Neisseria meningitidis strains express a diverse range of lipopolysaccharide (LPS) structures tha... more Neisseria meningitidis strains express a diverse range of lipopolysaccharide (LPS) structures that have been classified into 12 immunotypes. A feature of meningococcal LPS is the reversible, high-frequency switching of expression (phase variation) of terminal LPS structures. A number of studies are strongly suggestive of a key role for these terminal structures, and their phase-variable expression, in pathogenesis. In a previous study, a locus of three LPS biosynthetic genes, lgtABE, involved in the biosynthesis of one of these terminal structures, lacto-N-neotetraose, was described. The molecular mechanism of phase-variable expression of this structure is by high-frequency mutation in a homopolymeric tract of G residues in the lgtA gene. To investigate the genetic basis of the structural differences between the immunotypes, and the potential for strains to express alternative immunotypes, this locus was examined in all of the immunotype strains. Initially, the lgt locus of strain 126E, an L1 immunotype strain, was cloned and sequenced, revealing two active genes, lgtC and lgtE. The remnants of the lgtA and lgtB genes and an inactive lgtD gene were also present, indicating that the locus may have once contained five active genes, similar to a locus previously reported in Neisseria gonorrhoeae strain F62. Probes based on each of the lgt genes (ABCDE), and the recently reported lgtG gene, were used to determine the presence or absence of lgt genes within individual strains, allowing the prediction of the phase variation repertoire of these strains. Sequencing to determine the nature of homopolymeric tract regions within the lgt genes was carried out to establish the potential for LPS switching. In general, the set of strains examined could be sorted into two distinct groups : one group which phase-vary the α-chain extension via lgtA or lgtC but cannot make β-chain; the second group phase-vary the β-chain extension via lgtG but do not vary α-chain (lacto-N-neotetraose).
By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the... more By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the icsA gene, which encodes the glycosyltransferase required for adding GlcNAc to Hep-II in the inner core of meningococcal LPS. This gene has homology to several LPS glycosyltransferases, notably to rfaK from Salmonella typhimurium and bplH from Bordetella pertussis, both of which encode GlcNAc transferases. Directly upstream of icsA is an ORF showing significant homology to the hypothetical protein HI0653 from the Haemophilus influenzae genome sequence, and to a lesser degree to putative glycosyltransferases from Streptococcus thermophilus and Yersinia enterocolitica. Insertional inactivation of this ORF resulted in a meningococcal strain with truncated LPS. We have named this new LPS-involved gene icsB. Differences in binding of monoclonal antibodies and in mobility on Tricine-SDS-PAGE showed that LPS from icsA and icsB mutants is similar but not identical. On the basis of these results, we postulated that the new gene encodes the glycosyltransferase required for adding Glc to Hep-I. Structural analysis of purified mutant LPS by electrospray mass spectrometry was used to verify this hypothesis. The composition determined for icsA and icsB is lipidA-(KDO)2-(Hep)2.PEA and lipidA-(KDO)2-(Hep)2.PEA-GlcNAc, respectively. The icsA and icsB genes thus form an operon encoding the glycosyltransferases required for chain elongation from the lipidA-(KDO)2-(Hep)2 basal structure, with IcsA first adding GlcNAc to Hep-II and IcsB subsequently adding Glc to Hep-I. Only then is completion of the lacto-N-neotetraose structure possible through the action of the lgtA–E genes.
Neisseria meningitidis strains express a diverse range of lipopolysaccharide (LPS) structures tha... more Neisseria meningitidis strains express a diverse range of lipopolysaccharide (LPS) structures that have been classified into 12 immunotypes. A feature of meningococcal LPS is the reversible, high-frequency switching of expression (phase variation) of terminal LPS structures. A number of studies are strongly suggestive of a key role for these terminal structures, and their phase-variable expression, in pathogenesis. In a previous study, a locus of three LPS biosynthetic genes, lgtABE, involved in the biosynthesis of one of these terminal structures, lacto-N-neotetraose, was described. The molecular mechanism of phase-variable expression of this structure is by high-frequency mutation in a homopolymeric tract of G residues in the lgtA gene. To investigate the genetic basis of the structural differences between the immunotypes, and the potential for strains to express alternative immunotypes, this locus was examined in all of the immunotype strains. Initially, the lgt locus of strain 126E, an L1 immunotype strain, was cloned and sequenced, revealing two active genes, lgtC and lgtE. The remnants of the lgtA and lgtB genes and an inactive lgtD gene were also present, indicating that the locus may have once contained five active genes, similar to a locus previously reported in Neisseria gonorrhoeae strain F62. Probes based on each of the lgt genes (ABCDE), and the recently reported lgtG gene, were used to determine the presence or absence of lgt genes within individual strains, allowing the prediction of the phase variation repertoire of these strains. Sequencing to determine the nature of homopolymeric tract regions within the lgt genes was carried out to establish the potential for LPS switching. In general, the set of strains examined could be sorted into two distinct groups : one group which phase-vary the α-chain extension via lgtA or lgtC but cannot make β-chain; the second group phase-vary the β-chain extension via lgtG but do not vary α-chain (lacto-N-neotetraose).
By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the... more By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the icsA gene, which encodes the glycosyltransferase required for adding GlcNAc to Hep-II in the inner core of meningococcal LPS. This gene has homology to several LPS glycosyltransferases, notably to rfaK from Salmonella typhimurium and bplH from Bordetella pertussis, both of which encode GlcNAc transferases. Directly upstream of icsA is an ORF showing significant homology to the hypothetical protein HI0653 from the Haemophilus influenzae genome sequence, and to a lesser degree to putative glycosyltransferases from Streptococcus thermophilus and Yersinia enterocolitica. Insertional inactivation of this ORF resulted in a meningococcal strain with truncated LPS. We have named this new LPS-involved gene icsB. Differences in binding of monoclonal antibodies and in mobility on Tricine-SDS-PAGE showed that LPS from icsA and icsB mutants is similar but not identical. On the basis of these results, we postulated that the new gene encodes the glycosyltransferase required for adding Glc to Hep-I. Structural analysis of purified mutant LPS by electrospray mass spectrometry was used to verify this hypothesis. The composition determined for icsA and icsB is lipidA-(KDO)2-(Hep)2.PEA and lipidA-(KDO)2-(Hep)2.PEA-GlcNAc, respectively. The icsA and icsB genes thus form an operon encoding the glycosyltransferases required for chain elongation from the lipidA-(KDO)2-(Hep)2 basal structure, with IcsA first adding GlcNAc to Hep-II and IcsB subsequently adding Glc to Hep-I. Only then is completion of the lacto-N-neotetraose structure possible through the action of the lgtA–E genes.
Uploads
Papers by Marco Krämer