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In this study, we investigated the utility of glycoconjugates based on a linear α-1,6-glucan chain synthesized using a recombinant α-1,6-glucosyltransferase from the 26695 strain of Helicobacter pylori. Capillary electrophoresis–mass spectrometry analysis confirmed the main product to contain 9–10 sequentially added α-1,6-linked glucose residues. This was consistent with a length of α-1,6-glucan structure present in the outer core region of H. pylori lipopolysaccharide (LPS) from strains 26695 and 26695 HP0826::Kan. The synthetic α-1,6-glucan was conjugated to either bovine serum albumin or tetanus toxoid and immunological properties of resultant glycoconjugates investigated. The conjugates were immunogenic in rabbits and mice and induced strong and specific IgG responses against purified LPS from typeable and nontypeable α-1,6-glucan-positive H. pylori strains. Furthermore, the post-immune sera from rabbits that received the conjugates were bactericidal and cross-reacted with selected clarithromycin-resistant and clarithromycin-susceptible clinical isolates of H. pylori. This technology offers a novel approach to the design of a synthetic carbohydrate-based vaccine against H. pylori. [ABSTRACT FROM AUTHOR]

Highlights: [•] We determined the LPS structures of non-typeable H. influenzae strain 1232. [•] The strain expresses d,d-Hep in the outer core of LPS in a novel manner. [•] The branching at d,d-Hep was indicated by MS4 experiments and confirmed by NMR. [•] d,d-Hep is substituted at both O-4 and O-7. [ABSTRACT FROM AUTHOR]

Abstract: A heterogeneous population of glycoforms expressed by NTHi strains 1158 and 1159 has been elucidated using NMR spectroscopy and capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) materials, as well as HPLC-ESI-MS n on dephosphorylated and methylated OS samples. The most abundant glycoform contained a disaccharide chain: PCho→7)-d-α-d-Hepp-(1→6)-β-d-Glcp linked to HepI from the common structural element of H. influenzae LPS: l-α-d-HepIIIp-(1→2)-[PEtn→6]-l-α-d-HepIIp-(1→3)-l-α-d-HepIp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. Phosphocholine (PCho) was found at two positions in the LPS glycoforms; PCho substituted the 6-position of β-d-Glcp attached to HepIII and was also located at a novel position linked to d-α-d-Hepp; this latter position was determined by structural analysis of LPS from a 1158lpsA mutant strain. Additionally, HPLC-ESI-MS n experiments indicated glycoforms that have chain elongation from HepII, this was found only in glycoforms, which lack the additional heptose in the outer core region. Structural details of these glycoforms were confirmed by analyses of LPS from a 1158losB2 mutant strain; the losB2 gene is required for addition of the d,d-Hep to the outer core region in strain 1158. [Copyright &y& Elsevier]

The outer core region of Helicobacter pylori lipopolysaccharide of the majority of isolates contains an α-1,6-glucan polymer synthesized by the product of the HP0159 ORF. Structural studies carried out on HP0159 lipopolysaccharide mutants by a combination of chemical methods, mass spectrometry and nuclear magnetic resonance spectroscopy confirmed that insertional inactivation of HP0159 gene in H. pylori strains 26695 and SS1 resulted in formation of a truncated lipopolysaccharide molecule characterized by the presence of a terminaldd-heptose residue in the side-chain outer core fragment and maintaining an inner core backbone structure compared with the wild-type Lewis antigen-expressing strains. Colonization studies with HP0159 mutants of two mouse-colonizing strains, SS1 and M6, confirmed their inability to successfully colonize the murine stomach. [ABSTRACT FROM AUTHOR]

Abstract: The lipid A components of Aeromonas salmonicida subsp. salmonicida from strains A449, 80204-1 and an in vivo rough isolate were isolated by mild acid hydrolysis of the lipopolysaccharide. Structural studies carried out by a combination of fatty acid, electrospray ionization-mass spectrometry and nuclear magnetic resonance analyses confirmed that the structure of lipid A was conserved among different isolates of A. salmonicida subsp. salmonicida. All analyzed strains contained three major lipid A molecules differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising 4′-monophosphorylated β-2-amino-2-deoxy-d-glucopyranose-(1→6)-2-amino-2-deoxy-d-glucopyranose disaccharide, where the reducing end 2-amino-2-deoxy-d-glucose was present primarily in the α-pyranose form. Electrospray ionization-tandem mass spectrometry fragment pattern analysis, including investigation of the inner-ring fragmentation, allowed the localization of fatty acyl residues on the disaccharide backbone of lipid A. The tetraacylated lipid A structure containing 3-(dodecanoyloxy)tetradecanoic acid at N-2′,3-hydroxytetradecanoic acid at N-2 and 3-hydroxytetradecanoic acid at O-3, respectively, was found. The pentaacyl lipid A molecule had a similar fatty acid distribution pattern and, additionally, carried 3-hydroxytetradecanoic acid at O-3′. In the hexaacylated lipid A structure, 3-hydroxytetradecanoic acid at O-3′ was esterified with a secondary 9-hexadecenoic acid. Interestingly, lipid A of the in vivo rough isolate contained predominantly tetra- and pentaacylated lipid A species suggesting that the presence of the hexaacyl lipid A was associated with the smooth-form lipopolysaccharide. [Copyright &y& Elsevier]

Abstract: The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: ▪ where R=α-d-Galp-(1→4)-β-d-GalpNAc-(1→ or α-d-Galp-(1→ (approx. ratio 4:3). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida. [Copyright &y& Elsevier]

Abstract: The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MS n on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-Lipid A, in which the β-d-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEtn at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by β-d-Galp-(1→4)-β-d-Glcp-(1→ or β-d-Glcp-(1→. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MS n in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS. [Copyright &y& Elsevier]

The structure of the carbohydrate backbone of the lipopolysaccharide from Pectinatus frisingensis strain VTT E-79104 was analyzed using chemical degradations, NMR spectroscopy, mass spectrometry, and chemical methods. The LPS contains two major structural variants, differing in the presence or absence of an octasaccharide fragment. The largest structure of the carbohydrate backbone of the LPS, that could be deduced from experimental results, consists of 20 monosaccharides arranged in a nonrepetitive sequence: where R is H or 4-O-Me-α-L-Fuc-(1-2)-4-O-Me-β-Hep-(1-3)-α-GlcNAc-(1-2)-β-Man-(1-3)-β-ManNAc-(1-4)-α-Gal-(1-4)-β-Hep-(1-3)-β-GalNAc-(1- where Hep is a residue of D-glycero-D-galacto-heptose; all monosaccharides have the D-configuration except for 4-O-Me-L-Fuc and L-Ara4N. This structure is architecturally similar to the oligosaccharide system reported previously in P. frisingensis VTT E-82164 LPS, but differs from the latter in composition and also in the size of the outer region. [Copyright &y& Elsevier]

The amino acid glycine is identified as a component of the inner core oligosaccharide in meningococcal lipopolysaccharide (LPS). Ester-linked glycine residues were consistently found by mass spectrometry experiments to be located on the distal heptose residue (HepII) in LPS from several strains of Neisseria meningitidis . Nuclear magnetic resonance studies confirmed and extended this observation locating the glycine residue at the 7-position of the HepII molecule in L3 and L4 immunotype strains. [ABSTRACT FROM AUTHOR]

Structural analysis of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae strain 1003 has been achieved by the application of high-field NMR techniques, ESI-MS, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS␣and core oligosaccharide material. It was found that␣the LPS contains the common structural element of␣H. influenzae , l-α-d-Hepp -(1→2)-[P Etn→6]-l-α-d-Hepp -(1→3)-[β-d-Glcp -(1→4)]-l-α-d-Hepp -(1→5)-[PP Etn→4]-α-Kdop -(2→6)-Lipid A, in which the β-d-Glcp residue is substituted by phosphocholine at O-6 and an acetyl group at O-4. A second acetyl group is located at O-3 of the distal heptose residue (HepIII). HepIII is chain elongated at O-2 by either a β-d-Glcp residue (major), lactose or sialyllactose (minor, i.e. α-Neu5Ac-(2→3)-β-d-Galp -(1→4)-β-d-Glcp ), where a third minor acetylation site was identified at the glucose residue. Disialylated species were also detected. In addition, a minor substitution of ester-linked glycine at HepIII and Kdo was observed. [ABSTRACT FROM AUTHOR]

Abstract: We describe a re-investigation of the structure of the lipopolysaccharide (LPS) from Helicobacter pylori genomic strain 26695 and its corresponding HP0826::Kan mutant lacking the O-chain component based on the in-depth NMR analysis of the oligosaccharide products obtained through the use of various degradation procedures performed on the purified LPS from both strains, as well as CE–MS data. New structural evidence indicates the presence of the linear arrangement of glucan and heptan portions of the LPS attached through -6-α-ddHep-3-α-l-Fuc-3-β-GlcNAc- fragment to the inner core dd-heptose residue. This structure differs from previously reported structures of the H. pylori 26695 LPS in several aspects. [Copyright &y& Elsevier]

Abstract: The structure of lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strains 1008 and 1247 has been investigated by mass spectrometry and NMR analyses on O-deacylated LPS and core oligosaccharide material. Both strains express the conserved triheptosyl inner core, [l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-Lipid A] with PCho→6)-β-d-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses the common structural motifs of H. influenzae; globotetraose [β-d-GalpNAc-(1→3)-α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and its truncated versions globoside [α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and lactose [β-d-Galp-(1→4)-β-d-Glcp-(1→] linked to the terminal heptose of the inner core and GlcI. A genetically distinct NTHi strain, 1008, expresses identical structures to strain 1247 with the exception that it lacks GalNAc. A lpsA mutant of strain 1247 expressed LPS of reduced complexity that facilitated unambiguous structural determination of the oligosaccharide from HepI. By CE-ESI-MS/MS we identified disialylated glycoforms indicating disialyllactose [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)-β-d-Gal-(1→4)-β-d-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS. [Copyright &y& Elsevier]

Abstract: We have investigated the structure of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 2019, a prototype strain that is used for studies of NTHi biology and disease. Analysis of LPS from wild type and lex2B, lpt3 and pgm mutant strains using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MS n on permethylated dephosphorylated OS, confirmed the previously established structure in which lactose is linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. Importantly, our data provide further structural detail whereby extensions from the middle heptose (HepII) are now characterized as β-d-Galp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp-(1→3 and truncated versions thereof. PEtn substitutes O-3 of the distal heptose (HepIII) of the inner-core moiety. This PEtn substituent was absent in the lpt3 mutant indicating that Lpt3 is the transferase required to add PEtn to the distal heptose. Interestingly, in the lex2B mutant strain HepIII was found to be substituted at O-2 by β-d-Glcp which, in turn, can be further extended. Contrary to previous findings, LPS of the pgm mutant strain contained minor glycoforms having β-d-Glcp linked to O-4 of HepI and also glycoforms with an additional PEtn which could be assigned to HepIII. Acetate groups and one glycine residue further substitute HepIII in NTHi 2019. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-l-glycero-d-manno-heptosyl inner-core moiety (l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-GlcIp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdop) to which addition of β-d-Glcp to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a β-d-Galp is linked to O-4 of GlcI. In order to test the hypothesis that the lex2 locus is involved in the expression of β-d-Galp-(1→4-β-d-Glcp-(1→ from HepI, lex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESIMS n on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only β-d-Glcp extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a β-d-Galp to O-4 of GlcI in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product. [Copyright &y& Elsevier]

Abstract: The O-chain polysaccharide produced by a mild acid degradation of Aeromonas caviae ATCC 15468 lipopolysaccharide was found to be composed of l-rhamnose, 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose and phosphoglycerol. Subsequent methylation and CE–ESIMS analyses and 1D/2D NMR (1H, 13C and 31P) spectroscopy showed that the O-chain polysaccharide is a high-molecular-mass acidic branched polymer of tetrasaccharide repeating units with a phosphoglycerol substituent having the following structure: Display Omitted where Gro represents glycerol and P represents a phosphate group. [Copyright &y& Elsevier]

Non-typeable Haemophilus influenzae (NTHi) is a significant cause of otitis media in children. We have employed single and multiple step electrospray ionization mass spectrometry (ESIMS) and NMR spectroscopy to profile and elucidate lipopolysaccharide (LPS) structural types expressed by NTHi strain 162, a strain obtained from an epidemiological study in Finland. ESIMS on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples of LPS provided information on the composition and relative abundance of glycoforms differing in the number of hexoses linked to the conserved inner-core element, l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-Lipid A of H. influenzae LPS. The strain examined was found to elaborate Hex2 to Hex5 LPS glycoform populations having structures identical to those observed for H. influenzae strain Rd [Risberg, A.; Masoud, H.; Martin, A.; Richards, J.C.; Moxon, E.R.; Schweda, E.K.H. Eur. J. Biochem. 1999, 261, 171–180], the strain for which the complete genome has been sequenced. In addition, sialyllactose-containing glycoforms previously identified in strain Rd as well as several NTHi strains, were identified as minor components. Multiple step tandem ESIMS (MSn) on dephosphorylated and permethylated OS provided information on the arrangement of glycoses within the major population of glycoforms and on the existence of additional isomeric glycoforms. Minor Hex1 and Hex6 glycoforms were detected and characterized where the Hex6 glycoform was comprised of a dihexosamine-containing pentasaccharide chain attached at the proximal heptose residue of the inner-core unit. LPS structural motifs present in the NTHi strain 162 are expressed by a genetically diverse set of disease causing isolates, providing the basis for a vaccine strategy against NTHi otitis media. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae involves genes from the lic2 locus that are required for chain extension from the middle heptose (HepII) of the conserved triheptosyl inner-core moiety. Lic2C initiates the process by attaching the first glucose to HepII, but the gene encoding for the enzyme adding the next β-d-Glcp- is uncharacterized. Lic2B is the candidate glucosyltransferase; however, in previous investigations, mutation of lic2B resulted in no hexose extension from HepII, likely due to a polar effect on the lic2C gene. In this study we complemented a lic2B knock-out mutant of H. influenzae strain Eagan with a functional lic2C gene and investigated its LPS by mass spectrometry and 2D NMR spectroscopy. Lic2B was found to encode a glucosyltransferase responsible for the linkage of β-d-Glcp-(1→4)-α-d-Glcp-(1→ extending from O-3 of the central heptose of the triheptosyl inner-core moiety, l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. [Copyright &y& Elsevier]

Abstract: The aim of the present investigation was to develop a novel approach for rapid identification and differentiation of Pseudomonas aeruginosa clones from Canadian cystic fibrosis patients by capillary electrophoresis-mass spectrometry. We screened P. aeruginosa isolates for lipopolysaccharide structure and presence/absence of alginate and correlated these findings with antibiotic resistance patterns. [Copyright &y& Elsevier]

Abstract: The cell envelope of Aeromonas salmonicida contains a lipopolysaccharide (LPS) essential for the physical integrity and functioning of bacterial cell membrane. Using a recently developed in-source fragmentation technique, we screened 39 typical and atypical isolates of A. salmonicida and established their O-chain polysaccharide structure by capillary electrophoresis–mass spectrometry (CE–MS), compositional and linkage analyses and comparison to the previously determined O-chain polysaccharide structure of A. salmonicida strain A449. These studies have demonstrated that A. salmonicida isolates fall into three distinct structural types, types A–C, based on chemical structures of their respective O-chain polysaccharide components. Subsequent immunoblotting and serological studies with salmon polyclonal antisera produced to formalin-fixed cells of A. salmonicida strains A449, N4705 and 33659 representing three structural types A–C revealed that variations in the O-chain polysaccharide structure have led to significant serological differences between strains belonging to type A and non-type A, where non-type A species include chemically separated structural types B and C. Due to the presence of common antigenic determinants shared by their respective O-chain polysaccharide components, serological cross-reactions were observed between A. salmonicida strains belonging to structural types B and C. These findings suggest the possibility of developing LPS-based classification system of A. salmonicida sub-species consisting of two serologically distinct types, type A and non-type A. [Copyright &y& Elsevier]

Abstract: The majority of hetero-polysaccharide biosynthesis in Gram-negative bacteria utilizes the wzy-dependent pathway, in which repeating O-units are first synthesized in the cytosol and then subsequently translocated to the periplasmic face of the inner membrane where polymerization is initiated by the Wzy polymerase. Wzy proteins share little primary sequence homology and are specific for their cognate O-unit structures. Our previous studies on O-polysaccharide biosynthesis in Escherichia coli O86 identified the wbnI gene, which encodes a galactosyltransferase responsible for the introduction of α-(1→3)-Galp residues as side chains of the polysaccharide. In this work, we functionally inactivated the wbnI gene and showed that the mutant strain produced a different polysaccharide without the side chain Galp residue. The yield of the polysaccharide was substantially lower than the one produced by the wild-type strain. This study indicated that the complete O-unit structure is the preferred substrate for the polymerization, thus further confirming the specificity of Wzy. On the other hand, these studies also suggest that the Wzy polymerase might have moderate tolerance of side-chain truncated O-unit substrates. [Copyright &y& Elsevier]

Abstract: The O-chain polysaccharide (O-PS) of Aeromonas salmonicida was studied by a combination of compositional, methylation, CE-ESMS and one- and two-dimensional NMR analyses. It was found to be a branched polymer of trisaccharide-repeating units composed of l-rhamnose (Rha), d-glucose (Glc), 2-acetamido-2-deoxy-d-mannose (ManNAc) and O-acetyl group (OAc) and having the following structure: CE-ESMS analysis of A. salmonicida cells from strains A449, 80204 and 80204-1 grown under different conditions confirmed that the O-PS structure was conserved. ELISA-based serological study with native LPS-specific antisera performed on the native O-PS and its O-deacetylated and periodate-oxidized derivatives confirmed the importance of the O-PS backbone structure as an immunodominant determinant. [Copyright &y& Elsevier]