Liana Steeghs

Meningitis bacterium is viable without endotoxin

The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS) as its outer monolayer. This is anchored to the membrane by lipid A, which is responsible for LPSs activity as an endotoxin. In Escherichia coli, conditionally lethal mutants have been reported for the genes involved in the early steps of lipid A biosynthesis, suggesting that this part of the LPS molecule is essential for bacterial growth. However, we have isolated a mutant of Neisseria meningitidis which is viable in spite of an early block in lipid A biosynthesis that causes a loss of endotoxin activity.

Modification of Lipid A Biosynthesis in Neisseria meningitidis lpxL Mutants: Influence on Lipopolysaccharide Structure, Toxicity, and Adjuvant Activity

ABSTRACT Two genes homologous to lpxL andlpxM 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 agalE 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 lpxL1mutant 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 eitherlpxL 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.

A Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis Elicits Attenuated Cytokine Release by Human Macrophages and Signals via Toll-like Receptor (TLR) 2 but Not via TLR4/MD2

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.

Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant

In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)‐deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP‐GlcNAc acyltransferase required for the first 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‐deficient 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.

The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane.

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.

Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis

The 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 (β1–4) Gal (α1–3) 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 well‐defined 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 (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto‐N‐neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties  towards  DC.  These  properties  were  mediated by interaction of the GlcNAc(β1‐3)‐Gal(β1‐4)‐Glc‐R oligosaccharide outer core of lgtB LPS with the dendritic‐cell‐specific ICAM‐3 grabbing non‐integrin (DC‐SIGN) lectin receptor. Activation of DC‐SIGN with this novel oligosaccharide ligand skewed T‐cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.

Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response

To determine the relative contribution of lipopolysaccharide (LPS) and non‐LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild‐type serogroup B meningococci (strain H44/76), and LPS‐deficient mutant meningococci (strain H44/76[pLAK33]). Stimulation of human peripheral‐blood mononuclear cells with wild‐type and LPS‐deficient meningococci showed that non‐LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β production and virtually all interferon (IFN)‐γ production. Based on tricine sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of LPS in proteinase K‐treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine‐inducing capacity of isolated and purified LPS and LPS‐containing meningococci. At concentrations of >107 bacteria/mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non‐LPS components was additive to that by LPS. Experiments with mice showed that non‐LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non‐LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF‐α, IL‐1β, and IFN‐γ production.

SHORTENED HYDROXYACYL CHAINS ON LIPID A OF ESCHERICHIA COLI CELLS EXPRESSING A FOREIGN UDP-N-ACETYLGLUCOSAMINE O-ACYLTRANSFERASE

The first reaction of lipid A biosynthesis in Gram-negative bacteria is catalyzed by UDP-N-acetylglucosamine (UDP-GlcNAc)O-acyltransferase, the product of the lpxAgene. 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 colicontains (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 thelpxA gene from Neisseria meningitidis encodes a similar acyltransferase that selectively utilizes 3-hydroxylauroyl-ACP. Strains of E. coli harboring the temperature-sensitivelpxA2 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 theN. 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.

Differential Activation of Human and Mouse Toll-Like Receptor 4 by the Adjuvant Candidate LpxL1 of Neisseria meningitidis

ABSTRACT Neisseria meningitidis LpxL1 lipopolysaccharide (LPS) bearing penta-acylated lipid A is considered a promising adjuvant candidate for inclusion in future N. meningitidis vaccines, as it elicits a markedly reduced endotoxic response in human macrophages relative to that in wild-type (hexa-acylated) LPS, while it is an equally effective adjuvant in mice. As dendritic cells (DC) and Toll-like receptors (TLR) are regarded as central mediators in the initiation of an immune response, here we evaluated the ability of LpxL1 LPS to mature and to activate human DC and examined its TLR4-/MD-2-activating properties. Unexpectedly, purified LpxL1 LPS displayed minimal human DC-stimulating properties compared to wild-type LPS. Although whole bacteria induced DC maturation and activation irrespective of their type of LPS, the LpxL1 mutant failed to activate the human recombinant TLR4/MD-2 complex expressed in HeLa cells. Similarly, purified LpxL1 LPS was unable to activate human TLR4/MD-2 and it even acted as an antagonist of wild-type LPS. Both wild-type and LpxL1 LPSs activated the murine TLR4/MD-2 complex, consistent with their abilities to induce maturation and activation of murine DC. Assays with cells transfected with different combinations of human and murine TLR4 and MD-2 indicated that TLR4 was a more-major determinant of the LPS response than MD-2. The species-specific activation of the TLR4/MD-2 complex by LpxL1 LPS may have an impact on the use of LpxL1 LPS as an adjuvant and the use of murine immunization models in human meningococcal vaccine development.