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. 2001 Oct;69(10):5981-90.
doi: 10.1128/IAI.69.10.5981-5990.2001.

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

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

P van der Ley et al. Infect Immun. .
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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.

Figures

FIG. 1
FIG. 1
Tricine-SDS-PAGE analysis of LPS from H44/76 wild type (lane 1) and its lpxL1 derivative (lane 2) and from H44/76 galE (lane 3) and its lpxL2 derivative (lane 4).
FIG. 2
FIG. 2
Structural analysis by MS of lipid A from wild-type H44/76 and its lpxL1 and lpxL2 mutants. In positive ion mass spectra of lipid A, oxonium ions have been shown to be formed (6). These oxonium ions are considered to be the distal ion due to charge transfer to the left sugar ring and can therefore be used to discriminate between substitutions at the two GlcN residues. (A) In the positive-ion ms/ms spectrum of the parent ion 1757 of H44/76 wild type, the oxonium ion is 848 atomic mass units. (B) In the positive-ion ms/ms spectrum of the parent ion 1654 of the lpxL1 mutant, the oxonium ion is 666 atomic mass units, showing that the C12 acyloxyacyl chain is missing from the left sugar ring. (C) In the positive-ion mass spectrum of the lpxL2 mutant, a major tetra-acylated lipid A species corresponding to a mass of 1596 and a minor penta-acylated lipid A species corresponding to a mass of 1611 were found. In the positive-ion ms/ms spectrum of the 1611 ion no oxonium ion could be found, so the position of the remaining C12 chain in this minor species could not be established (results not shown).
FIG. 2
FIG. 2
Structural analysis by MS of lipid A from wild-type H44/76 and its lpxL1 and lpxL2 mutants. In positive ion mass spectra of lipid A, oxonium ions have been shown to be formed (6). These oxonium ions are considered to be the distal ion due to charge transfer to the left sugar ring and can therefore be used to discriminate between substitutions at the two GlcN residues. (A) In the positive-ion ms/ms spectrum of the parent ion 1757 of H44/76 wild type, the oxonium ion is 848 atomic mass units. (B) In the positive-ion ms/ms spectrum of the parent ion 1654 of the lpxL1 mutant, the oxonium ion is 666 atomic mass units, showing that the C12 acyloxyacyl chain is missing from the left sugar ring. (C) In the positive-ion mass spectrum of the lpxL2 mutant, a major tetra-acylated lipid A species corresponding to a mass of 1596 and a minor penta-acylated lipid A species corresponding to a mass of 1611 were found. In the positive-ion ms/ms spectrum of the 1611 ion no oxonium ion could be found, so the position of the remaining C12 chain in this minor species could not be established (results not shown).
FIG. 2
FIG. 2
Structural analysis by MS of lipid A from wild-type H44/76 and its lpxL1 and lpxL2 mutants. In positive ion mass spectra of lipid A, oxonium ions have been shown to be formed (6). These oxonium ions are considered to be the distal ion due to charge transfer to the left sugar ring and can therefore be used to discriminate between substitutions at the two GlcN residues. (A) In the positive-ion ms/ms spectrum of the parent ion 1757 of H44/76 wild type, the oxonium ion is 848 atomic mass units. (B) In the positive-ion ms/ms spectrum of the parent ion 1654 of the lpxL1 mutant, the oxonium ion is 666 atomic mass units, showing that the C12 acyloxyacyl chain is missing from the left sugar ring. (C) In the positive-ion mass spectrum of the lpxL2 mutant, a major tetra-acylated lipid A species corresponding to a mass of 1596 and a minor penta-acylated lipid A species corresponding to a mass of 1611 were found. In the positive-ion ms/ms spectrum of the 1611 ion no oxonium ion could be found, so the position of the remaining C12 chain in this minor species could not be established (results not shown).
FIG. 3
FIG. 3
Antibiotic susceptibility of strain H44/76, its lpxL1 and lpxL2 mutants, and the LPS-deficient lpxA mutant. Shown are the diameters of the inhibition zones around filter paper disks containing each of four different hydrophobic antibiotics.
FIG. 4
FIG. 4
TNF-α induction in MM6 cells by whole bacteria of strain H44/76, its lpxL1 and lpxL2 mutants, and the LPS-deficient lpxA mutant. The horizontal axis gives the dilutions made from a bacterial suspension with an OD620 of 1.0. The data shown represent one of several experiments with similar results.
FIG. 5
FIG. 5
Comparison of adjuvant activity of LPS preparations from strain H44/76 and its lpxL1 and lpxL2 mutants and from R. sphaeroides (Rs) when used for immunization of mice together with LPS-deficient OMCs. Shown are antibody titers measured by whole-cell ELISA and bactericidal assay against wild-type strain H44/76. The data represent the average of five mice in each group. Error bars, standard deviations.
FIG. 6
FIG. 6
Schematic representation of the meningococcal lipid A biosynthesis pathway. After acylation of UDP-GlcN by LpxA and LpxD, dimerization by LpxB takes place. Secondary acylation by LpxL2 and LpxL1 (in that order) follows later, presumably after addition of KDO to GlcN II.

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