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.     

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.     

Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis

The pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post-translationally modified by the addition of an O-linked trisaccharide, Gal (β1–4) Gal (α1–3) 2,4-diacetimido-2,4,6-trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with well-defined lipopolysaccharide (LPS) or pilin-linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin-linked trisaccharide structure. The pglA mutation, like the galE mutation reported previously, had no effect on pilus-mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.     

Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis

We have identified a homologue of the adhesin AIDA-I of Escherichia coli in Neisseria meningitidis. This gene was designated nhhA (Neisseria hia homologue), as analysis of the complete coding sequence revealed that it is more closely related to the adhesins Hia and Hsf of Haemophilus influenzae. The sequence of nhhA was determined from 10 strains, and found to be highly conserved. Studies of the localisation by Western immunoblot analysis of total cell proteins and outer membrane complex preparations and by immunogold electron microscopy revealed that NhhA is located in the outer membrane. A strain survey showed that nhhA is present in 85/85 strains of N. meningitidis representative of all the major disease-associated serogroups, based on Southern blot analysis. It is expressed in the majority of strains tested by Western immunoblot.     

NarE: a novel ADP-ribosyltransferase from Neisseria meningitidis

Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co-transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed.     

Identification of an outer-membrane haemoglobin-binding protein in Neisseria meningitidis.

Although Neisseria meningitidis can use haemoglobin as an iron source in vitro, the mechanism of haemoglobin-iron uptake is unknown. Using a biotinylated human haemoglobin probe in a solid-phase dot-binding assay, haemoglobin-binding activity was detected in total membranes derived from meningococci grown under iron-limited but not iron-sufficient conditions. In competition binding experiments, bovine and human haemoglobin could abrogate binding. In contrast, no binding inhibition was seen with ferric nitrate, protoporphyrin IX, and iron-loaded human transferrin. The ability of both haemin and catalase, a nonhaemoglobin haem-containing compound, to inhibit binding competitively suggested that the ligand recognized by the binding protein is the haem moiety. Scatchard plot analysis revealed a heterogeneous receptor population. Limited proteolysis with proteinase K abolished binding activity, suggesting a haemoglobin-protein interaction. Detection of activity in a whole-cell binding assay demonstrated that this haemin-binding protein was surface exposed. In a limited survey of meningococcal strains, the presence of haemoglobin-binding activity in all isolates indicated that expression of this binding protein is not serogroup specific.     

Structure of the L2 lipopolysaccharide core oligosaccharides of Neisseria meningitidis.

Three different oligosaccharides were identified following mild acid hydrolysis of the lipopolysaccharide obtained from Neisseria meningitidis serotype 2 and their structures elucidated by combined chemical and physical techniques. The use of 500 MHz 1H nmr in both one- and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The oligosaccharides were all triantennary nonasaccharides in which the longest antenna terminates in lacto-N-neotetraose. Two of the nonasaccharides (major components), not separable by column chromatography, were distinguishable only by their different patterns of phosphorylethanolamine substitution and the third minor component by the absence of this substituent.     

Identification of a cation transport pathway in Neisseria meningitidis PorB

Neisseria meningitidis is the main causative agent of bacterial meningitis. In its outer membrane, the trimeric Neisserial porin PorB is responsible for the diffusive transport of essential hydrophilic solutes across the bilayer. Previous molecular dynamics simulations based on the recent crystal structure of PorB have suggested the presence of distinct solute translocation pathways through this channel. Although PorB has been electrophysiologically characterized as anion‐selective, cation translocation through nucleotide‐bound PorB during pathogenesis is thought to be instrumental for host cell death. As a result, we were particularly interested in further characterizing cation transport through the pore. We combined a structural approach with additional computational analysis. Here, we present two crystal structures of PorB at 2.1 and 2.65 Å resolution. The new structures display additional electron densities around the protruding loop 3 (L3) inside the pore. We show that these electron densities can be identified as monovalent cations, in our case Cs⁺, which are tightly bound to the inner channel. Molecular dynamics simulations reveal further ion interactions and the free energy landscape for ions inside PorB. Our results suggest that the crystallographically identified locations of Cs⁺ form a cation transport pathway inside the pore. This finding suggests how positively charged ions are translocated through PorB when the channel is inserted into mitochondrial membranes during Neisserial infection, a process which is considered to dissipate the mitochondrial transmembrane potential gradient and thereby induce apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Studies on the chemical composition of lipopolysaccharide from Neisseria meningitidis group B.

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.     

Response to antigenic determinants of Neisseria meningitidis lipopolysaccharide investigated with a new radioactive antigen-binding assay.

Adaptations of the Farr technique have resulted in a specific and reproducible radioactive antigen-binding assay for antibodies directed against the lipopolysaccharide (LPS) of Neisseria meningitidis. LPS was intrinsically labeled with 14C acetate during 16-hr growth in a modified Frantz media, extracted by hot phenol-H2O, and purified by dialysis, ultracentrifugation, and ethanol precipitation. LPS, which aggregates in aqueous solutions, was maintained in a monomeric form in 3% sodium deoxycholate (NaD) as determined by gel filtration on Sephadex G-75. Since NaD is insoluble in (NH4)2SO4, polyethylene glycol, 20%, was used to precipitate immunoglobulins of all three major classes.     

Identification and cloning of a fur homologue from Neisseria meningitidis

The iron response in a number of bacterial systems is mediated by fur (f erric u ptake r egulation)-like regulatory systems. We have cloned and characterized a gene from Neisseria meningitidis that was homologous to Escherichia coli fur. This clone was capable of modulating expression from both E. coli and neisserial iron-regulated promoters in response to iron, and it produced a protein that reacted with anti-E. coli fur serum. Although the DNA and predicted amino acid sequences were very similar to those of four other published fur homologues, meningococcal fur was the most divergent of the group. Inability to construct a meningococcal fur mutant suggested that fur may be essential in this species.     

Structure of the L5 lipopolysaccharide core oligosaccharides of Neisseria meningitidis

Three different oligosaccharides were isolated by mild acid hydrolysis of the lipopolysaccharides, obtained from Neisseria meningitidis serotype 5, and their structures were elucidated by combined chemical and physical techniques. The use of 500-MHz 1H NMR in both one-dimensional and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the purified oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The largest of the three original oligosaccharides is a triantennary partially O-acetylated decasaccharide in which the largest antenna terminates in a lacto-N-neotetraose unit. The smaller oligosaccharides (heptasaccharide and octasaccharide) except for terminal glycose deletions from the longest antenna are structural replicas of the larger.     

Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis

Lipopolysaccharide (LPS) is a major determinant of Neisseria meningitidis virulence. A key feature of meningococcal LPS is the phase-variable expression of terminal structures which are proposed to have disparate roles in pathogenesis. In order to identify the biosynthetic genes for terminal LPS structures and the control mechanisms for their phase-variable expression, the lic2A gene, which is involved in LPS biosynthesis in Haemophilus influenzae , was used as a hybridization probe to identify a homologous gene in N. meningitidis strain MC58. The homologous region of DNA was cloned and nucleotide sequence analysis revealed three open reading frames (ORFs), two of which were homologous to the H. influenzae lic2A gene. All three ORFs were mutagenized by the insertion of antibiotic-resistance cassettes and the LPS from these mutant strains was analysed to determine if the genes had a role in LPS biosynthesis. Immunological and tricine—SDS—PAGE analysis of LPS from the mutant strains indicated that all three genes were probably transferases in the biosynthesis of the terminal lacto- N -neotetraose structure of meningococcal LPS. The first ORF of the locus contains a homopolymeric tract of 14 guanosine residues within the 5'-end of the coding sequence. As the lacto- N -neotetraose structure in meningococcal LPS is subject to phase-variable expression, colonies that no longer expressed the terminal structure, as determined by monoclonal antibody binding, were isolated. Analysis of an 'off' phase variant revealed a change in the number of guanosine residues resulting in a frameshift mutation, indicating that a slipped-strand mispairing mechanism, operating in the first ORF, controls the phase-variable expression of lacto- N -neotetraose.     

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.     

Identification of the iroA gene product of Neisseria meningitidis as a lactoferrin receptor.

The iroA gene product is an iron limitation-inducible outer membrane protein of Neisseria meningitidis. A spontaneous mutant lacking the gene was unable to bind lactoferrin. Furthermore, Escherichia coli strains expressing the IroA protein were capable of binding lactoferrin. Apparently, the IroA protein functions as a lactoferrin receptor.     

A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.     

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto-N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(beta1-3)-Gal(beta1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.     

Neisseria lactamicus sp. n., a Lactose-fermenting Species Resembling Neisseria meningitidis

The biochemical and serological characteristics of lactose-utilizing strains of Neisseria were determined. These organisms were found in the nasopharynx of man and grew well on Thayer-Martin Selective Medium. They were compared with N. meningitidis to ascertain whether they were variants of this species. Differences between the lactose-using strains and the recognized species of Neisseria were considered significant enough to warrant designation of a new species, Neisseria lactamicus. This group has not been widely recognized as being separate from N. meningitidis; therefore, the normal incidence and clinical significance of these organisms has not been fully established. These organisms are oxidase-positive and positive for beta-D-galactosidase activity; they demonstrate fermentation in King Oxidation-Fermentation Medium; and they produce acid from only glucose, lactose, and maltose, of the 27 substrates incorporated in Cystine Trypticase Agar. Individual strains vary in their ability to grow on Nutrient Agar at both 25 and 37 C and in their pigmentation on Loeffler Medium. Results indicated that these organisms are serologically distinct from the N. meningitidis serogroups. Only 34 of 116 strains of N. lactamicus were smooth and could be tested by slide agglutination. None of the 34 could be grouped as N. meningitidis group A, B, C, D, X, Y, or Z. Thirty-one of these strains could, however, be specifically grouped with antisera prepared with N. lactamicus strains. Cross absorptions confirmed that N. lactamicus is serologically distinguishable from N. meningitidis.