Article

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

Molecular Infectious Diseases Group, John Radcliffe Hospital, Headington, Oxford, UK.
Molecular Microbiology (Impact Factor: 5.03). 12/1995; DOI: 10.1111/j.1365-2958.1995.mmi_18040729.x

ABSTRACT 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 Type: JOURNAL ARTICLE Language: Eng 96414473

0 Bookmarks
 · 
34 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.
    Molecular Microbiology 01/2004; 39(5):1259-1271. · 5.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: NhhA (Neisseria hia homologue) is an outer membrane protein from Neisseria meningitidis, the causative agent of meningococcal disease. The protein is surface exposed and its expression in a wide range of meningococcal strains suggests it is a promising vaccine candidate. In addition, immunization of mice with outer membrane vesicles of strains that overexpress NhhA in conjunction with one of TbpA, Omp85 or NspA results in synergistic bactericidal responses. We previously showed that the NhhA sequence is highly conserved between strains, with the majority of the differences localized to four distinct variable regions located in the amino-terminal region of the mature protein. In this study, N. meningitidis strains were constructed that over-express wild-type NhhA. Strains expressing truncated versions of NhhA, with deletions from the amino-terminal region that removed the most variable regions, were also made. These expression strains were also modified so that immunodominant, phase- and antigenically-variable outer membrane proteins were not expressed, truncated lipooligosaccharide (LOS) expression was genetically fixed (no phase variability), and capsular polysaccharide expression abolished. Outer membrane vesicles derived from these strains were used to immunize mice. As previously observed, a synergistic effect involving another antigen, TbpA, was required to demonstrate bactericidal activity. The highest bactericidal response against a heterologous strain was obtained with a truncated variant of NhhA. These results indicate that removal of (a) variable region(s) does not reduce bactericidal responses against NhhA, and that bactericidal targets exist in regions other than the variable N-teminus. This provides the basis for future examination of responses against truncated NhhA in protecting against heterologous NhhA strains, and further evaluation of truncated NhhA as a candidate for inclusion in a vaccine against all serogroups of N. meningitidis.
    PLoS ONE 01/2013; 8(9):e72003. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We evaluated the adjuvant effect of a modified glycoform of LPS (LgtB-LpxL1) as compared to the non-modified glycoform Lpxl1 serogroup B meningococcal H44/76 native outer membrane vesicles (nOMVs), on immune responses to vaccination with recombinant meningococcal protein, rPorA, tetanus toxoid or meningococcal serogroup C capsular polysaccharide. We used LgtB-LpxL1 LPS because the disruption of lgtB, which results in the exposure of N-acetylglucosamine-galactose-glucose residues in the LPS outer core, has been shown to enhance the activation of human dendritic cells in vitro. Responses were compared to a monophosphoryl lipid A (MPL)-based adjuvant and to aluminium hydroxide suspension. The nOMVs induced serum IgG responses against each of the three antigens comparable to those obtained with MPL or aluminium salt. However, nOMVs elicited (i) a lower IgG1/IgG2a ratio against rPorA, and (ii) serum bactericidal antibody titres superior to those achieved with aluminium salt, reaching similar titers to those obtained with MPL. Similarly, bactericidal antibody titres induced following immunisation with meningococcal serogroup C polysaccharide and nOMVs were similar to those obtained using MPL, but better than with aluminium salt. Immunisation with tetanus toxoid and nOMVs resulted in similar levels of tetanus toxoid-specific IgG as when adjuvanted with aluminium salt. These results highlight the potential utility of meningococcal LpxL1 LPS-containing nOMVs as an adjuvant for recombinant meningococcal protein vaccines, as well as suggest their possible use with a variety of other antigens.
    Clinical and vaccine Immunology: CVI 12/2013; · 2.60 Impact Factor