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.

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial outer membrane, is sensed by mammalian cells through Toll-like receptor 4 (TLR4), resulting in activation of proinflammatory cytokine pathways. TLR4 recognizes the lipid A moiety of the LPS molecule, and the chemical composition of the lipid A determines how well it is recognized by TLR4. N. meningitidis has been reported to produce lipid A with six acyl chains, the optimal number for TLR4 recognition. Indeed, meningococcal sepsis is generally seen as the prototypical endotoxin-mediated disease. In the present study, we screened meningococcal disease isolates from 464 patients for their ability to induce cytokine production in vitro. We found that around 9% of them were dramatically less potent than wild-type strains. Analysis of the lipid A of several of the low-activity strains by mass spectrometry revealed they were penta-acylated, suggesting a mutation in the lpxL1 or lpxL2 genes required for addition of secondary acyl chains. Sequencing of these genes showed that all the low activity strains had mutations that inactivated the lpxL1 gene. In order to see whether lpxL1 mutants might give a different clinical picture, we investigated the clinical correlate of these mutations in a prospective nationwide observational cohort study of adults with meningococcal meningitis. Patients infected with an lpxL1 mutant presented significantly less frequently with rash and had higher thrombocyte counts, consistent with reduced cytokine induction and less activation of tissue-factor mediated coagulopathy. In conclusion, here we report for the first time that a surprisingly large fraction of meningococcal clinical isolates have LPS with underacylated lipid A due to mutations in the lpxL1 gene. The resulting low-activity LPS may have an important role in virulence by aiding the bacteria to evade the innate immune system. Our results provide the first example of a specific mutation in N. meningitidis that can be correlated with the clinical course of meningococcal disease.

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Opacity-associated (Opa) proteins are outer membrane proteins which play a critical role in the adhesion of pathogenic Neisseria spp. to epithelial and endothelial cells and polymorphonuclear neutrophils. The adherence is mainly mediated by the CD66-epitope-containing members of the carcinoembryonic-antigen family of human cell-adhesion molecules (CEACAM). For the analysis of the specific interactions of individual Opa proteins with their receptors, pure protein is needed in its native conformation. In this study, we describe the isolation and structural analysis of opacity proteins OpaJ129 and OpaB128 derived from Neisseria meningitidis strain H44/76. When the Opa proteins were produced with the phoE signal sequence in Escherichia coli, they were localized at the cell surface and the recombinant bacteria were found to specifically interact with CEACAM1. For refolding and purification, the proteins were overproduced without their signal sequences in E. coli, resulting in its cytoplasmic accumulation in the form of inclusion bodies. After solubilization of the inclusion bodies in urea, the proteins could be folded efficiently in vitro, under alkaline conditions by dilution in ethanolamine and the detergent n-dodecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate (SB12). The structure of the refolded and purified proteins, determined by circular dichroism, indicated a high content of b-sheet conformation, which is consistent with previously proposed topology models for Opa proteins. A clear difference was found between the binding of refolded vs. denatured OpaJ protein to the N-A1 domain of CEA-CAM1. Almost no binding was found with the denatured Opa protein, showing that the Opa-receptor interaction is conformation-dependent.

cMeningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL) on the in vitro induction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs and lpxL1 OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1␤ but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment. G ram-negative bacteria have the ability to naturally shed vesicles from the outer membrane. These spontaneous outer membrane vesicles (sOMVs) are composed of lipids, outer membrane proteins, lipopolysaccharide (LPS), and some periplasmic proteins. Shedding of sOMVs occurs without significant disruption in the integrity of the cell, and release of vesicles is increased by bacterial stress responses (1, 2). Some of the proposed functions of shedding of outer membrane blebs include protection and transport of secreted molecules, interaction with host cells, evasion of immunity by action as a decoy for antibodies and other antibacterial molecules, and action as nucleators in biofilm formation (3).The fact that OMVs are nonreplicating structures that contain many crucial surface components and virulence factors, in combination with pathogen-associated molecular patterns (PAMPs) that trigger innate immune responses, makes them attractive as candidate vaccines. This is evidenced by the successful control of meningococcal serogroup B epidemics using OMV vaccines (4-6) and experimental studies that have explored the vaccine potential of OMVs against other bacterial pathogens (7, 8). The isolation of OMVs from Neiss...