Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia

Abstract

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.

Keywords: Burkholderia; Gram-negative bacteria; TLR4/MD-2; cystic fibrosis; innate immunity; lipopolysaccharide (LPS); molecular modeling.

FIGURE 1.

Structure of B. cenocepacia LPS inner core and lipid A. B. cenocepacia lipid A is heterogeneous, being composed of a mixture of penta- and tetra-acylated species (30, 40). Lipid A fatty acids are two 3-(R)-hydroxyhexadecanoic acids, two 3-(R)-hydroxytetradecanoic acids, and one tetradecanoic acid (30, 40). The dotted lines indicate the non-stoichiometric substitution.

FIGURE 2.

LPS_BC activates the murine and human TLR4·MD-2 complexes. NF-κB activation upon stimulation of HEK293 mTLR4·mMD-2 and hTLR4·hMD-2 after 6 h with 5 ng/ml LPS_BC and LPS_BCΔAra. Stimulation for 6 h with E. coli LPS was used as the control. The data are pooled from three independent experiments done in triplicate. Bars indicate S.D.; significance was calculated in comparison with stimulation with E. coli LPS (*, p < 0.05; **, p < 0.01). Curly brackets indicate significance calculated comparing LPS_BC and LPS_BCΔAra (*, p < 0.05; **, p < 0.01). NS, no stimulation.

FIGURE 3.

Lipid A activation of murine and human TLR4·MD-2 complexes. A, NF-κB activation upon stimulation of HEK293 mTLR4·mMD-2 after 6 h with LA_BC and LA_BCΔAra. Stimulations for 6 h with E. coli LPS, hexa-acylated lipid A, and lipid IV_A were used as controls. The data are pooled from three independent experiments done in triplicate. Bars indicate S.D.; significance was calculated in comparison with stimulation with hexa-acylated E. coli lipid A (*, p < 0.05; **, p < 0.01; ***, p < 0.001); curly brackets indicate significance calculated comparing LPS_BC and LPS_BCΔAra (**p < 0.01). B, NF-κB activation upon stimulation of HEK293 hTLR4·hMD-2 after 6 h with LA_BC and LA_BCΔAra. Stimulations for 6 h with E. coli LPS, hexa-acylated lipid A, and lipid IV_A were used as controls. The data are pooled from three independent experiments done in triplicate. Bars indicate S.D.; significance was calculated in comparison with stimulation with hexa-acylated E. coli lipid A (*, p < 0.05; ***, p < 0.001); curly brackets indicate significance calculated comparing LPS_BC and LPS_BCΔAra (*, p < 0.05). NS, no stimulation.

FIGURE 4.

B. cenocepacia LPS_BCΔAra effects on the E. coli LPS agonist activity. The potential antagonist activity of LPS_BC and LPS_BCΔAra on hexa-acylated E. coli LPS was assayed. NF-κB activation upon stimulation of HEK293 hTLR4 after 1 h with LPS_BC (1, 10, and 100 ng/ml) and LPS_BCΔAra (1, 10, and 100 ng/ml) followed by exposure to E. coli LPS (1 and 10 ng/ml) for 4 h is shown. The data are pooled from three independent experiments done in triplicate. Bars indicate S.D.; significance was calculated in comparison with stimulation with E. coli LPS (1 and 10 ng/ml) (*, p < 0.05; **, p < 0.01). NS, no stimulation.

FIGURE 5.

Proinflammatory and endotoxic potential of LPS_BC in C57Bl/6 mice. From three to five mice per group were challenged via intraperitoneal injection with 300 μg/mouse LPS from E. coli and P. aeruginosa RP73, LPS_BC, and LPS_BCΔAra. TNF-α levels in sera were quantified after 5 h of treatment. Treatment with sterile saline solution was used as control (Ctrl). The data are pooled from two independent experiments. Bars indicate mean ± S.D. Statistical analysis was made for pair wise comparisons (*, p < 0.05; **, p < 0.01).

FIGURE 6.

B. cenocepacia LPS acylation pattern is responsible for the NF-κB activation. NF-κB activation upon stimulation of HEK293 hTLR4·hMD-2 after 6 h with LPS_BC and LPS_BCΔAra is shown. Stimulation for 6 h with E. coli LPS was used as the control. The same protocol was used to stimulate HEK293 hTLR4·hMD-2 V82F cells. The data are pooled from three independent experiments done in triplicate. Bars indicate S.D.; significance was calculated in comparison with E. coli LPS (*, p < 0.05; **, p < 0.01; ***, p < 0.001). NS, no stimulation.

FIGURE 7.

Lys-122 and Lys-125 in the B. cenocepacia LPS signaling on murine and human TLR4·MD-2 complexes. An NF-κB-luciferase reporter assay stimulating HEK293 hTLR4·hMD-2, HEK293 mTLR4·mMD-2, and mTLR4·hMD-2 after 6 h with LPS_BC and LPS_BCΔAra was carried out. Stimulations for 6 h with E. coli LPS and with lipid IV_A were used as controls. The same protocol was used to stimulate HEK293 mTLR4·mMD-2 E1222K and HEK293 mTLR4·mMD-2 E122K/L125K cells. The data are pooled from two independent experiments done in triplicate. Bars indicate S.D.; statistical analysis was calculated for pairwise comparisons (*, p < 0.05; ***, p < 0.001). NS, no stimulation.

FIGURE 8.

Predicted binding mode of the LPS_BC core to hTLR4·MD-2. The computational model from docking followed by MDS is shown. LPS_BC docked to MD-2 is shown in Corey-Pauling-Koltun colors. Superimposed fatty acids chains from E. coli lipid A are shown in different colors. Some representative residues from the MD-2 binding site are represented with carbon atoms in green.

FIGURE 9.

Predicted binding mode of the LPS_BC core to hTLR4·MD-2. Details of LPS_BC (Corey-Pauling-Koltun colors) docked to MD-2 are shown. Superimposed fatty acids chains from E. coli lipid A are shown with carbon atoms in blue.

FIGURE 10.

Predicted binding mode of the LPS_BC core to hTLR4·MD-2. Detail of some H-bond interactions between the LPS_BC inner core and TLR4·MD-2 involving Tyr-102 is shown. LPS_BC is represented in Corey-Pauling-Koltun colors, and MD-2 protein is represented in green.

FIGURE 11.

Superimposition of best dock results of LPS_BC core. The docking was obtained using AutoDock Vina (depicted in red) and E. coli LPS core from Protein Data Bank code 3fxi (depicted in green). TLR4 is not shown for the sake of clarity.

FIGURE 12.

Predicted binding mode of the LPS_BC core to hTLR4·MD-2. The computational model from docking followed by MDS is shown. Left panel, three-dimensional model of the dimer of LPS_BC core in complex with TLR4·MD-2. Right panel, detail of the dimer of LPS_BC inner core in complex with TLR4·MD-2.

FIGURE 13.

Predicted binding mode of the LPS_BC core to hTLR4·MD-2. Details of some interactions between the LPS_BC inner core and TLR4·MD-2 from the computational model are shown.