Burkholderia mallei expresses a unique lipopolysaccharide mixture that is a potent activator of human Toll-like receptor 4 complexes

Abstract

Burkholderia mallei, the aetiologic agent of glanders, causes a variety of illnesses in animals and humans ranging from occult infections to acute fulminating septicaemias. To better understand the role of lipopolysaccharide (LPS) in the pathogenesis of these diseases, studies were initiated to characterize the structural and biological properties of lipid A moieties expressed by this organism. Using a combination of chemical analyses and MALDI-TOF mass spectrometry, B. mallei was shown to express a heterogeneous mixture of tetra- and penta-acylated lipid A species that were non-stoichiometrically substituted with 4-amino-4-deoxy-arabinose residues. The major penta-acylated species consisted of bisphosphorylated d-glucosamine disaccharide backbones possessing two amide linked 3-hydroxyhexadecanoic acids, two ester linked 3-hydroxytetradecanoic acids [C14:0(3-OH)] and an acyloxyacyl linked tetradecanoic acid, whereas, the major tetra-acylated species possessed all but the 3'-linked C14:0(3-OH) residues. In addition, although devoid of hexa-acylated species, B. mallei LPS was shown to be a potent activator of human Toll-like receptor 4 complexes and stimulated human macrophage-like cells (THP-1 and U-937), monocyte-derived macrophages and dendritic cells to produce high levels of TNF-alpha, IL-6 and RANTES. Based upon these results, it appears that B. mallei LPS is likely to play a significant role in the pathogenesis of human disease.

Fig. 1. Analysis of purified LPS preparations

A. SDS-PAGE. The B. mallei ATCC 23344 and GM3773 LPS preparations (1 μg lane⁻¹) were electrophoresed on 12% Express Gels and visualized by silver staining. B. CPS-specific ELISA. Microtiter plate wells were coated with the ATCC 23344 or GM3773 LPS preparations (500 ng well⁻¹) and then incubated with the MCA147 mAb to assay for the presence of CPS. Error bars represent the standard deviation of samples assayed in quadruplicate. The figure is representative of two independent experiments.

Fig. 2

Negative-ion MALDI-TOF mass spectra of lipid A species isolated from purified LPS antigens. (A) B. mallei GM3773 and (B) ATCC 23344 mass spectra. The molecular ion peaks (1–5) are described in Table 1 or in the text. Sodium adducts (Δm/z + 22) are not labelled.

Fig. 3

Positive-ion MALDI-TOF mass spectra of lipid A isolated from B. mallei GM3773. The significance of the major (m/z 956) and minor (m/z 984) oxonium ion peaks are described in the text.

Fig. 4

Predicted structures of the major lipid A species identified in this study. Non-stoichiometric substitutions are depicted in blue (Ara4N) and red [C14:0(3-OH)].

Fig. 5

B. mallei LPS is a potent hTLR4 agonist. HEK 293-hTLR4/MD2-CD14 cells were stimulated with varying concentrations of B. mallei GM3773 (open squares) or E. coli O55:B5 (open circles) LPS following which the culture supernatants were assayed for IL-8 production. HEK 293-hTLR2/CD14 were similarly stimulated with B. mallei (filled squares) or E. coli (filled circles) LPS and the culture supernatants assayed for IL-8 production. Values represent the means ± SD of one experiment assayed in triplicate. The figure is representative of three independent experiments.

Fig. 6

Production of TNF-α, IL-6 and RANTES by LPS stimulated THP-1 cells. THP-1 cells were stimulated for 24 h with 10 ng ml⁻¹ of B. mallei GM3773 or E. coli O55:B5 LPS following which the culture supernatants were assayed for the production of (A) TNF-α (B) IL-6 or (C) RANTES. Unstimulated cells were included as a control. Values represent the means ± SD of four independent experiments assayed in duplicate.

Fig. 7

Production of TNF-α, IL-6 and RANTES by LPS stimulated MDM. MDM cells were stimulated for 24 h with 10 ng ml⁻¹ of B. mallei GM3773 or E. coli O55:B5 LPS following which the culture supernatants were assayed for the production of (A) TNF-α (B) IL-6 or (C) RANTES. Unstimulated cells were included as a control. Values represent the means of two readings. The figures are representative of three independent experiments.

Fig. 8

Production of TNF-α, IL-6, RANTES and IP-10 by LPS stimulated MDDC is CD14-Dependent. Prior to stimulation, the MDDC were incubated for 30 min with an anti-CD14 antibody (black bars) or a non-specific isotype control (grey bars). The cells were then stimulated for 24 h with 10 ng ml⁻¹ of B. mallei GM3773 or E. coli O55:B5 LPS following which the culture supernatants were assayed for the production of (A) TNF-α, (B) IL-6, (C) RANTES or (D) IP-10. Unstimulated cells were included as a control. Values represent the means of two readings. The figures are representative of three independent experiments.