Meningococcal Lipooligosaccharide Research Articles

Antibiotics modulates the virulence of Neisseria meningitidis by regulating capsule synthesis

The opportunistic pathogens residing are frequently exposed to range of antimicrobials which affects virulence attributes. Neisseria meningitidis, is a host-restricted commensal of human upper respiratory tract which is subjected to a variety of stresses within the host, including antibiotic exposure. One of the most important virulence factors for pathogenesis is the meningococcal lipo-oligosaccharide capsule. Role of capsules in antimicrobial resistance and persistence is not yet established. In this study, different virulence factors of N. meningitidis were examined in presence of sub-MIC of four antibiotics: penicillin, ciprofloxacin, erythromycin and chloramphenicol. We observed increased production of the capsule by N. meningitidis when grown in the presence of penicillin, erythromycin, and chloramphenicol at sub-inhibitory concentration. Capsular production increase concurrently with increased resistance to inducing antibiotic which also confers increased survival in human serum. Finally, we show that increased capsule production in response to antibiotic exposure is aided by siaC, ctrB, lipA gene expression. These findings show that capsule synthesis, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our findings support a model in which gene expression changes caused by ineffective antibiotic treatment cause N. meningitidis transition between states of low and high virulence potential, contributing to pathogen's opportunistic nature.

Predominant phosphorylation patterns in Neisseria meningitidis lipid A determined by top-down MS/MS

Among the virulence factors in Neisseria infections, a major inducer of inflammatory cytokines is the lipooligosaccharide (LOS). The activation of NF-κB via extracellular binding of LOS or lipopolysaccharide (LPS) to the toll-like receptor 4 and its coreceptor, MD-2, results in production of pro-inflammatory cytokines that initiate adaptive immune responses. LOS can also be absorbed by cells and activate intracellular inflammasomes, causing the release of inflammatory cytokines and pyroptosis. Studies of LOS and LPS have shown that their inflammatory potential is highly dependent on lipid A phosphorylation and acylation, but little is known on the location and pattern of these posttranslational modifications. Herein, we report on the localization of phosphoryl groups on phosphorylated meningococcal lipid A, which has two to three phosphate and zero to two phosphoethanolamine substituents. Intact LOS with symmetrical hexa-acylated and asymmetrical penta-acylated lipid A moieties was subjected to high-resolution ion mobility spectrometry MALDI-TOF MS. LOS molecular ions readily underwent in-source decay to give fragments of the oligosaccharide and lipid A formed by cleavage of the ketosidic linkage, which enabled performing MS/MS (pseudo-MS3). The resulting spectra revealed several patterns of phosphoryl substitution on lipid A, with certain species predominating. The extent of phosphoryl substitution, particularly phosphoethanolaminylation, on the 4'-hydroxyl was greater than that on the 1-hydroxyl. The heretofore unrecognized phosphorylation patterns of lipid A of meningococcal LOS that we detected are likely determinants of both pathogenicity and the ability of the bacteria to evade the innate immune system.

Conjugation of Meningococcal Lipooligosaccharides Through Their Non-Reducing Terminus Results in Improved Induction a Protective Immune Response

The present studies prove that conjugation of meningococcal lipooligosaccharides through their non-reducing terminus conserves their inner epitopes resulting in conjugates potent to induce a protective immune response. Four different oligosaccharides were obtained by specific degradations of the same L7 lipooligosaccharide (L7-LOS), and each was linked to tetanus toxoid by direct reductive amination. Two were truncated oligosaccharides with incomplete inner epitopes and were obtained by mild acid hydrolysis of lipooligosaccharide. The terminal galactose of one oligosaccharide was additionally enzymatically oxidized. These oligosaccharides were conjugated through a newly exposed terminal Kdo in reducing end or through oxidized galactose localized at non-reducing end of the core, respectively. The third was a full-length oligosaccharide obtained by O-deacylation of the L7-LOS and subsequent enzymatic removal of phosphate substituents from its lipid A moiety. The fourth one was also a full-length O-deacylated lipooligosaccharide, but treated with galactose oxidase. This allowed direct conjugation to tetanus toxoid through terminal 2-N-acyl-2-deoxy-d-glucopyranose or through oxidized galactose, respectively. Comparison of the immune performance of four conjugates in mice revealed, that while each was able to induce significant level of L7-LOS-specific IgG antibody, the conjugates made with the full-length saccharides were able to induce antibodies with increased bactericidal activity against homologous meningococci. Only full-length oligosaccharides were good inhibitors of the binding of L7-LOS to the bactericidal antiserum. Moreover, induction of the significant level of the L7-LOS-specific antibody by full-length lipooligosaccharide conjugated from non-reducing end, provided also the direct evidence that internal core epitopes are fully responsible for the immunorecognition and immunoreactivity.

Induction of Endotoxin Tolerance by Pathogenic Neisseria Is Correlated with the Inflammatory Potential of Lipooligosaccharides and Regulated by MicroRNA-146a

In this article, we report that retreatment of human monocytic THP-1 cells and primary monocytes with pathogenic Neisseria or with purified lipooligosaccharides (LOS) after previous exposure to LOS induced immune tolerance, as evidenced by reduced TNF-α and IL-1β cytokine expression. LOS that we have previously shown to vary in their potential to activate TLR4 signaling, which was correlated with differences in levels of lipid A phosphorylation, had similarly variable ability to induce tolerance. Efficacy for induction of tolerance was proportional to the level of lipid A phosphorylation, as LOS from meningococcal strain 89I with the highest degree of phosphorylation was the most tolerogenic following retreatment with LOS or whole bacteria, compared with LOS from gonococcal strains 1291 and GC56 with reduced levels of phosphorylation. Hydrogen fluoride treatment of 89I LOS to remove phosphates rendered the LOS nontolerogenic. Tolerance induced by the more highly inflammatory meningococcal LOS was correlated with significantly greater downregulation of p38 activation, greater induction of the expression of A20 and of microRNA-146a, and greater reductions in IL-1R-associated kinase 1 and TRAF6 levels following LOS retreatment of cells. The role of miR-146a in regulation of induction of TNF-α was confirmed by transfecting cells with an inhibitor and a mimic of miR-146a. Our results provide a mechanistic framework for understanding the variable pathophysiology of meningococcal and gonococcal infections given that after an initial exposure, greater upregulation of microRNA-146a by more highly inflammatory LOS conversely leads to the suppression of immune responses, which would be expected to facilitate bacterial survival and dissemination.

Improvement of Immunogenicity of Meningococcal Lipooligosaccharide by Coformulation with Lipidated Transferrin-Binding Protein B in Liposomes: Implications for Vaccine Development

Among various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group B Neisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluated in vitro using the Limulus Amebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, an in vivo study demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

Neisserial Lipooligosaccharides (LOS) Differentially Modulate Innate Immune Responses in Human Monocytes throughTLR4 Signaling (135.17)

Abstract Neisseria gonorrhoeae and N. meningitidis engage innate immune receptors TLR4 and TLR2, as well as TREM1 and TREM2. In this study, we report differential innate immune activation by Neisserial LOS in monocytes. LOS differentially induced proinflammatory mediators TNF-alpha, IL-1 beta, IL-6 and IL-8 in THP1 monocytic cells as well as IL-10, IL-12, IFN-alpha, MIP-1 alpha, MIP-1 beta, and RANTES in human monocytes. Interestingly, while gonococcal LOS was not able to induce GM-CSF in monocytes, GM-CSF expression was enhanced by meningococcal LOS in both THP1 cells and monocytes. In THP1 cells, the induction of TNF-alpha by LOS was inhibited by anti-TLR4 and anti-CD14 antibody, but not by anti-TLR2, suggesting that the TLR4, but not TLR2, was responsible for the induction of cytokines. The ability of LOS to directly activate the TLR4 pathway was demonstrated by using TLR4-MD2 transfected HEK reporter cells, the activation of which was blocked by lipid IVa, a TLR4-MD2 antagonist. In monocytes, lipid IVa also inhibited the induction of all cytokines as well as CD80 expression by gonococcal LOS, but it only inhibited the activation of TNF-alpha, IL-10 and GM-CSF by meningococcal LOS. These results suggest that Neisserial LOS differentially activate innate immunity in monocytes through the TLR4 pathway, but that the induction of innate immune responses by meningococcal LOS may rely on the engagement of additional pattern recognition receptors.

Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4

MD-2, a eukaryotic accessory protein, is an essential component for the molecular pattern recognition of bacterial endotoxins. MD-2 interacts with lipid A of endotoxins [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] to activate human toll-like receptor (TLR) 4. The structure of lipid A influences the subsequent activation of human TLR4 and the immune response, but the basis for the discrimination of lipid A structures is unclear. A recombinant human MD-2 (rMD-2) protein was produced in the Pichia pastoris yeast expression system. Human embryonic kidney (HEK293) cells were transfected with human TLR4 and were stimulated with highly purified LOS (0.56 pmol) from Neisseria meningitidis or LPS from other structurally defined bacterial endotoxins in the presence or absence of human rMD-2. Human rMD-2 restored, in a dose-dependent manner, interleukin (IL-8) responsiveness to LOS or LPS in TLR4-transfected HEK293 cells. The interaction of endotoxin with human rMD-2 was then assessed by enzyme-linked immunosorbent assays. Wild-type meningococcal LOS (Wt m LOS) bound human rMD-2, and binding was inhibited by an anti-MD-2 antibody to MD-2 dose-dependently (P < 0.005). Wt m LOS or meningococcal KDO(2)-lipid A had the highest binding affinity for human rMD-2; unglycosylated meningococcal lipid A produced by meningococci with defects in the 3-deoxy-d-manno-2-octulosonic acid (KDO) biosynthesis pathway did not appear to bind human rMD-2 (P < 0.005). The affinity of meningococcal LOS with a penta-acylated lipid A for human rMD-2 was significantly less than that for hexa-acylated LOS (P < 0.05). The hierarchy in the binding affinity of different lipid A structures for human rMD-2 was directly correlated with differences in TLR4 pathway activation and cytokine production by human macrophages.

Differential Role of Lipooligosaccharide ofNeisseria meningitidisin Virulence and Inflammatory Response during Respiratory Infection in Mice

Meningococcal lipooligosaccharide (LOS) induces a strong proinflammatory response in humans during meningococcal infection. We analyzed the role of LOS in the inflammatory response and virulence during the early infectious process in a mouse model of meningococcal respiratory challenge. An lpxA mutant strain (serogroup B) devoid of LOS (strain Z0204) could not persist in the lungs and did not invade the blood. The persistence in the lungs and invasion of the bloodstream by a rfaD mutant expressing truncated LOS with only lipid A and 3-deoxy-d-manno-2-octulosonic acid molecules (strain Z0401) was intermediate between those of the wild-type and Z0204 strains. Both LOS mutants induced acute pneumonia with the presence of infiltrating polymorphonuclear leukocytes in lungs. Although tumor necrosis factor alpha production was reduced in mice infected with the mutant of devoid LOS, both LOS mutants induced production of other proinflammatory cytokines, such as interleukin-1beta (IL-1beta), IL-6, and the murine IL-8 homolog KC. Together, these results suggest that meningococcal LOS plays a role during the early infectious and invasive process, and they further confirm that other, nonlipopolysaccharide components of Neisseria meningitidis may significantly contribute to the inflammatory reaction of the host.

Non-lipooligosaccharide-mediated signalling via Toll-like receptor 4 causes fatal meningococcal sepsis in a mouse model

Meningococcal lipooligosaccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis with disease severity correlating with circulating concentrations of LOS and proinflammatory cytokines. In this study we show that the proinflammatory response to live meningococci in a mouse model of sepsis involves TLR4, but can develop independently of the expression of LOS. This is supported by data showing that in vivo an isogenic LOS-deficient strain, lpxA, induced equivalent disease severity and similar proinflammatory responses as the serogroup C wild-type parent strain FAM20. This response was abolished in TLR4-/- mice, and neither the wild-type strain of meningococci nor the LOS-deficient mutant was able to cause fatal sepsis in these mice. Mouse survival correlated with low levels of cytokines and chemokines, the chemotactic complement factor C5a and neutrophil levels in blood at 24 h post infection. These data suggest that during meningococcal sepsis the recognition of one or more unidentified non-LOS component(s) by TLR4 is important in stimulating proinflammatory responses, and that fatality associated with meningococcal sepsis in mice is induced by the proinflammatory host response.

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.

The Immunogenicity of a Conformationally Restricted Peptide Mimetic of Meningococcal Lipooligosaccharide

Life-threatening meningitis and septicaemia caused by Neisseria meningitidis are a public health priority, and their prevention by vaccination is a major objective. Meningococcal capsular polysaccharide-based vaccines are effective against the major invasive serogroups, except for serogroup B, the capsule of which mimics human polysaccharides and is poorly immunogenic. An alternative vaccine candidate that has the potential to offer cross-protection against antigenically diverse meningococci is the lipooligosaccharide (LOS). The structurally constrained peptide mimetic, C22, of a bactericidal antibody epitope within LOS was previously shown to elicit cross-reactive antibodies to meningococcal LOS when complexed to NeutrAvidintrade mark as a carrier protein. The immunogenicity of this antigen in H-2(d) (BALB/c) and H-2(k) (C3H/HeN) haplotype mice was further investigated. Anti-LOS immunoglobulin G (IgG) antibody titres increased with the vaccine dose and correlated with the anti-C22 peptide antibody titres in both haplotypes. Antigen-stimulated Th1/Th2 cytokine secretion by splenocytes and antibody isotypes indicated a Th2-type immune response with IgG1 antibodies and a low titre of IgG2b. There was no serum bactericidal activity observed against the meningococcus.

Regulation of interactions of endotoxin with host cells

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin requires lipopolysaccharide-binding protein (LBP) and CD14-dependent delivery of endotoxin to cells containing MD-2 and TLR4. We have used metabolically labeled [14C] meningococcal lipooligosaccharide (LOS), purified recombinant endotoxin-binding proteins, and cultured endothelial cells to better define protein: endotoxin intermediates key in cell activation in the absence of functional membrane (m) CD14. Protein:endotoxin complexes or aggregates ( agg) were purified by gel sieving and characterized by immunocapture and bio-assays. Cell activation closely correlated with LBP, albumin and soluble (s) CD14-dependent conversion of endotoxin agg (Mr≥ 20 × 106) to monomeric (M ~55 × 103) endotoxin:sCD14 complexes. Ordered interaction of LBP (+ albumin) and sCD14 withrLOS agg was required for the efficient formation of a bioactive endotoxin:sCD14 complex and potent cell activation. Increasing the ratio of LBP/sCD14 or addition of bactericidal/permeability-increasing protein (BPI) reduced accumulation of endotoxin:sCD14 complexes and instead yielded aggregates of endotoxin (Mr~1—20 × 106) containing LBP or BPI that were taken up by cells in a CD14- and TLR4-independent manner without inducing pro-inflammatory responses. These findings strongly suggest that host machinery linked to TLR4-dependent cellular activation or TLR4-independent cellular clearance of endotoxin selectively recognizes different protein:endotoxin complexes. At the outset of infection, the low concentrations of LBP present and absence of extracellular BPI favor formation of pro-inflammatory endotoxin:CD14 complexes. The mobilization of LBP and BPI that is triggered by inflammation directs endotoxin for clearance and hence resolution of endotoxin-triggered inflammation.

Regulation of interactions of endotoxin with host cells.

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin requires lipopolysaccharide-binding protein (LBP) and CD14-dependent delivery of endotoxin to cells containing MD-2 and TLR4. We have used metabolically labeled [(14)C] meningococcal lipooligosaccharide (LOS), purified recombinant endotoxin-binding proteins, and cultured endothelial cells to better define protein:endotoxin intermediates key in cell activation in the absence of functional membrane (m) CD14. Protein:endotoxin complexes or aggregates (agg) were purified by gel sieving and characterized by immunocapture and bio-assays. Cell activation closely correlated with LBP, albumin and soluble (s) CD14-dependent conversion of endotoxin agg (M(r) > or = 20 x 10(6)) to monomeric (M(r) approximately 55 x 10(3)) endotoxin:sCD14 complexes. Ordered interaction of LBP (+ albumin) and sCD14 with LOSagg was required for the efficient formation of a bioactive endotoxin:sCD14 complex and potent cell activation. Increasing the ratio of LBP/sCD14 or addition of bactericidal/permeability-increasing protein (BPI) reduced accumulation of endotoxin:sCD14 complexes and instead yielded aggregates of endotoxin (M(r) approximately 1-20 x 10(6)) containing LBP or BPI that were taken up by cells in a CD14- and TLR4-independent manner without inducing pro-inflammatory responses. These findings strongly suggest that host machinery linked to TLR4-dependent cellular activation or TLR4-independent cellular clearance of endotoxin selectively recognizes different protein:endotoxin complexes. At the outset of infection, the low concentrations of LBP present and absence of extracellular BPI favor formation of pro-inflammatory endotoxin:CD14 complexes. The mobilization of LBP and BPI that is triggered by inflammation directs endotoxin for clearance and hence resolution of endotoxin-triggered inflammation.

Phase-variation of the truncated lipo-oligosaccharide of Neisseria meningitidis NMB phosphoglucomutase isogenic mutant NMB-R6

The detection of antibodies specific to meningococcal lipo-oligosaccharides (LOSs; outer-core→inner-core→lipid A) in sera of patients convalescent from meningococcal infection suggests the potential use of LOS as a vaccine to combat pathogenic Neisseria spp. Removal of the outer-core region, which expresses glycans homologous to human blood-group antigens, is a required first-step in order to avoid undesirable immunological reactions following vaccination. To this end, we describe here the structural makeup of the LOS produced by serogroup B N. meningitidis NMB isogenic phosphoglucomutase (Pgm) mutant (NMB-R6). The dominant LOS types produced by NMB-R6 expressed a deep-truncated inner-core region, GlcNAc-(1→2)- ldHepII-(1→3)- ldHepI-(1→5)-[Kdo-2→4]-Kdo→lipid A, with one PEA unit attached at either O-6 or O-7 of ldHepII, or with two simultaneously PEA moieties attached at O-3 and O-6 or O-3 and O-7 of the same unit. Unexpectedly, this mutation did not completely deactivate the production of Glc, as some LOS molecules were observed to carry Glc at O-4 of ldHepI and at O-3 of ldHepII. A glycoconjugate vaccine comprised of NMB-R6 LOSs is currently being evaluated in our laboratory.

Conjugation of meningococcal lipooligosaccharides through their lipid A terminus conserves their inner epitopes and results in conjugate vaccines having improved immunological properties

The importance of conserved inner saccharide epitopes to the immune performance of meningococcal lipooligosaccharide–protein conjugate vaccines was demonstrated in the following experiments. Two different oligosaccharides were obtained by chemical degradations of the same L7 lipooligosaccharide, and both were linked terminally to tetanus toxoid. One was a truncated oligosaccharide in which the inner epitopes were incomplete and was obtained by mild acid hydrolysis of the L7 lipooligosaccharide. This oligosaccharide was conjugated by direct reductive amination through its newly exposed terminal Kdo residue. The second, a full-length oligosaccharide, was obtained by O-deacylation of the L7 lipooligosaccharide, with subsequent removal of phosphate substituents from its lipid A moiety using alkaline phosphatase. This permitted the full-length oligosaccharide to be conjugated directly to tetanus toxoid by reductive amination through its newly exposed terminal 2- N-acyl-2-deoxy- d-glucopyranose residue. Comparison of the immune performance of the two conjugates in mice revealed, that while both were able to induce significant levels of L7-lipooligosaccharide-specific IgG antibody, the conjugate made with the full-length saccharide was able to induce antibodies with increased bactericidal activity against homologous meningococci.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or lgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes.

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or lgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host.

KpsF Is the Arabinose-5-phosphate Isomerase Required for 3-Deoxy-d-manno-octulosonic Acid Biosynthesis and for Both Lipooligosaccharide Assembly and Capsular Polysaccharide Expression in Neisseria meningitidis

We have identified and defined the function of kpsF of Neisseria meningitidis and the homologues of kpsF in encapsulated K1 and K5 Escherichia coli. KpsF was shown to be the arabinose-5-phosphate isomerase, an enzyme not previously identified in prokaryotes, that mediates the interconversion of ribulose 5-phosphate and arabinose 5-phosphate. KpsF is required for 3-deoxy-d-manno-octulosonic acid (Kdo) biosynthesis in N. meningitidis. Mutation of kpsF or the gene encoding the CMP-Kdo synthetase (kpsU/kdsB) in N. meningitidis resulted in expression of a lipooligosaccharide (LOS) structure that contained only lipid A and reduced capsule expression in the five invasive disease-associated meningococcal serogroups (A, B, C, Y, and W-135). The step linking meningococcal capsule and LOS biosynthesis was shown to be Kdo production as the expression of capsule was wild type in a Kdo transferase (kdtA) mutant. Thus, in addition to lipooligosaccharide assembly, Kdo is required for meningococcal capsular polysaccharide expression. Furthermore, N. meningitidis, unlike enteric Gram-negative bacteria, can survive and synthesize only unglycosylated lipid A.

Characterization of Oligopeptides That Cross-react with Carbohydrate-specific Antibodies by Real Time Kinetics, In-solution Competition Enzyme-linked Immunosorbent Assay, and Immunological Analyses

Phage displaying random cyclic 7-mer, and linear 7-mer and 12-mer peptides at the N terminus of the coat protein, pIII, were panned with the murine monoclonal antibody, 9-2-L379 specific for meningococcal lipo-oligosaccharide. Five cyclic peptides with two sequence motifs, six linear 7-mers, and five linear 12-mers with different sequence motifs were identified. Only phage displaying cyclic peptides were specifically captured by and were antigenic for 9-2-L379. Monoclonal antibody 9-2-L379 exhibited "apparent" binding affinities to the cyclic peptides between 11 and 184 nm, comparable with lipo-oligosaccharide. All cyclic peptides competed with the binding of 9-2-L379 to lipo-oligosaccharide with EC(50) values in the range 10-105 microm, which correlated with their apparent binding affinities. Structural modifications of the cyclic peptides eliminated their ability to bind and compete with monoclonal antibody 9-2-L379. Mice (C3H/HeN) immunized with the cyclic peptide with optimal apparent binding affinity and EC(50) of competition elicited cross-reactive antibodies to meningococcal lipo-oligosaccharide with end point dilution serum antibody titers of 3200. Cyclic peptides were converted to T-cell-dependent immunogens without disrupting these properties by C-terminal biotinylation and complexing with NeutrAvidin. The data indicate that constrained peptides can cross-react with a carbohydrate-specific antibody with greater specificity than linear peptides, and critical to this specificity is their structural conformation.

Lipooligosaccharide and polysaccharide capsule: virulence factors of Neisseria meningitidis that determine meningococcal interaction with human dendritic cells.

In this work we analyzed the roles of meningococcal lipooligosaccharide (LOS) and capsule expression in the interaction of Neisseria meningitidis with human dendritic cells (DC). Infection of DC with serogroup B wild-type meningococci induced a strong burst of the proinflammatory cytokines and chemokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. In contrast, a serogroup B mutant strain lacking LOS expression barely led to cytokine induction, demonstrating that meningococcal LOS is the main mediator of the proinflammatory response in human DC. Sialylation of meningococcal LOS did not influence cytokine secretion by DC. However, we found the phagocytosis of N. meningitidis by human DC to be inhibited by LOS sialylation. In addition, the expression of the meningococcal serogroup A, B, and C capsules dramatically reduced DC adherence of N. meningitidis and phagocytosis to some extent. Hence, LOS sialylation and capsule expression are independent mechanisms protecting N. meningitidis from the phagocytic activity of human DC.