Characterization of novel structural features in the lipopolysaccharide of nondisease associated nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a common commensal of the human upper respiratory tract and is associated with otitis media in children. The structures of the oligosaccharide portions of NTHi lipopolysaccharide (LPS) from several otitis media isolates are now well characterized but it is not known whether there are structural differences in LPS from colonizing, nondisease associated strains. Structural analysis of LPS from nondisease associated NTHi strains 11 and 16 has been achieved by the application of high-field NMR techniques, ESI-MS, ESI-MSn, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. This is the first study to report structural details on LPS from strains taken from the nasopharynx from healthy individuals. Both strains express identical structures and contain the common element of H. influenzae LPS, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-lipid A, in which each heptose is elongated by a single hexose residue with no further oligosaccharide extensions. In the major Hex3 glycoform, the terminal Hepp residue (HepIII) is substituted at the O-2 position by a beta-D-Galp residue and the central Hepp residue (HepII) is substituted at O-3 by a alpha-D-Glcp residue. Notably, the strains express two phosphocholine (PCho) substituents, one at the O-6 position of alpha-D-Glcp and the other at the O-6 position of beta-D-Galp. Major acetylation sites were identified at O-4 of Gal and O-3 of HepIII. Additionally, both strains express glycine, and strain 11 also expresses detectable amounts of N-acetylneuraminic acid.     

Structural analysis of the lipopolysaccharide from Neisseria meningitidis strain BZ157 galE: localisation of two phosphoethanolamine residues in the inner core oligosaccharide

The structure of the phase-variable lipopolysaccharide (LPS) from the group B Neisseria meningitidis strain BZ157 galE was elucidated. The structural basis for the LPS's variation in reactivity with a monoclonal antibody (MAb) B5 that has specificity for the presence of phosphoethanolamine (PEtn) at the 3-position of the distal heptose residue (HepII) was established. The structure of the O-deacylated LPS was deduced by a combination of monosaccharide analyses, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. These analyses revealed the presence of a novel inner core oligosaccharide (OS) structure in the MAb B5 reactive (B5+) LPS that contained two PEtn residues simultaneously substituting the 3- and 6-positions of the HepII residue. The determination of this structure has identified a further degree of variability within the inner core OS of meningococcal LPS that could contribute to the interaction of meningococcal strains with their host.     

Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide

We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease-associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an alpha-d-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material, as well as electrospray ionization-multiple-step tandem mass spectrometry on permethylated dephosphorylated oligosaccharide material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis-ESI-MS/MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same position.     

lpt6, a gene required for addition of phosphoethanolamine to inner-core lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae

We previously described a gene, lpt3, required for the addition of phosphoethanolamine (PEtn) at the 3 position on the beta-chain heptose (HepII) of the inner-core Neisseria meningitidis lipopolysaccharide (LPS), but it has long been recognized that the inner-core LPS of some strains possesses PEtn at the 6 position (PEtn-6) on HepII. We have now identified a gene, lpt6 (NMA0408), that is required for the addition of PEtn-6 on HepII. The lpt6 gene is located in a region previously identified as Lgt-3 and is associated with other LPS biosynthetic genes. We screened 113 strains, representing all serogroups and including disease and carriage strains, for the lpt3 and lpt6 genes and showed that 36% contained both genes, while 50% possessed lpt3 only and 12% possessed lpt6 only. The translated amino acid sequence of lpt6 has a homologue (72.5% similarity) in a product of the Haemophilus influenzae Rd genome sequence. Previous structural studies have shown that all H. influenzae strains investigated have PEtn-6 on HepII. Consistent with this, we found that, among 70 strains representing all capsular serotypes and nonencapsulated H. influenzae strains, the lpt6 homologue was invariably present. Structural analysis of LPS from H. influenzae and N. meningitidis strains where lpt6 had been insertionally inactivated revealed that PEtn-6 on HepII could not be detected. The translated amino acid sequences from the N. meningitidis and H. influenzae lpt6 genes have conserved residues across their lengths and are part of a family of proven or putative PEtn transferases present in a wide range of gram-negative bacteria.     

Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f. Structural diversity observed in three strains.

Structural elucidation of the lipopolysaccharide (LPS) from three serotype f Haemophilus influenzae clinical isolates RM6255, RM7290 and RM6252 has been achieved using NMR spectroscopy techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. This is the first study to report structural details on LPS from serotype f strains. We found that the LPSs of all strains were highly heterogeneous mixtures of glycoforms expressing the common H. influenzae structural element l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A with variable length of OS chains linked to each of the heptoses. The terminal heptose (HepIII) in RM6255 is substituted at the O-3 position by a beta-d-Glcp residue whereas HepIII in strains RM7290 and RM6252 is substituted at O-2 by the globoside unit (alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glc) or truncated versions thereof. The central heptose (HepII) is substituted by an alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp-(1-->4)-alpha-d-Glcp unit in RM7290 and RM6252 or truncated versions thereof. Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elongating from HepII (beta-d-Glcp-(1-->4)-alpha-d-Glcp). ESI-MSn on dephosphorylated and permethylated OS provided information on the existence of additional minor isomeric glycoforms.     

Characterization of the phosphocholine-substituted oligosaccharide in lipopolysaccharides of type b Haemophilus influenzae.

Haemophilus influenzae expresses heterogeneous populations of short-chain lipopolysaccharide (LPS) which exhibit extensive antigenic diversity among multiple oligosaccharide epitopes. These LPS oligosaccharide epitopes can carry phosphocholine (PCho) substituents, the expression of which is subject to high frequency phase variation mediated by genes in the lic1 genetic locus. The location and site of attachment of PCho substituents were determined by structural analysis of LPS from two type b H. influenzae strains, Eagan and RM7004. The lic2 locus is involved in phase variation of oligosaccharide expression. LPS obtained from the parent strains, from mutants generated by insertion of antibiotic resistance cassettes in the lic2 genetic locus, and from phase-variants showing high levels of PCho expression was characterized by electrospray ionization-mass spectrometry (ESI-MS) and 1H NMR spectroscopy of derived O-deacylated samples. ESI-MS of O-deacylated LPS from wild-type strains revealed mixtures of related glycoform structures differing in the number of hexose residues. Analysis of LPS from PCho-expressing phase-variants revealed similar mixtures of glycoforms, each containing a single PCho substituent. O-Deacylated LPS preparations from the lic2 mutants were much less complex than their respective parent strains, consisting only of Hex3 and/or Hex2 glycoforms, were examined in detail by high-field NMR techniques. It was found that the LPS samples contain the phosphoethanolamine (PEtn) substituted inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1--> 3)-L-alpha-D-He pp-(1-->5)-alpha-Kdo in which the major glycoforms carry a beta-D-Glcp or beta-D-Glcp-(1-->4)-beta-D-Glcp at the O-4 position of the 3-substituted heptose (HepI) and a beta-D-Galp at the O-2 position of the terminal heptose (HepIII). LPS from the lic2 mutants of both type b strains were found to carry PCho groups at the O-6 position of the terminal beta-D-Galp residue attached to HepIII. In the parent strains, the central heptose (HepII) of the LPS inner-core element is also substituted by hexose containing oligosaccharides. The expression of the galabiose epitope in LPS of H. influenzae type b strains has previously been linked to genes comprising the lic2 locus. The present study provides definitive evidence for the role of lic2 genes in initiating chain extension from HepII. From the analysis of core oligosaccharide samples, LPS from the lic2 mutant strain of RM7004 was also found to carry O-acetyl substituents. Mono-, di-, and tri-O-acetylated LPS oligosaccharides were identified. The major O-acetylated glycoforms were found to be substituted at the O-3 position of HepIII. A di-O-acetylated species was characterized which was also substituted at the O-6 postion of the terminal beta-D-Glc in the Hex3 glycoform. This is the first report pointing to the occurrence of O-acetyl groups in the inner-core region of H. influenzae LPS. We have previously shown that in H. influenzae strain Rd, a capsule-deficient type d strain, PCho groups are expressed in a different molecular environment, being attached at the O-6 position of a beta-D-Glcp, which is in turn attached to HepI.     

Neisserial lipooligosaccharide is a target for complement component C4b. Inner core phosphoethanolamine residues define C4b linkage specificity

We identified Neisseria meningitidis lipooligosaccharide (LOS) as an acceptor for complement component C4b (C4b). Phosphoethanolamine (PEA) residues on the second heptose (HepII) residue in the LOS core structure formed amide linkages with C4b. PEA at the 6-position of HepII (6-PEA) was more efficient than 3-PEA in binding C4b. Strains bearing 6-PEA bound more C4b than strains with 3-PEA and were more susceptible to complement-mediated killing in serum bactericidal assays. Deleting 3-PEA from a strain that expressed both 3- and 6-PEA simultaneously on HepII did not decrease C4b binding. Glycose chain extension of the first heptose residue (HepI) influenced the nature of the C4b-LOS linkage. Predominantly ester C4b-LOS bonds were seen when lacto-N-neotetraose formed the terminus of the glycose chain extension of HepI with 3-PEA on HepII in the LOS core. Related LOS species with more truncated chain extensions from HepI bound C4b via amide linkages to 3-PEA on HepII. However, 6-PEA in the LOS core bound C4b even when the glycose chain from HepI bore lacto-N-neotetraose at the terminus. The C4A isoform exclusively formed amide linkages, whereas C4B bound meningococci preferentially via ester linkages. These data may serve to explain the preponderance of 3-PEA-bearing meningococci among clinical isolates, because 6-PEA enhances C4b binding that may facilitate clearance of 6-PEA-bearing strains resulting from enhanced serum killing by the classical pathway of complement.     

Identification of a gene (lpt-3) required for the addition of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria meningitidis and its role in mediating susceptibility to bactericidal killing and opsonophagocytosis

We have identified a gene, lpt-3, that is required for the addition of phosphoethanolamine to the 3-position (PEtn-3) on the beta-chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn-3 substituent is characteristic of the LPS of a majority ( approximately 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface-accessible epitope. All strains of Nm that have PEtn-3 possess the lpt-3 gene. In some lpt-3-containing strains, the 3-position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt-3 resulted in loss of PEtn-3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt-3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a candidate immunogen for inclusion in an LPS-based vaccine to protect against invasive meningococcal disease.     

Asparagine 405 of heparin lyase II prevents the cleavage of glycosidic linkages proximate to a 3-O-sulfoglucosamine residue

Heparin and heparan sulfate contain a rare 3-O-sulfoglucosamine residue critical for anticoagulation and virus recognition, respectively. The glycosidic linkage proximate to this 3-O-sulfoglucosamine is resistant to cleavage by all heparin lyases (Heps). HepII has a broad specificity. The crystal structure of the wild type HepII identified its active site and showed a close spatial proximity between Asn405 and the 3-OH group of the bound glucosamine residue. In this study, we mutated Asn405 to the less sterically demanding Ala405 or Gly405, which broadened the substrate specificity of HepII and caused it to cleave the resistant linkage proximate to the 3-O-sulfoglucosamine residue.     

Effects of lipopolysaccharide biosynthesis mutations on K1 polysaccharide association with the Escherichia coli cell surface

The 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.     

Glycine is a common substituent of the inner core in Haemophilus influenzae lipopolysaccharide.

A survey of both typeable and nontypeable strains of Haemophilus influenzae indicated that they contain glycine (Gly) in their lipopolysaccharide (LPS). Significant amounts (30-250 pmol Gly/microg LPS) were determined by high-performance anion-exchange chromatography using pulsed amperometric detection after treatment of the LPS with mild alkali. Oligosaccharides obtained from LPS after mild acid hydrolysis and gel filtration chromatography were investigated by electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis (CE) ESI-MS. In all cases, molecular ions corresponding to the major glycoforms were identified and were accompanied by ions differing by 57 Da, thus indicating the presence of glycine. The position of glycine in these glycoforms was determined by CE-ESI-MS/MS analyses. It was found that, depending on strain, glycine can substitute each of the heptoses of the inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp-(1-->5)-alpha-Kdo of H. influenzae LPS as well as Kdo. In some strains, mixtures of monosubstituted Gly-containing glycoforms having different substitution patterns were identified.     

Inner core assembly and structure of the lipooligosaccharide of Neisseria meningitidis: capacity of strain NMB to express all known immunotype epitopes

Neisseria meningitidis expresses a heterogeneous populationof lipooligosaccharide (LOS) inner cores variously substitutedwith 1-3-linked glucose and O-3, O-6, and O-7 linked phosphoethanolamine(PEA), as well as glycine, attached to HepII. Combinations ofthese attachments to the LOS inner core represent immunodominantepitopes that are being exploited as future vaccine candidates.Historically, each LOS immunotype was structurally assessedand prescribed a certain unique inner core epitope. We reportthat a single isolate, strain NMB, possesses the capacity toproduce all of the known neisserial LOS inner core immunotypestructures. Analysis of the inner cores from parental LOS revealedthe presence or absence of 1,3-linked glucose, O-6 and/or O-7linked PEA, in addition to glycine attached at the 7 positionof the HepII inner core. Identification and inactivation oflpt-6 in strain NMB resulted in the loss of both O-6 and O-7linked PEA groups from the LOS inner core, suggesting that Lpt-6of strain NMB may have bifunctional transferase activities orthat the O-6 linked PEA groups once attached to the inner coreundergo nonenzymatic transfer to the O-7 position of HepII.Although O-3 linked PEA was not detected in parental LOS innercores devoid of 1-3-linked glucose residues, LOS glycoformsbearing O-3 PEA groups accumulated in a truncated mutant, NMBlgtK(Hep₂Kdo₂-lipid A). Because these structures disappeared uponinactivation of the lpt-3 locus, strain NMB expresses a functionalO-3 PEA transferase. The LOS glycoforms expressed by NMBlgtKwere also devoid of glycine attachments, indicating that glycinewas added to the inner core after the completion of the -chainby LgtK. In conclusion, strain NMB has the capability to expressall known inner core structures, but in in vitro culture L2and L4 immunotype structures are predominantly expressed.     

Structural investigation of lipopolysaccharides from nontypeable Haemophilus influenzae: investigation of inner-core phosphoethanolamine addition in NTHi strain 981

LPS of NTHi comprises a conserved tri-l-glycero-D-manno-heptosyl inner-core moiety (l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-alpha-Kdop) in which addition of PEtn to the central heptose (HepII) in strain Rd is controlled by the gene lpt6. It was recently shown that NTHi strain 981 contains an additional PEtn linked to O-3 of the terminal heptose of the inner-core moiety (HepIII). In order to establish whether lpt6 is also involved in adding PEtn to HepIII, lpt6 in strain 981 was inactivated. The structure of the LPS of the resulting mutant strain 98llpt6 was investigated by MS and NMR techniques by which it was confirmed that the lpt6 gene product is responsible for addition of PEtn to O-6 of HepII in strain 981. However, it is not responsible for adding PEtn to O-3 of HepIII since the 981lpt6 mutant still had full substitution with PEtn at HepIII.     

Identification of a novel inner-core oligosaccharide structure in Neisseria meningitidis lipopolysaccharide.

The structure of the lipopolysaccharide (LPS) from three Neisseria meningitidis strains was elucidated. These strains were nonreactive with mAbs that recognize common inner-core epitopes from meningococcal LPS. It is well established that the inner core of meningococcal LPS consists of a diheptosyl-N-acetylglucosamine unit, in which the distal heptose unit (Hep II) can carry PEtn at the 3 or 6 position or not at all, and the proximal heptose residue (Hep I) is substituted at the 4 position by a glucose residue. Additional substitution at the 3 position of Hep II with a glucose residue is also a common structural feature in some strains. The structures of the O-deacylated LPSs and core oligosaccharides of the three chosen strains were deduced by a combination of monosaccharide analysis, NMR spectroscopy and MS. These analyses revealed the presence of a structure not previously identified in meningococcal LPS, in which an additional beta-configured glucose residue was found to substitute Hep I at the 2 position. This provided the structural basis for the nonreactivity of LPS with these mAbs. The determination of this novel structural feature identified a further degree of variability within the inner-core oligosaccharide of meningococcal LPS which may contribute to the interaction of meningococcal strains with their host.     

A new structural type for Haemophilus influenzae lipopolysaccharide. Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486.

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha-D-Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.     

The occurrence of glycine in bacterial lipopolysaccharides

Abstract The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [¹⁴C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100°C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.     

Structural analysis of the lipopolysaccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa.

Lipopolysaccharide (LPS) expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis patients lacks the O-polysaccharide chain but the degree to which the rest of the molecule changes has not been determined. We analyzed, for the first time, the core structure of an LPS from a rough, cystic fibrosis isolate of P. aeruginosa. The products of mild acid hydrolysis and strong alkaline degradation of the LPS were studied by ESI MS, MALDI MS, and NMR spectroscopy. The following structure was determined for the highest-phosphorylated core-lipid A backbone oligosaccharide isolated after alkaline deacylation of the LPS: [structure: see text] where Kdo and Hep are 3-deoxy-D-manno-octulosonic acid and L-glycero-D-manno-heptose, respectively; all sugars are in the pyranose form and have the D configuration unless stated otherwise. The outer core region occurs as two isomeric glycoforms differing in the position of rhamnose (Rha). The inner core region carries four phosphorylation sites at two Hep residues, HepI being predominantly bisphosphorylated and HepII monophosphorylated. In the intact LPS, both Hep residues carry monophosphate and diphosphate groups in nonstoichiometric quantities, GalN is N-acylated by an L-alanyl group, HepII is 7-O-carbamoylated, and the outer core region is nonstoichiometrically O-acetylated at four sites. Therefore, the switch to the LPS-rough phenotype in cystic fibrosis isolates of P. aeruginosa is not accompanied by losses of core monosaccharide, phosphate or acyl components. The exact positions of the O-acetyl groups and the role of the previously undescribed O-acetylation in the LPS core of P. aeruginosa remain to be determined.     

Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4.

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough-type LPS. Additional faint high molecular-mass bands revealed that the O-chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel-permeation chromatography and high-performance anion-exchange liquid chromatography. The oligosaccharide contained two residues of L-glycero-D-manno-heptose (Hep) and one residue each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and GalNAc. Upon matrix-assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M-H]- at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one- and two-dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide: [equation: see text] Two unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide-linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.