lipid-a and Meningitis--Meningococcal

Antimicrobial peptides (AMPs) play an important role as a host defense against microbial pathogens and are key components of the human innate immune response. Neisseria meningitidis frequently colonizes the human nasopharynx as a commensal but also is a worldwide cause of epidemic meningitis and rapidly fatal sepsis. In the human respiratory tract, the only known reservoir of N. meningitidis, meningococci are exposed to human endogenous AMPs. Thus, it is not surprising that meningococci have evolved effective mechanisms to confer intrinsic and high levels of resistance to the action of AMPs. This article reviews the current knowledge about AMP resistance mechanisms employed by N. meningitidis. Two major resistance mechanisms employed by meningococci are the constitutive modification of the lipid A head groups of lipooligosaccharides by phosphoethanolamine and the active efflux pump mediated excretion of AMPs. Other factors influencing AMP resistance, such as the major porin PorB, the pilin biogenesis apparatus, and capsular polysaccharides, have also been identified. Even with an inherently high intrinsic resistance, several AMP resistance determinants can be further induced upon exposure to AMPs. Many well-characterized AMP resistance mechanisms in other Gram-negative bacteria are not found in meningococci. Thus, N. meningitidis utilizes a limited but highly effective set of molecular mechanisms to mediate antimicrobial peptide resistance. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Proteins; Drug Resistance, Bacterial; Host-Pathogen Interactions; Humans; Immune Evasion; Immunity, Innate; Lipid A; Membrane Transport Proteins; Meningitis, Meningococcal; Microbial Viability; Neisseria meningitidis; Signal Transduction

Topics: Animals; Bacterial Adhesion; Disease Outbreaks; Ethanolamines; Evolution, Molecular; Glutamyl Aminopeptidase; Humans; Japan; Lipid A; Lipopolysaccharides; Meningitis, Meningococcal; Neisseria meningitidis

Neisseria meningitidis (Nm) and N. gonorrhoeae (Ng) express a Macrophage Infectivity Potentiator (MIP, NMB1567/NEIS1487) protein in their outer membrane (OM). In this study, we prepared independent batches of liposomes (n = 3) and liposomes + MonoPhosphoryl Lipid A (MPLA) (n = 3) containing recombinant truncated Nm-MIP protein encoded by Allele 2 (rT-Nm-MIP, amino acids 22-142), and used these to immunize mice. We tested the hypothesis that independent vaccine batches showed similar antigenicity, and that antisera could recognise both meningococcal and gonococcal MIP and induce cross-species bactericidal activity. The different batches of M2 rT-Nm-MIP-liposomes ± MPLA showed no significant (P > 0.05) batch-to-batch variation in antigenicity. Anti-rT-Nm-MIP sera reacted equally and specifically with Nm-MIP and Ng-MIP in OM and on live bacterial cell surfaces. Specificity was shown by no antiserum reactivity with Δmip bacteria. Using human complement/serum bactericidal assays, anti-M2 rT-Nm-MIP sera killed homologous meningococcal serogroup B (MenB) strains (median titres of 32-64 for anti-rT-Nm-MIP-liposome sera; 128-256 for anti-rT-Nm-MIP-liposome + MPLA sera) and heterologous M1 protein-expressing MenB strains (titres of 64 for anti rT-Nm-MIP-liposome sera; 128-256 for anti-rT-Nm-MIP-liposome + MPLA sera). Low-level killing (P < 0.05) was observed for a MenB isolate expressing M7 protein (titres 4-8), but MenB strains expressing M6 protein were not killed (titre < 4-8). Killing (P < 0.05) was observed against MenC and MenW bacteria expressing homologous M2 protein (titres of 8-16) but not against MenA or MenY bacteria (titres < 4-8). Antisera to M2 rT-Nm-MIP showed significant (P < 0.05) cross-bactericidal activity against gonococcal strain P9-17 (expressing M35 Ng-MIP, titres of 64-512) and strain 12CFX_T_003 (expressing M10 Ng-MIP, titres 8-16) but not against FA1090 (expressing M8 Ng-MIP). As an alternative to producing recombinant protein, we engineered successfully the Nm-OM to express M2 Truncated-Nm-MIP, but lipooligosaccharide-extraction with Na-DOC was contra-indicated. Our data suggest that a multi-component vaccine containing a select number of Nm- and Ng-MIP type proteins would be required to provide broad coverage of both pathogens.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; Cross Reactions; Gonorrhea; Humans; Immune Sera; Immunization; Lipid A; Liposomes; Meningitis, Meningococcal; Mice; Mice, Inbred BALB C; Neisseria gonorrhoeae; Neisseria meningitidis; Recombinant Proteins

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.

Topics: DNA-Binding Proteins; Endotoxins; Enzyme Activation; Gonorrhea; Humans; Hydrofluoric Acid; Immune Tolerance; Inflammation; Interleukin-1 Receptor-Associated Kinases; Interleukin-1beta; Intracellular Signaling Peptides and Proteins; Lipid A; Lipopolysaccharides; Meningitis, Meningococcal; MicroRNAs; Monocytes; Neisseria gonorrhoeae; Neisseria meningitidis; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4; Tumor Necrosis Factor alpha-Induced Protein 3; Tumor Necrosis Factor-alpha

Engineering the lipopolysaccharide (LPS) biosynthetic pathway offers the potential to obtain modified derivatives with optimized adjuvant properties. Neisseria meningitidis strain H44/76 was modified by expression of the pagL gene encoding lipid A 3-O-deacylase from Bordetella bronchiseptica and by inactivation of the lgtB gene encoding the terminal oligosaccharide galactosyltransferase. Mass spectrometry analysis of purified mutant LPS was used for detailed compositional analysis of all present molecular species. This determined that the modified LPS was mainly pentaacylated, demonstrating high efficiency of conversion from the hexaacyl to the 3-O-deacylated form by heterologous lipid A 3-O-deacylase (PagL) expression. MS analyses also provided evidence for expression of only one major oligosaccharide glycoform, which lacked the terminal galactose residue as expected from inactivation of the lgtB gene. The immunomodulatory properties of PagL-deacylated LPS were compared with another pentaacyl form obtained from an lpxL1(-) mutant, which lacks the 2' secondary acyl chain. Although both LPS mutants displayed impaired capacity to induce production of the pro-inflammatory cytokine IL-6 in the monocytic cell line Mono Mac 6, induction of the Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β-dependent chemokine interferon-γ-induced protein 10 was largely retained only for the lgtB(-)/pagL(+) mutant. Removal of remaining hexaacyl species exclusively present in lgtB(-)/pagL(+) LPS demonstrated that these minor species potentiate but do not determine the activity of this LPS. These results are the first to indicate a qualitatively different response of human innate cells to pentaacyl lpxL1(-) and pagL(+) LPS and show the importance of detailed structure-function analysis when working with modified lipid A structures. The pagL(+) LPS has significant potential as immune modulator in humans.

Topics: Bordetella; Carbohydrate Sequence; Cell Line; Cytokines; Genes, Bacterial; Genetic Engineering; Host-Pathogen Interactions; Humans; Immunologic Factors; Lipid A; Meningitis, Meningococcal; Molecular Sequence Data; Monocytes; Mutation; Neisseria meningitidis

The opacity (Opa) proteins of Neisseria meningitidis are outer membrane proteins involved in adhesion and invasion of host epithelial cells and are therefore expected to play an important role in colonisation of the nasopharynx. The majority of meningococcal Opa proteins bind to members of the CEACAM receptor family, such as CEA. Blocking of the Opa-CEACAM interaction by mucosal anti-Opa antibodies could thus constitute an important protective mechanism for novel meningococcal vaccines. In this study we analysed the specific anti-Opa antibody responses after intranasal immunisation of mice with liposomes containing purified and native OpaB (recognising the CEA receptor) and OpaJ (no affinity for CEA) proteins. These antigens were combined with or without one of three different adjuvants, i.e. purified meningococcal LPS, monophosphoryl lipid A (MPL) or the B-subunit of Escherichia coli heat-labile enterotoxin (EtxB). After intranasal immunisation with any of these formulations, anti-Opa IgA antibodies were found in nasal lavages and in some cases anti-Opa IgA and IgG antibodies were also found in lung lavages. With OpaJ but not OpaB, significant bactericidal serum titres were obtained. Of the different adjuvants used, meningococcal LPS gave the strongest overall immune response. Non-adjuvated liposomal Opa formulations were poorly immunogenic. No differences were found between the immune response in transgenic mice expressing the CEA-receptor and non-transgenic mice, showing that the CEA-Opa interaction does not influence the antibody response.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Toxins; Bronchoalveolar Lavage Fluid; Enterotoxins; Escherichia coli Proteins; Immunoglobulin A; Immunoglobulin G; Lipid A; Lipopolysaccharides; Liposomes; Meningitis, Meningococcal; Meningococcal Vaccines; Mice; Mice, Transgenic; Nasal Lavage Fluid; Neisseria meningitidis; Vaccination

Topics: Bacteremia; Carbohydrate Sequence; Chemical Phenomena; Chemistry, Physical; Cytokines; Female; Humans; Lipid A; Lipopolysaccharides; Male; Meningitis, Meningococcal; Meningococcal Infections; Molecular Sequence Data; Molecular Structure; Neisseria meningitidis; Shock, Septic