving gonococcal LOS.

Acetylglucosamine↗

PubMed1990

G.l.c.-m.s. of partially methylated and acetylated derivatives of L-glycero-D-manno- and D-glycero-D-manno-heptopyranoses and -heptitols.

Methylated or acetylated heptopyranose derivatives were prepared variously from L-glycero-D-manno- and D-glycero-D-manno-heptopyranose, O-L- glycero-alpha-D-manno-heptopyranosyl-(1----3)-L-glycero-D-manno- heptopyranose, O-L-glycero-alpha-D-manno-heptopyranosyl-(1----7)-L-glycero- D-manno-heptopyranose, and O-L-glycero-alpha-D-manno- heptopyranosyl-(1----7)-O-L-glycero-alpha-D-manno- heptopyranosyl-(1----3)-L-glycero-D-manno- heptopyranose, which are structural elements of the heptose region of enterobacterial lipopolysaccharides. Each derivative was investigated by g.l.c. and g.l.c.-m.s., and the retention times and fragmentation patterns were used to identify partial structures of the heptose region of the core oligosaccharide of bacterial LPS.

Acetylation↗

PubMed1993

Structural studies of the saccharide part of the cell envelope lipopolysaccharide from Haemophilus influenzae strain AH1-3 (lic3+).

The structure of the saccharide part of the lipopolysaccharide from Haemophilus influenzae strain AH1-3 (lic3+) has been investigated. The saccharide was obtained from the lipopolysaccharide by mild acid hydrolysis followed by high-performance anion-exchange chromatography, and isolated fractions were studied by methylation analysis, NMR spectroscopy, and FAB mass spectrometry. The major saccharide is a heptasaccharide with the following structure, [formula: see text] in which Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid and PEA is 2-aminoethyl phosphate. Hep is identified as L-glycero-D-manno-heptose. The absolute configuration of the phosphorylated heptose is tentative only.

Carbohydrate Sequence↗

PubMed2004

A convenient synthesis of GDP D-glycero-alpha-D-manno-heptopyranose.

GDP D-glycero-alpha-D-manno-Heptopyranose has been prepared in good overall yield from 2,3,4,6,7-penta-O-acetyl-D-glycero-D-manno-heptopyranose by a short-step synthesis. Phosphitylation using the phosphoramidite procedure afforded the alpha-anomer in high selectivity. Subsequent oxidation and partial deprotection gave the acetylated phosphate derivative, which was subjected to the coupling reaction with GMP-morpholidate to furnish the acetylated heptose nucleoside diphosphate in good yield. De-O-acetylation and final purification afforded the target GDP D-glycero-alpha-D-manno-heptopyranose, which serves as the substrate of the heptosyl transferase in Aneurinibacillus thermoaerophilus DSM 10155 and occurs as an intermediate in the biosynthesis of GDP 6-deoxy-heptose in Yersinia pseudotuberculosis.

Acetylation↗

PubMed1976

Structural studies of the polysaccharide antigen of Eubacterium saburreum, strain 49.

The polysaccharide antigen produced by Eubacterium saburreum, strain L 49, is composed of D-glycero-D-galacto-heptose and a new sugar, tentatively identified as 6-deoxy-D-altro-heptose. It contains chains of alternating (1 leads to 3)- and (1 leads to 6)- linked beta-D-glycero-D-galacto-heptopyranosyl residues, the latter being substituted with 6-deoxy-alpha-heptofuranosyl groups at O-3. The polysaccharide further contains 0-acetyl groups, linked to O-7 of part of the heptosyl residues and to O-2 of part of the 6-deoxyheptosyl groups.

Chromatography, Gas↗

PubMed1971

Immunochemistry of the common antigen of Enterobacteriaceae (Kunin). Relation to lipopolysaccharide core structure.

Preparations of E. coli 014 lipopolysaccharide (LPS) contain a common enterobacterial antigen (CA) in large amounts or in an immunogenic form. Chemical analysis revealed, in addition to o-acetyl groups, only those sugars which are present in the basal core structure of the E. coli or Salmonella LPS (e.g., galactose, glucose, glucosamine, heptose, and ketodeoxyoctonate). On treatment with acetic acid (pH 3.2) at 100 degrees C for 1.5 hr, a fragment was liberated which after gel filtration on Sephadex G-50 appeared in the molecular weight range of 2-3 x 10(3). The fragment inhibited precipitation of alkali-treated E. coli 014 LPS by antibodies to CA from anti-E. coli 014 serum. It also inhibited hemagglutination between anti-CA antibodies and red cells coated with E. coli 08 LPS. Chemical analysis of the fragment indicated that it corresponded to the core region of E. coli 014 LPS. It contained a heptose and ketodeoxyoctonate in addition to glucose and galactose. However the fraction lacked glucosamine. Enterobacterial CA has previously been found to cross-react with colon antigen of ulcerative colitis. These results should provide a chemical basis for further studies of this cross-reactivity.

Acetates↗

PubMed1993

A quantitative microanalysis of bacterial endotoxin using [3H]-labeled L-glycero-D-mannoheptitol as a marker.

Quantitative microanalysis of bacterial endotoxin was performed using [3H]-labeled L-glycero-D-mannoheptitol (LD-Heptitol) as a marker. Several different amounts of authentic L-glycero-D-mannoheptose (LD-Heptose) were reduced with 20 micrograms of cold NaBH4 containing 2 micrograms of NaB3H4 (40 Ci/mmol) in 20 microliters of 1 mM NaOH at 4 C for 48 hr. The product, [1-3H]-labeled LD-Heptitol, has high specific activity, and was purified by HPLC and detected using a liquid-scintillation counter. As little as 50 pg of LD-Heptose was detectable, and the radioactivity increased dose-dependently in the 100 pg to 80 ng range tested. More than 2 ng of Salmonella abortus equi endotoxin could be accurately determined by this method. It is possible to detect 50 pg of endotoxin by this method, if 100% hot material (NaB3H4) is used for [3H]-labeling.

Chromatography, High Pressure Liquid↗

PubMed1992

Immunochemical and structural characterization of the antigenic polysaccharide from Eubacterium saburreum T18.

An antigenic surface polysaccharide produced by Eubacterium saburreum strain T18, isolated from human dental plaque, was purified from formamide extract of whole cells. Methylation analysis, Smith degradation, optical rotation data and nuclear magnetic resonance spectra demonstrated that the purified antigen was a homopolysaccharide composed of D-glycero-D-galacto-heptose (Hep.) residues. The structure of the repeating unit in the polysaccharide was: -[----6)-[alpha-Hep.furanosyl-(1----4)]-beta-Hep.pyranosyl- (1----6)-[alpha-Hep.furanosyl-(1----2), alpha-Hep.furanosyl-(1----4)]-beta- Hep.pyranosyl-(1-)4----6)-beta-Hep.pyranosyl-(1----. No heptose residues were acetylated. Immunodiffusion reactions in agar gel suggested that the immunodeterminant of the antigenic polysaccharide was D-glycero-D-galacto-heptofuranosyl residues as branched nonreducing terminals.

Antigens, Bacterial↗

PubMed1975

Cell-wall lipopolysaccharide from Escherichia coli B.

The lipopolysaccharide of Escherichia coli BB and a number of R-phage selected (e.g. T3, T4) cell-wall-defective mutants were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation, the oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. This revealed the sugar sequence in the hexose-heptose region of the core. The linkage of heptose (Hep) to 2-keto-3-deoxyoctonate (KDO) was established as ... Hep 1,5 leads to KDO ... by methylation analysis. The substituted derivative of KDO was identified by gas chromatography and mass spectrometry. The KDO region contains three KDO units. Its structure was elaborated by (a) selective removal and identification of 7-phosphoryl ethanolamine-KDO (KDO-PN), (b) periodate oxidation and thiobarbituric acid reaction in conjunction with mild hydrolysis, (c) a modified methylation analysis. Phosphate substitution of E. coli BB core was studied by beta-elimination and using the information obtained with KDO-PN. The structures of the cell wall lipopolysaccharides from E. coli BB and cell-wall-defective mutants are given.

Chromatography, Gas↗

PubMed1988

Structural studies on the core and lipid A region of a 4-amino-L-arabinose-lacking Rc-type mutant of Proteus mirabilis.

The structure of the 4-amino-L-arabinose-lacking lipopolysaccharide of the Proteus mirabilis Rc-type mutant R4, derived from wild-type O28, was elucidated. The lipopolysaccharide core structure has previously been partially characterized. The linkage between heptose and deoxyoctulosonic acid(dOclA) is now reported, as well as the structure of the lipid A moiety of this mutant strain. Besides the tentative identification of an alpha-linked glucosamine disaccharide in the lipid A backbone accompanying the usual beta 1----6-linked glucosamine-disaccharide, the only significant structural variation to previous studies was the lack of substitution of the C-4' phosphate by 4-amino-L-arabinose. In addition, the substitution at C-8 of one dOclA unit by 4-amino-L-arabinose, previously reported for the R45 mutant of P. mirabilis 1959, is lacking in this R mutant. Also in addition to previous findings, the terminal unit of heptose was found to be substituted at C-7 with phosphorylethanolamine (PEtN) and not only with phosphate, although this substitution is not complete as demonstrated by the relevant signals in 31P-NMR. Additional studies with the wild-type strain P. mirabilis O28 revealed the presence of 4-amino-L-arabinose in both the core and the lipid A regions suggesting that the R4 mutant is defective in the biosynthesis of this amino sugar rather than in its transfer. Otherwise the lipid A regions of the mutant and the wild-type strain show no structural differences. The following formula is proposed for the lipopolysaccharide of 4-amino-L-arabinose-lacking mutant R4/O28 P. mirabilis: (Formula; see text)

Electrophoresis, Polyacrylamide Gel↗

PubMed1991

Investigation of the structure of lipid A from Actinobacillus actinomycetemcomitans strain Y4 and human clinical isolate PO 1021-7.

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021-7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR-type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A. actinomycetemcomitans to consist of short sugar chains. Chemical analysis revealed the presence of thiobarbituric-acid-positive material (3-deoxy-D-manno-octulosonic acid equivalents) and four neutral sugars: glucose, galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose. Phosphate, glucosamine, glycine, and the fatty acids, 3-hydroxymyristic acid, myristic acid and palmitic acid, comprised the remainder of the molecule. The structure of the free lipid A revealed it to consist of a 1,6-glucosamine disaccharide esterified at C4' by a phosphomonoester. The hydroxyl group at C3 and the amide group of the non-reducing glucosamine were both acylated by 3-myristoylmyristic acid; analogous sites on the reducing glucosamine were acylated by 3-hydroxymyristic acid. Hydroxyl groups at C4 and C6' in the free lipid A were unsubstituted, with C6 being the proposed attachment site of the polysaccharide moiety. Chemical analysis revealed the presence of glycine in the intact LPS; its exact location in the A. actinomycetemcomitans LPS is still to be determined. Both intact LPS and free lipid A were highly lethal to galactosamine-sensitized mice, comparable to that of Salmonella.

Actinobacillus↗

PubMed1985

Composition and antigenic activity of the oligosaccharide moiety of Haemophilus influenzae type b lipooligosaccharide.

The oligosaccharide moiety of the lipooligosaccharide of Haemophilus influenzae type b strain Eag was isolated from the lipid component by mild acid hydrolysis and purified by gel filtration. Fast atom bombardment-mass spectrometry indicated that the lipid-free oligosaccharide had a basic molecular weight of 1,768; polysaccharides comparable to high-molecular-weight O side chains were not found. Glucose, galactose, galactosamine, heptose, 3-deoxy-D-manno-2-octulosonic acid (KDO), ethanolamine, and phosphate were identified in the lipid-free oligosaccharide by colorimetric assays, gas chromatography-mass spectrometry, or an amino acid analyzer. The presence of KDO was not clearly established by a thiobarbituric acid assay or by growth inhibition by a diazaborine derivative thought to block KDO synthesis. However, the semicarbizide assay and gas chromatography-mass spectrometry confirmed the presence of KDO. Lectin precipitation by Eag lipooligosaccharide in gels indicated that beta-D-galactose was present and that some of this monosaccharide was a terminal, nonreducing residue linked to N-acetyl-D-galactosamine. The lipid-free oligosaccharide was antigenic and completely inhibited lipooligosaccharide antibody (predominantly immunoglobulin G [IgG] and IgM) in an enzyme-linked immunosorbent assay, whereas the solubilized lipid A moiety did not. H. influenzae type b lipid-free oligosaccharide differed from core oligosaccharide of Salmonella lipooligosaccharide by the presence of galactosamine and a smaller percentage of heptose and KDO.

Ethanolamines↗

PubMed2002

Functional substitution of the TibC protein of enterotoxigenic Escherichia coli strains for the autotransporter adhesin heptosyltransferase of the AIDA system.

The plasmid-encoded AIDA (adhesin involved in diffuse adherence) autotransporter protein derived from diffuse-adhering clinical Escherichia coli isolate 2787 and the TibA (enterotoxigenic invasion locus B) protein encoded by the chromosomal tib locus of enterotoxigenic E. coli (ETEC) strain H10407 are posttranslationally modified by carbohydrate substituents. Analysis of the AIDA-I adhesin showed that the modification involved heptose residues. AIDA-I is modified by the heptosyltransferase activity of the product of the aah gene, which is located directly upstream of adhesin-encoding gene aidA. The carbohydrate modification of the TibA adhesin/invasin is mediated by the TibC protein but has not been elucidated. Based on the sequence similarities between TibC and AAH (autotransporter adhesin heptosyltransferase) and between the TibA and the AIDA proteins we hypothesized that the AIDA system and the Tib system encoded by the tib locus are structurally and functionally related. Here we show that (i) TibC proteins derived from different ETEC strains appear to be highly conserved, (ii) recombinant TibC proteins can substitute for the AAH heptosyltransferase in introducing the heptosyl modification to AIDA-I, (iii) this modification is functional in restoring the adhesive function of AIDA-I, (iv) a single amino acid substitution at position 358 completely abolishes this activity, and (v) antibodies directed at the functionally active AIDA-I recognize a protein resembling modified TibA in ETEC strains. In summary, we conclude that, like AAH, TibC represents an example of a novel class of heptosyltransferases specifically transferring heptose residues onto multiple sites of a protein backbone. A potential consensus sequence for the modification site is suggested.

Adhesins, Escherichia coli↗