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.     

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.     

Damage-repair error-prone polymerases of eubacteria: association with mobile genome elements

Lipopolysaccharide (LPS) is important for the virulence of Neisseria meningitidis, and is the target of immune responses. We took advantage of a monoclonal antibody (Mab B5) that recognises phosphoethanolamine (PEtn) attached to the inner core of meningococcal LPS to identify genes required for the addition of PEtn to LPS. Insertional mutants that lost Mab B5 reactivity were isolated and characterised, but failed to yield genes directly responsible for PEtn substitution. Subsequent genetic linkage analysis was used to define a region of DNA containing a single intact open reading frame which is sufficient to confer B5 reactivity to a B5 negative meningococcal isolate. The results provide an initial characterisation of the genetic basis of a key, immunodominant epitope of meningococcal LPS.     

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 and localization of glycine in the inner core lipopolysaccharide of Neisseria meningitidis.

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.     

Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation

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-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural 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-Kdo-(2-->6)-Lipid A, in which the beta-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 beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> or beta-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-MSn 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.     

Evidence that WapB is a 1,2-glucosyltransferase of Pseudomonas aeruginosa involved in Lipopolysaccharide outer core biosynthesis

Pseudomonas aeruginosa is an important opportunistic pathogen infecting debilitated individuals. One of the major virulence factors expressed by P. aeruginosa is lipopolysaccharide (LPS), which is composed of lipid A, core oligosaccharide (OS), and O-antigen polysaccharide. The core OS is divided into inner and outer regions. Although the structure of the outer core OS has been elucidated, the functions and mechanisms of the glycosyltransferases involved in core OS biogenesis are currently unknown. Here, we show that a previously uncharacterized gene, pa1014, is involved in outer core biosynthesis, and we propose to rename this gene wapB. We constructed a chromosomal mutant, wapB::Gm, in a PAO1 (O5 serotype) strain background. Characterization of the LPS from the mutant by Western immunoblotting showed a lack of reactivity to PAO1 outer core-specific monoclonal antibody (MAb) 5c-101. The chemical structure of the core OS of the wapB mutant was elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry techniques and revealed that the core OS of the wapB mutant lacked the terminal β-1,2-linked-d-glucose residue. Complementation of the mutant with wapB in trans restored the core structure to one that is identical to that of the wild type. Eleven of the 20 P. aeruginosa International Antigenic Typing Scheme (IATS) serotypes produce LPSs that lack the terminal d-glucose residue (Glc(IV)). Interestingly, expressing wapB in each of these 11 serotypes modifies each of their outer core OS structures, which became reactive to MAb 5c-101 in Western immunoblotting, suggesting the presence of a terminal d-glucose in these core OS structures. Our results strongly suggested that wapB encodes a 1,2-glucosyltransferase.     

Structural analysis of the lipooligosaccharide-derived oligosaccharide of Histophilus somni (Haemophilus somnus) strain 8025

Previous structural studies in our laboratory on lipooligosaccharide (LOS) inner core oligosaccharide (OS) had identified structures from several strains of Histophilus (Haemophilus) somni (738, 2336, 1P, 129Pt). Recently a type strain 8025 was proposed for this species and we therefore sought to determine the core OS structure of this H. somni strain. Core OS was isolated by standard methods from Westphal purified LOS. Structural information was established by a combination of monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the core OS was determined on the basis of the combined data from these experiments: [carbohydrates: see text]. The structure determined contains aspects of other Histophilus somni core OS structures, such as the beta-Gal attached at the 2-position of Hep II (2336), PEtn only at the 6-position of Hep II (738, 129Pt) and a lactose extension from Hep I (1P). Since genetic manipulation has been achieved with this strain, the identification of the core OS structure will enable experiments designed to identify the role of glycosyltransferases involved in LOS biosynthesis.     

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.     

Genetic and Structural Characterization of L11 Lipooligosaccharide from Neisseria meningitidis Serogroup A Strains

The lipooligosaccharide (LOS) of immunotype L11 is unique within serogroup A meningococci. In order to resolve its molecular structure, we conducted LOS genotyping by PCR analysis of genes responsible for α-chain sugar addition (lgtA, -B, -C, -E, -H, and -F) and inner core substituents (lgtG, lpt-3, and lpt-6). For this study, we selected seven strains belonging to subgroup III, a major clonal complex responsible for meningococcal meningitis epidemics in Africa. In addition, we sequenced the homopolymeric tract regions of three phase-variable genes (lgtA, lgtG, and lot-3) to predict gene functionality. The fine structure of the L11 LOS of each strain was determined using composition and glycosyl linkage analyses, NMR, and mass spectrometry. The masses of the dephosphorylated oligosaccharides were consistent with an oligosaccharide composed of two hexoses, one N-acetyl-hexosamine, two heptoses, and one KDO, as proposed previously. The molar composition of LOS showed two glucose residues to be present, in agreement with lgtH sequence prediction. Despite phosphoethanolaminetransferase genes lpt-3 and lpt-6 being present in all seven Neisseria meningitidis strains, phosphoethanolamine (PEtn) was found at both O-3 and O-6 of HepII among the three ST-5 strains, whereas among the four ST-7 strains, only one PEtn was found and located at O-3 of the HepII. The L11 LOS was found to be O-acetylated, as was indicated by the presence of the lot-3 gene being in-frame in all of the seven N. meningitidis strains. To our knowledge, these studies represent the first full genetic and structural characterization of the L11 LOS of N. meningitidis. These investigations also suggest the presence of further regulatory mechanisms affecting LOS structure microheterogeneity in N. meningitidis related to PEtn decoration of the inner core.     

Structural characterization of a novel branching pattern in the lipopolysaccharide from nontypeable Haemophilus influenzae.

Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as by ESI-MSn on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcxp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-d-Glcp residue was either substituted by PCho or the disaccharide branch beta-d-Galp-(1-->4)-d-alpha-d-Hepp, while the O-4 position was substituted by the globotetraose unit, beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp, or sequentially truncated versions thereof. This is the first time a branching sugar residue has been reported in the outer-core region of H. influenzae LPS. Additionally, a PEtn group was identified at O-3 of the distal heptose residue in the inner-core.     

O-Acetylation of the terminal N-acetylglucosamine of the lipooligosaccharide inner core in Neisseria meningitidis. Influence on inner core structure and assembly

O-Acetylation is a common decoration on endotoxins derived from many Gram-negative bacterial species, and it has been shown to be instrumental (e.g. in Salmonella typhimurium) in determining the final tertiary structure of the endotoxin and the immunogenicity of the molecule. Structural heterogeneity of endotoxins produced by mucosal pathogens such as Neisseria meningitidis is determined by decorations on the heptose inner core, including O-acetylation of the terminal N-acetylglucosamine (GlcNAc) attached to HepII. In this report, we show that O-acetylation of the meningococcal lipooligosaccharide (LOS) inner core has an important role in determining inner core assembly and immunotype expression. The gene encoding the LOS O-acetyltransferase, lot3, was identified by homology to NodX from Rhizobium leguminosarum. Inactivation of lot3 in strain NMB resulted in the loss of the O-acetyl group located at the C-3 position of the terminal GlcNAc of the LOS inner core. Inactivation of either lot3 or lgtG, which encodes the HepII glucosyltransferase, did not result in the appearance of the O-3-linked phosphoethanolamine (PEA) groups on the LOS inner core. Construction of a double mutant in which both lot3 and lgtG were inactivated resulted in the appearance of O-3-linked PEA groups on the LOS inner core. In conclusion, O-acetylation status of the terminal GlcNAc of the gamma-chain of the meningococcal LOS inner core is an important determinant for the appearance or exclusion of the O-3-linked PEA group on the LOS inner core and contributes to LOS structural diversity. O-Acetylation also likely influences resistance to complement-mediated lysis and may be important in LOS conjugate vaccine design.     

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: chemical strategies to prepare glycoconjugates with good carbohydrate loading

In previous studies protective antibodies that could facilitate bactericidal killing of Neisseria meningitidis (Nm) serogroup B strains were derived from immunisation with glycoconjugates prepared from O-deacylated lipopolysaccharide (LPS-OH) via direct reductive amination between the reducing end of the oligosaccharide molecule, created by treatment with alkaline phosphatase, and amino functionalities on the CRM₁₉₇ carrier protein. These glycoconjugates proved difficult to prepare because the presence of amide linked fatty-acyl groups results in glycolipids that are relatively insoluble and aggregate. Therefore, we have examined several strategies to prepare glycoconjugates in order to identify a robust, consistently reproducible strategy that produces glycoconjugates with a high loading of LPS derived oligosaccharides. Initially we used completely deacylated LPS molecules, but lacking phosphoethanolamine (PEtn) from the core OS as the strong basic conditions required to completely deacylate the LPS would modify the PEtn residue. We utilised a squarate linker and conjugated via the reducing end of the carbohydrate antigen following removal of the glycosidic phosphate to amino groups on CRM₁₉₇, however carbohydrate loading on the carrier protein was low. Glycoconjugates were then produced utilising amidases produced by Dictyostelium discoideum (Dd), which partially remove N-linked fatty acids from the lipid A region of the Nm LPS molecule, which enabled the retention of the PEtn residue. LPS-OH was treated with Dd amidase, the reducing glycosidic phosphate removed, and using a cystamine linker strategy, conjugated to the carrier protein. Carbohydrate loading was somewhat improved but still not high. Finally, we have developed a novel conjugation strategy that targets the amino functionality created by the amidase activity as the attachment point. The amino functionality on the PEtn residue of the inner core was protected via a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy, targeting lysine residues on the carrier protein did not result in high loading of the carbohydrate molecules, however when we targeted the carboxyl residues we have consistently obtained a high loading of carbohydrate antigens per CRM₁₉₇, which can be controlled by variation in the amount of activated carbohydrate utilised in the conjugation reaction.     

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.     

Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae.

A sialylated lacto-N-neotetraose (Sial-lNnT) structural unit was identified and structurally characterized in the lipopolysaccharide (LPS) from the genome-sequenced strain Rd [corrected] (RM118) of the human pathogen Haemophilus influenzae grown in the presence of sialic acid. A combination of molecular genetics, MS and NMR spectroscopy techniques showed that this structural unit extended from the proximal heptose residue of the inner core region of the LPS molecule. The structure of the Sial-lNnT unit was identical to that found in meningococcal LPS, but glycoforms containing truncations of the Sial-lNnT unit, comprising fewer residues than the complete oligosaccharide component, were not detected. The finding of sialylated glycoforms that were either fully extended or absent suggests a novel biosynthetic feature for adding the terminal tetrasaccharide unit of the Sial-lNnT to the glycose acceptor at the proximal inner core heptose.     

Structure and immunochemistry of meningococcal lipopolysaccharides

The structures of the largest dephosphorylated oligosaccharides (OS) obtained by mild acid hydrolyses of the L2, L3 and L5 serotype lipopolysaccharides (LPS) of Neisseria meningitidis have been elucidated. The OS have extensive regions of structural similarity of which some are responsible for cross-reactivity among the meningococcal LPS. However, the fact that the LPS are predominantly serotype-specific antigens implies that the terminal lacto-N-neotetraose unit, common to all the above OS, is not immunodominant, and that the major LPS serotype specificity originates in the inner core region of the OS.This is N.R.C.C. No. 26392.     

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: conjugates based on core oligosaccharides

In this study we have prepared glycoconjugates with core oligosaccharides (OS) from the lipopolysaccharide (LPS) of Neisseria meningitidis, thus avoiding the neo-epitopes of the deacylated lipid A region of the derived LPS molecule identified in our previous studies. A comprehensive investigation was performed with glycoconjugates prepared from the most extended to the most truncated core OS still maintaining the conserved inner core epitope. As previously, we have established reproducible bactericidal killing of the homologous antigen elaborating strain, but a failure to kill wild-type strains. In these studies it was evident that the linker molecules used in the conjugation methodologies were dominating the immune response. However, when galE core OS based conjugates were prepared without utilizing linkers, via direct reductive amination, we failed to generate an immune response to even the homologous antigen. We also identified that immunisation with the galE antigen via linker methodologies provoked an immune response that was dependent upon key residues of the conserved inner core OS structure, whereas the immune responses to lgtB and lgtA antigens did not involve the inner core OS. This comprehensive study has, despite our best efforts, cast significant doubt as to the utility of the conserved inner core region of the meningococcal LPS as a potential vaccine antigen.     

The structure of the lipooligosaccharide (LOS) from the alpha-1,2-N-acetyl glucosamine transferase (rfaK(NMB)) mutant strain CMK1 of Neisseria meningitidis: implications for LOS inner core assembly and LOS-based vaccines

The inner core structures of the lipooligosaccharides (LOS) of Neisseria meningitidis are potential vaccine candidates because both bactericidal and opsonic antibodies can be generated against these epitopes. In an effort to better understand LOS biosynthesis and the potential immunogenicity of the LOS inner core, we have determined the LOS structure from a meningococcal rfaK mutant CMK1. The rfaK gene encodes the transferase that adds an alpha-N-acetylglucosaminosyl residue to O-2 of the inner core heptose (Hep) II of the LOS. The LOS oligosaccharide from this mutant was previously shown to contain only Hep, 3-deoxy-D-manno-2-octulosonic acid (Kdo), and multiple phosphoethanolamine (PEA) substituents (Kahler et al., 1996a, J. Bacteriol., 178, 1265-1273). The complete structure of the oligosaccharide (OS) component of the LOS from mutant CMK1 was determined using glycosyl composition and linkage analyses, and 1H, 13C, and 31P nuclear magnetic resonance spectroscopy. The CMK1 OS structure contains a PEA group at O-3 of Hep II in place of the usual glucosyl residue found at this position in the completed L2 LOS glycoform from the parent NMB strain. The PEA group at O-6 of Hep II, however, is present in both the CMK1 mutant LOS and parental NMB L2 LOS structures. The structure of the OS from CMK1 suggests that PEA substituents are transferred to both the O-3 and O-6 positions of Hep II prior to: (1) the incorporation of the alpha-GlcNAc on Hep II; (2) the synthesis of the alpha-chain on Hep I; and (3) the substitution of the glycosyl residue at the O-3 Hep II, which distinguishes L2 and L3 immunotypes. The LOS structure of the CMK1 mutant makes it a candidate immunogen that could generate broadly cross-reactive inner-core LOS antibodies.