No abstract

Two genes homologous to lpxL and lpxM from Escherichia coli and other gram-negative bacteria, which are involved in lipid A acyloxyacylation, were identified in Neisseria meningitidis strain H44/76 and insertionally inactivated. Analysis by tandem mass spectrometry showed that one of the resulting mutants, termed lpxL1, makes lipopolysaccharide (LPS) with penta-instead of hexa-acylated lipid A, in which the secondary lauroyl chain is specifically missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (lpxL2) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but could be readily achieved in a galE mutant expressing a truncated oligosaccharide chain. Structural analysis of lpxL2 mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species. The lpxL1 mutant LPS has retained adjuvant activity similar to wild-type meningococcal LPS when used for immunization of mice in combination with LPS-deficient outer membrane complexes from N. meningitidis but has reduced toxicity as measured in a tumor necrosis factor alpha induction assay with whole bacteria. In contrast, both adjuvant activity and toxicity of the lpxL2 mutant LPS are strongly reduced. As the combination of reduced toxicity and retained adjuvant activity has not been reported before for either lpxL or lpxM mutants from other bacterial species, our results demonstrate that modification of meningococcal lipid A biosynthesis can lead to novel LPS species more suitable for inclusion in human vaccines.

Meningococcal disease severity correlates with circulating concentrations of lipopolysaccharide (LPS) and proinflammatory cytokines. Disruption of the lpxA gene of Neisseria meningitidis generated a viable strain that was deficient of detectable LPS. The potency of wild-type N. meningitidis to elicit tumor necrosis factor (TNF)-alpha production by human monocyte-derived macrophages was approximately 10-fold greater than that of the lpxA mutant. Killed wild-type N. meningitidis and its soluble products induced interleukin (IL)-8 and TNF-alpha secretion by transfected HeLa cells expressing Toll-like receptor (TLR) 4/MD2, but the lpxA mutant was inactive via this pathway. In contrast, both strains induced IL-8 promoter activity in TLR2-transfected HeLa cells. These data provide evidence that N. meningitidis contains components other than LPS that can elicit biological responses via pathways that are independent of the TLR4/MD2 receptor system, and TLR2 is one of these alternate pathways. These findings have implications for future therapeutic strategies against meningococcal disease on the basis of the blockade of TLRs and the modulation of LPS activity.

In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)-de®cient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the ®rst step of lipid A biosynthesis. To study how outer membrane structure and biogenesis are affected by the absence of this normally major component, inner and outer membranes were separated and their composition analysed. The expression and assembly of integral outer membrane proteins appeared largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins was greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards phosphatidylethanolamine and phosphatidylglycerol species containing mostly shorter chain, saturated fatty acids, one of which was unique to the LPS-de®cient outer membrane. The presence of the capsular polysaccharide turned out to be essential for viability without LPS, as demonstrated by using a strain in which LPS biosynthesis could be switched on or off through a tac promoter-controlled lpxA gene. Taken together, these results can help to explain why meningococci have the unique ability to survive without LPS.

SummaryThe pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post-translationally modified by the addition of an O-linked trisaccharide, Gal (␤1-4) Gal (␣1-3) 2,4-diacetimido-2,4,6-trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with welldefined lipopolysaccharide (LPS) or pilin-linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin-linked trisaccharide structure.The pglA mutation, like the galE mutation reported previously, had no effect on pilus-mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (␤1-4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.

The first reaction of lipid A biosynthesis in Gramnegative bacteria is catalyzed by UDP-N-acetylglucosamine (UDP-GlcNAc) O-acyltransferase, the product of the lpxA gene. The reaction involves the transfer of an acyl chain from hydroxyacyl-acyl carrier protein (ACP) to the glucosamine 3-OH position of UDP-GlcNAc. The lipid A isolated from Escherichia coli contains (R)-3-hydroxymyristate at the 3 and 3 positions. Accordingly, LpxA of E. coli is highly selective for (R)-3-hydroxymyristoyl-ACP over ACP thioesters of longer or shorter acyl chains. We now demonstrate that the lpxA gene from Neisseria meningitidis encodes a similar acyltransferase that selectively utilizes 3-hydroxylauroyl-ACP. Strains of E. coli harboring the temperature-sensitive lpxA2 mutation make very little lipid A and lose viability rapidly at 42°C. We have created an E. coli strain in which the chromosomal lpxA2 mutation is complemented by the N. meningitidis lpxA gene introduced on a plasmid. This strain, RO138/pTO6, grows similarly to wild type cells at 42°C and produces wild type levels of lipid A. However, the lipid A isolated from RO138/pTO6 contains mostly hydroxylaurate and hydroxydecanoate in the 3 and 3 positions. The strain RO138/pTO6 is more susceptible than wild type to certain antibiotics at 42°C. This is the first report of an E. coli strain growing with shortened hydroxyacyl chains on its lipid A. The lpxA gene product appears to be a critical determinant of the length of the ester-linked hydroxyacyl chains found on lipid A in living cells.

In Gram‐negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram‐negative bacteria. We first demonstrated that the omp85 gene is co‐transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter‐controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron‐dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.

Lipopolysaccharide (LPS) is one of the major constituents of the gram-negative bacterial cell envelope. Its endotoxic activity causes the relatively high reactogenicity of whole-cell vaccines. Several bacteria harbor LPS-modifying enzymes that modulate the endotoxic activity of the LPS. Here we evaluated whether two such enzymes, i.e., PagP and PagL, could be useful tools for the development of an improved and less reactogenic whole-cell pertussis vaccine. We showed that expression of PagP and PagL in Bordetella pertussis leads to increased and decreased endotoxic activity of the LPS, respectively. As expected, PagP activity also resulted in increased endotoxic activity of whole bacterial cells. However, more unexpectedly, this was also the case for PagL. This paradoxical result may be explained, in part, by an increased release of LPS, which we observed in the PagL-expressing cells.