Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide

Abstract

Neisseria meningitidis contains a very potent hexa-acylated LPS that is too toxic for therapeutic applications. We used systematic molecular bioengineering of meningococcal LPS through deletion of biosynthetic enzymes in combination with induction of LPS modifying enzymes to yield a variety of novel LPS mutants with changes in both lipid A acylation and phosphorylation. Mass spectrometry was used for detailed compositional determination of the LPS molecular species, and stimulation of immune cells was done to correlate this with endotoxic activity. Removal of phosphethanolamine in lipid A by deletion of lptA slightly reduces activity of hexa-acylated LPS, but this reduction is even more evident in penta-acylated LPS. Surprisingly, expression of PagL deacylase in a penta-acylated lpxL1 mutant increased LPS activity, contradicting the general rule that tetra-acylated LPS is less active than penta-acylated LPS. Further modification included expression of lpxP, an enzyme known to add a secondary 9-hexadecenoic acid to the 2' acyl chain. The LpxP enzyme is temperature-sensitive, enabling control over the ratio of expressed modified hexa- and penta-acylated LPS by simply changing the growth temperature. These LPS derivatives display a broad range of TLR4 activity and differential cytokine induction, which can be exploited for use as vaccine adjuvant or other TLR4-based therapeutics.

Figure 1. Applied lipid A modification enzymes.

The structural changes made by the various combinations of modifying enzymes are depicted in color (A). LpxL1 (green), LpxL2 (blue), LpxP (pink) and LptA (brown) all add the corresponding group to the molecule, whereas PagL (red) and LpxE (orange) remove the group. The abbreviation of the enzymes, organism source and activity are presented (B) and the acylation pattern and number of phosphate and phosphoethanolamine groups for each strain are indicated (C).

Figure 2. Charge deconvoluted ESI-FT mass spectra of LPS.

The charge deconvoluted ESI-FT mass spectra of the LPS isolated from twelve different strains of Neisseria meningitidis are shown as follows: parent HB-1 strain (A), ΔlpxL1 (B), ΔlpxL2 (C), pagL (D), ΔlpxL1-pagL (E), ΔlpxL2-pagL (F), ΔlpxL1-lpxP cultured at 30 °C for 5 h (G) or at 25 °C overnight (H), ΔlptA (I), ΔlptA-ΔlpxL1 (J), ΔlptA-pagL (K) and ΔlptA-lpxE (L). A simplified representation of the LPS structure assigned to the ion of 3408.507 u is included in mass spectrum (A). The vertical line at a mass of 3408.514 u, which corresponds to the calculated molecular mass of this latter LPS species, is used as a reference to indicate LPS composition assigned to other ion signals. See Supplementary Table 1 for detailed LPS composition proposals. All annotations refer to monoisotopic masses of the neutral molecules.

Figure 3. TLR4 activation by N. meningitidis strains.

HEK-blue hTLR4 cells were stimulated with 5-fold serial dilutions of heat-inactivated N. meningitidis for 20 h. TLR4 activation was measured by detection of secreted alkaline phosphatase. Results of serial dilutions are depicted in a line graph (A) and for a single OD_600 nm of 0.0004 in a bar graph (B). Data are expressed as mean values or mean ± SD of three independent experiments. Statistical significance was determined with an ANOVA test comparing against HB-1. *p < 0.05.; **p < 0.001.

Figure 4. TLR4 activation by purified LPS.

HEK-blue hTLR4 cells were stimulated with 10-fold serial dilutions of 12 different LPS mutants. TLR4 activation was measured by detection of secreted alkaline phosphatase. Data shown are from one representative experiment out of three independent experiments with similar outcomes and are depicted as the mean ± SEM of triplicates.

Figure 5. Cytokine release of MM6 cells stimulated with purified LPS.

MM6 cells were incubated with 10-fold serial dilution of different LPS mutants for 20 h. IL-6 (A), IL-1β (B), IP-10 (C), MCP-1 (D) production was measured by ELISA. IL-6 and IL-1β are considered MyD88 dependent cytokines and IP-10 and MCP-1 are more TRIF dependent. Cytokine levels of MM6 cells stimulated with 5 ng/ml LPS are also presented as percentages of the HB-1 strain (E) and cytokine ratios in concentration (F) and percentages (G). For the cytokine ratios (F,G) the background without LPS stimulation was subtracted. Data shown are depicted as the mean values of two independent experiments. Statistical significance was determined with a 2-way ANOVA test comparing against HB-1. *p < 0.05.