Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1–3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.     

Iron and outer membrane proteins in the susceptibility of Neisseria meningitidis to human serum

The proportion of carrier-isolated Neisseria meningitidis strains sensitive to human serum (37.2%) was found to be significantly higher than that of case-isolated ones (4.1%), although the difference is too low to consider serum-resistance responsible for invasion in this microorganism. Serum-susceptibility was not related to the existence of specific outer membrane proteins, as is the case of N. gonorrhoeae. Iron restriction induced iron-regulated outer membrane proteins in each strain (but not the same proteins in all strains) but without any detectable effect on serum-susceptibility. Iron excess was also unable to induce changes in the susceptibility of N. meningitidis to human serum.     

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 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.     

The class 3 outer membrane protein (PorB) of Neisseria meningitidis: gene sequence and homology to the gonococcal porin PIA

The class 3 protein (PorB) is an important component of the meningococcal outer membrane. The structural gene (porB) encoding the class 3 protein has been cloned using primers suitable for the amplification of the corresponding chromosomal fragment by the polymerase chain reaction (PCR). The complete nucleotide sequence was determined and predicts a mature protein of 310 amino acids, preceded by a signal peptide of 19 residues. The predicted protein sequence of the class 3 protein exhibits essential structural homology to the gonococcal porin PIA. The class 3 protein encoding gene was expressed in Escherichia coli under the control of an inducible promoter.     

In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.     

Post-translational processing of Neisseria meningitidis gamma-glutamyl aminopeptidase and its association with inner membrane facing to the cytoplasmic space

We previously demonstrated that gamma-glutamyl aminopeptidase (also called gamma-glutamyl transpeptidase) (GGT) of Neisseria meningitidis is involved in the bacterial multiplication in cerebrospinal fluid. To further understand the function of meningococcal GGT, the biochemical properties were investigated in this study. The deduced amino acid sequence in N. meningitidis GGT was 37% identical to that of Escherichia coli GGT and that of Helicobacter pylori GGT, respectively, while a typical signal sequence was not found at the N-terminus of meningococcal GGT. Western blotting using rabbit antiserum against recombinant meningococcal GGT protein demonstrated that the meningococcal GGT is processed into two subunits in N. meningitidis at the conserved amino acid, threonine 427. The experiments on subcellular fractionation suggested that the majority of meningococcal GGT is associated with inner membrane facing to the cytoplasmic side. This cell localization might be unique for N. meningitidis compared to other GGTs.     

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.     

Characterization of serogroup A Neisseria meningitidis strains by rRNA gene restriction patterns and PCR: Correlation with the results of serotyping, subtyping and multilocus enzyme electrophoresis

Abstract We studied 35 strains of Neisseria meningitidis serogroup A from different locations (France, Central African Republic, Sudan and Burkina Faso) using both ribotyping and a polymerase chain reaction (PCR). A non-radioactive probe label was used for ribotyping; detection consisted of an immunoenzymatic procedure using a bispecific antibody. The PCR was designed to amplify the 16S–23S rDNA internal transcribed spacer. These techniques were compared with other markers. The strains were identified as belonging to three clones (I, III-1, IV) by multilocus enzyme electrophoresis (MEE) and to three subtypes by serological methods. Ribotyping identified five groups and PCR identified four groups. Ribotyping gave more diversity between strains than either MEE or sero/subtyping, but confirmed the epidemiological data provided by the combination of these two techniques. The PCR provided a simple and convenient one-step procedure for the differentiation of strains of serogroup A.     

Molecular Cloning and Functional Characterization of Components of the Capsule Biosynthesis Complex of Neisseria meningitidis Serogroup A: TOWARD IN VITRO VACCINE PRODUCTION*

The human pathogen Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis globally. A major virulence factor of Nm is the capsular polysaccharide (CPS), which in Nm serogroup A consists of N-acetyl-mannosamine-1-phosphate units linked together by phosphodiester linkages [→6)-α-d-ManNAc-(1→OPO₃⁻→]_n. Acetylation in O-3 (to a minor extent in O-4) position results in immunologically active polymer. In the capsule gene cluster (cps) of Nm, region A contains the genetic information for CPSA biosynthesis. Thereby the open reading frames csaA, -B, and -C are thought to encode the UDP-N-acetyl-d-glucosamine-2-epimerase, poly-ManNAc-1-phosphate-transferase, and O-acetyltransferase, respectively. With the aim to use a minimal number of recombinant enzymes to produce immunologically active CPSA, we cloned the genes csaA, csaB, and csaC and functionally characterized the purified recombinant proteins. If recombinant CsaA and CsaB were combined in one reaction tube, priming CPSA-oligosaccharides were efficiently elongated with UDP-GlcNAc as the donor substrate, confirming that CsaA is the functional UDP-N-acetyl-d-glucosamine-2-epimerase and CsaB the functional poly-ManNAc-1-phosphate-transferase. Subsequently, CsaB was shown to transfer ManNAc-1P onto O-6 of the non-reducing end sugar of priming oligosaccharides, to prefer non-O-acetylated over O-acetylated primers, and to efficiently elongate the dimer of ManNAc-1-phosphate. The in vitro synthesized CPSA was purified, O-acetylated with recombinant CsaC, and proven to be identical to the natural CPSA by ¹H NMR, ³¹P NMR, and immunoblotting. If all three enzymes and their substrates were combined in a one-pot reaction, nature identical CPSA was obtained. These data provide the basis for the development of novel vaccine production protocols.