Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis
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
- SourceAvailable from: Charlene Kahler
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- "LNT formation then depends on the phase variable expression of the β1,3 N-acetylglucosamine transferase, LgtA, that transfers UDP-GlcNAc to the 3 position of the terminal Gal of Galβ1,4→Glcβ1,4→HepI of LOS. The β1,4 galactosyltransferase, LgtB, then transfers UDP-Galactose to the 4 position of the GlcNAc resulting in the LNT structure (Jennings et al., 1995; Wakarchuk et al., 1996). In the absence of lgtA expression, the α-chain can terminate at Galβ1,4→ or "
ABSTRACT: The Gram-negative bacterial cell envelope consists of an inner membrane (IM) that surrounds the cytoplasm and an asymmetrical outer-membrane (OM) that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS), phospholipids, outer membrane proteins (OMPs), and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella, and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation, and isomerization pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, the pathways required for the biosynthesis of the OM and the regulatory circuits that control them have evolved to suit the lifestyle of each organism.Frontiers in Cellular and Infection Microbiology 12/2012; 2:162. DOI:10.3389/fcimb.2012.00162 · 2.62 Impact Factor
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- "Opa outer membrane proteins mediate adherence to host cells and certain variants can promote cellular invasion additionally Opa protein expression can increase resistance to complement-mediated bacteriolysis (Makino et al., 1991, Bos et al., 1997). The LOS and Opa outer membrane protein each rely on ON/OFF phase variation of multiple genes to generate a more diverse repertoire of antigenic variants (Stern et al., 1986, Danaher et al., 1995, Jennings et al., 1995). "
ABSTRACT: Some pathogenic microbes utilize homologous recombination to generate antigenic variability in targets of immune surveillance. These specialized systems rely on the cellular recombination machinery to catalyse dedicated, high-frequency reactions that provide extensive diversity in the genes encoding surface antigens. A description of the specific mechanisms that allow unusually high rates of recombination without deleterious effects on the genome in the well-characterized pilin antigenic variation systems of Neisseria gonorrhoeae and Neisseria meningitidis is presented. We will also draw parallels to selected bacterial and eukaryotic antigenic variation systems, and suggest the most pressing unanswered questions related to understanding these important processes.Molecular Microbiology 08/2011; 81(5):1136-43. DOI:10.1111/j.1365-2958.2011.07773.x · 5.03 Impact Factor
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- "Multiple, independent mutant strains were isolated and confirmed by PCR using the primers preprx and kan_do (Table 2). Previous work has demonstrated that the pUC4Kan kanamycin cassette has no promoter or terminator that is active in Neisseria and will neither affect transcription nor have a polar effect on expression of adjacent genes (Jennings et al., 1995; van der Ley et al., 1997). To rule out the possibility that suppressor mutations may have arisen in 1291oxyR::kan, we constructed a wild-type oxyR 'knock-in' version 'wild type*' of the 1291oxyR::kan mutant strain. "
ABSTRACT: OxyR regulates the expression of the majority of H2O2 responses in Gram-negative organisms. In a previous study we reported the OxyR-dependent derepression of catalase expression in the human pathogen Neis-seria gonorrhoeae. In the present study we used microarray expression profiling of N. gonorrhoeae wild-type strain 1291 and an oxyR mutant strain to define the OxyR regulon. In addition to katA (encod-ing catalase), only one other locus displayed a greater than two-fold difference in expression in the wild type : oxyR comparison. This locus encodes an operon of two genes, a putative peroxiredoxin/ glutaredoxin (Prx) and a putative glutathione oxi-doreductase (Gor). Mutant strains were constructed in which each of these genes was inactivated. A pre-vious biochemical study in Neisseria meningitidis had confirmed function of the glutaredoxin/ peroxiredoxin. Assay of the wild-type 1291 cell free extract confirmed Gor activity, which was lost in the gor mutant strain. Phenotypic analysis of the prx mutant strain in H 2O2 killing assays revealed increased resistance, presumably due to upregula-tion of alternative defence mechanisms. The oxyR, prx and gor mutant strains were deficient in biofilm formation, and the oxyR and prx strains had decreased survival in cervical epithelial cells, indicat-ing a key role for the OxyR regulon in these processes.