Identification of a gene (lpt‐3) required for the addition of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria meningitidis and its role in mediating susceptibility to bactericidal killing and opsonophagocytosis

Abstract

SummaryWe have identified a gene, lpt-3, that is required for the addition of phosphoethanolamine to the 3-position (PEtn-3) on the b-chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn-3 substituent is characteristic of the LPS of a majority (ª 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface-accessible epitope. All strains of Nm that have PEtn-3 possess the lpt-3 gene. In some lpt-3-containing strains, the 3-position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt-3 resulted in loss of PEtn-3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt-3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a L9 (Jennings et al., 1983) have been elucidated. There has been significant success in identifying genes involved in LPS biosynthesis, including lgtA, lgtB, lgtE (Jennings et al., 1995a), lgtC (Gotschlich, 1994), rfaC (Stojiljkovic et al., 1997), lgtF, rfaK (Kahler et al., 1996a,b), rfaF (Jennings et al., 1995b) and lgtG (Banerjee et al., 1998). A characteristic of meningococcal LPS is reversible, highfrequency phase variation of its outer core structures mediated by slippage-like mechanisms in homopolymeric DNA tracts present in LPS biosynthetic genes . These homopolymeric tracts are absent in inner core LPS biosynthetic genes such as rfaC, rfaK, rfaF and lgtF, making this structure relatively stable and an attractive candidate for incorporation into a vaccine.In an attempt to investigate LPS epitopes for potential inclusion into a vaccine against serogroup B meningococcal disease, we have previously identified an inner core LPS epitope defined by a monoclonal antibody designated B5 (mAb B5). The cognate epitope of mAb B5 was found to be present on 70% of all Nm strains (Plested et al., 1999). mAb B5 recognises LPS inner core structures that contain phosphoethanolamine (PEtn) attached specifically at the 3-position (PEtn-3) on the b-chain heptose (HepII). L1, L3, L7, L8 and L9 LPS immunotypes of Nm containing PEtn-3 were mAb B5 reactive (B5+), whereas those containing PEtn in an exocyclic position (L2, L4 and L6 immunotypes) or glycoforms that completely lack PEtn (L5 immunotype) were mAb B5 nonreactive (B5-).Recent studies have revealed that antibodies specific for the mAb B5+ epitope are present in sera from infants recovering from invasive meningococcal disease (Plested et al., 2000). These antibodies exhibited functional activity towards meningococci in an in vitro opsonophagocytic (OP) assay . Taken together, these findings indicate that the mAb B5+ epitope is a target for protective antibodies, and that the inner core...

Modification of the Campylobacter jejuni N-Linked Glycan by EptC Protein-mediated Addition of Phosphoethanolamine

“…S4) Influence of pEtN Modification on the N-Linked Glycoproteome-The observation of pEtN on multiple N-linked glycoproteins, several of which are known targets of the human humoral immune response (47,49,52), suggested a possible association with protein immunogenicity. This would also be consistent with studies showing that pEtN is an immunodominant modification of Neisseria meningitidis LOS (53). Coupled with the association of the C. jejuni N-linked glycan itself in reactivity to patient sera (54), we attempted to determine whether pEtN modification influenced immunoreactivity to C. jejuni proteins.…”

“…The addition of pEtN to C. jejuni substrates by E. coli pEtN transferases does have precedent, as E. coli EptA is capable of modifying C. jejuni lipid A (15). It is unclear whether the addition of pEtN will have an influence on vaccine reactivity or efficiency; however, pEtN has been noted as an immunodominant epitope (53), and therefore, care must be taken to ensure glycoprotein vaccines derived from this system either do not display the pEtN moiety or that the pEtN-glycan is harmless to recipients, and immunological reactivity is minimal compared with the intended epitope. Alternatively, these concerns may be overcome by the generation of an E. coli CLM24 derivative, in which the genes encoding endogenous pEtN transferases have been removed to limit background addition of pEtN to glyco-conjugates.…”

Development, Characterization, and Functional Activity of a Panel of Specific Monoclonal Antibodies to Inner Core Lipopolysaccharide Epitopes inNeisseria meningitidis

“…Finally, we sought to produce MAbs that would react with inner core structures that did not require the presence of PEtn at either the 3 or 6 position of HepII. To this end, we immunized mice with the double mutant MC58 lpt3 galE strain, a strain known to be deficient in any addition of PEtn to HepII (24). Three hybridomas producing MAbs that did not require the presence of PEtn at HepII were established as a result of three fusions.…”

“…In previous studies of L3B5 reactivity, it was clear that two genes, lpt3 and lgtG, were critical in determining the structures of the inner core epitopes. The gene lpt3 codes for an LPS PEtn transferase that adds PEtn to position 3 of HepII and competes with lgtG, which encodes the enzyme that adds glucose (Glc) to position 3 of HepII (24). Whether lgtG is switched on or off depends on the number of cytidines in the homopolymeric tract that affects whether the gene is in or out of frame and therefore affects the nature and functionality of the translated product.…”

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…For example, the LPS of pmrAB-constitutive mutants of S. enterica serovar Typhimurium and E. coli contains more of its heptose-pyrophosphate moiety capped by ethanolamine (264,468). The gene (lpt-3) responsible for the addition of the phosphoethanolamine moiety to a heptose residue in the core region of N. meningitidis was identified (392); it is a homolog of the lptA gene involved in the addition of the phosphoethanolamine to lipid A, described above. Schnaitman and Klena (594) reported that a tolC mutant of E. coli produced a similar LPS, with an increased content of pyrophosphorylethanolamine.…”