The structure of Neisseria meningitidis lipid A determines outcome in experimental meningococcal disease

Abstract

Lipopolysaccharide (LPS), a major component of the meningococcal outer membrane, is sensed by the host through activation of Toll-like receptor 4 (TLR4). Recently, we demonstrated that a surprisingly large fraction of Neisseria meningitidis disease isolates are lipid A mutants, due to inactivating mutations in the lpxL1 gene. The lpxL1 mutants activate human TLR4 much less efficiently than wild-type bacteria, which may be advantageous by allowing them to escape from the innate immune system. Here we investigated the influence of lipid A structure on virulence in a mouse model of meningococcal sepsis. One limitation, however, is that murine TLR4 recognizes lpxL1 mutant bacteria much better than human TLR4. We show that an lpxL2 mutant, another lipid A mutant lacking an acyl chain at a different position, activates murine TLR4 less efficiently than the lpxL1 mutant. Therefore, the lpxL2 mutant in mice might be a better model for infections with lpxL1 mutants in humans. Interestingly, we found that the lpxL2 mutant is more virulent in mice than the wild-type strain, whereas the lpxL1 mutant is actually much less virulent than the wild-type strain. These results demonstrate the crucial role of N. meningitidis lipid A structure in virulence.

FIG. 1.

Characterization of N. meningitidis strains L8 H44/76, L8 lpxL1, and L8 lpxL2. (A) Schematic structure of LPS from the wild-type strain H44/76 (immunotype L8). The acyl chains missing in the lpxL1 and lpxL2 mutants are indicated with green and blue, respectively. (B and C) Growth of strain H44/76 and the lpxL1 and lpxL2 mutants in whole blood from C57BL/6JIco mice. Results of one of two independent experiments are shown. Data are expressed as means of triplicates, and error bars indicate standard errors of the means (SEM). The outcome for an initial dose of approximately 1 × 10³ CFU/ml is shown in panel B and that for an initial dose of approximately 1 × 10⁵ CFU/ml in panel C. (D) Outer membrane complex proteins of strain H44/76 and the lpxL1 and lpxL2 mutants separated by SDS-PAGE and stained with Coomassie blue.

FIG. 2.

Comparison of murine TLR4 activation by the lpxL mutants and the wild-type strain. HEK293 cells transfected with mouse TLR4, MD-2, and CD14 were stimulated with different concentrations of heat-inactivated whole H44/76 bacteria, lpxL1 bacteria, lpxL2 bacteria, lpxA bacteria, or purified LPS from these strains. After overnight incubation, the IL-8 concentration in the supernatant was measured by ELISA. Results of one representative experiment of three independent experiments are shown. Data are expressed as means of triplicates, and error bars indicate SEM.

FIG. 3.

Activation of J774A.1 cells with H44/76 and the lpxL1, lpxL2, and lpxA mutants. J774A.1 cells were stimulated overnight with titrations of heat-inactivated strains. Concentrations of IL-6 and IP-10 in the supernatant were determined by ELISA. Results of one representative experiment of three independent experiments are shown. Data are expressed as means of triplicates, and error bars indicate SEM.

FIG. 4.

Disease severity in mice infected with strain H44/76 or the lpxL1 or lpxL2 mutant. After infection, the health of the animals was monitored regularly, and a heath score was given at each time point. The score ranged from healthy (0) to death (6). There were three mice per group. (A and B) Health scores 19 h after infection (A) and 25 h after infection (B). (C) Percentage of survivors in each group after 25 h. Animals with a score of 5 that were killed at this time point were counted as nonsurvivors. Results of one representative experiment of two independent experiments are shown. Data are expressed as means for three mice, and error bars indicate SEM. Asterisks indicate that the group receiving the lpxL mutant was significantly different from the group receiving the same dose of wild-type strain H44/76. *, P < 0.05; **, P < 0.01; ***, indicates P < 0.001.

FIG. 5.

Number of bacteria in blood after infection. Samples of blood were taken from mice infected with strain H44/76 or the lpxL1 or lpxL2 mutant at 2 h (A) and 19 h (B) after administration. Serial dilutions of blood in PBS were plated to determine CFU. Results of one representative experiment of two independent experiments are shown. Data are expressed as means for three mice, and error bars indicate SEM. Asterisks indicate that the group that received an lpxL mutant was significantly different from the group that received the same dose of wild-type strain H44/76. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 6.

Cytokine levels in plasma of mice after infection with H44/76 or the lpxL1 or lpxL2 mutant. At 2 h (A) or 19 h (B) after infection with H44/76 or the lpxL1 or lpxL2 mutant, blood samples were taken from the mice. Cytokine levels in the plasma were determined with a six-plex Bio-Plex assay (Bio-Rad) containing beads for mouse IL-1β, IL-6, IL-10, IL-12p70, RANTES, and TNF-α. Data are expressed as means for three mice, and error bars indicate SEM. Asterisks indicate that the group that received an lpxL mutant was significantly different from the group that received the same dose of wild-type strain H44/76. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 6.

Cytokine levels in plasma of mice after infection with H44/76 or the lpxL1 or lpxL2 mutant. At 2 h (A) or 19 h (B) after infection with H44/76 or the lpxL1 or lpxL2 mutant, blood samples were taken from the mice. Cytokine levels in the plasma were determined with a six-plex Bio-Plex assay (Bio-Rad) containing beads for mouse IL-1β, IL-6, IL-10, IL-12p70, RANTES, and TNF-α. Data are expressed as means for three mice, and error bars indicate SEM. Asterisks indicate that the group that received an lpxL mutant was significantly different from the group that received the same dose of wild-type strain H44/76. *, P < 0.05; **, P < 0.01; ***, P < 0.001.