PMC

(A) Alphabetic list of enzymes required and the corresponding gene codes in the C. canimorsus 5 genome are listed. (B) Proposed enzymatic modification on lipid A by LpxF, LpxE and EptA. (C) Single steps in the biosynthesis of C. canimorsus lipid A-Kdo (adapted from KEGG map00540).

¹H,¹³C-HSQC spectrum (700 MHz) of lipid A in chloroform-methanol-water (20∶10∶1, v/v/v) at 27°C. The corresponding parts of the ¹³C and ¹H NMR spectra are displayed along the F1 and F2 axes, respectively. Numerals refer to atoms in sugar and acyl chain residues denoted by letters as shown in Supplementary and . Signals from an unidentified contaminating lipid are indicated by X.

Ionic interactions or hydrogen bonds involving the 4′ phosphate or the Kdo carboxy group in LPS lacking a 4′ phosphate enable the binding of lipid A to either LBP (1.), soluble CD14 (sCD14) (2.) or via an intermediate state to MD-2 (3.). Dependent on the type of lipid A bound to MD-2 this leads to TLR4 multimerization (4.), a downstream signaling cascade and finally release of proinflammatory cytokines (5.).

(A) Front and side view of the equilibrated complexes between MD-2 (gray) and C. canimorsus (yellow) and E. coli (green) lipid A. (B) Pairwise decomposition of the global total (Van der Waals+electrostatic+solvation) binding free energy calculated at MM-GBSA level. (C) Binding energy between MD-2 and the two lipid A molecules calculated using the MM-GBSA and MM-PBSA methods on 300 snapshots extracted from two 10 ns long equilibrated NPT molecular dynamics simulations.

(A–B) HEKBlue human TLR4 cells were preincubated for 3 h with purified lipid A, LA-core or LPS samples at the concentration indicated. Then the cells were stimulated with 5 ng/ml E. coli O111 LPS for further 20–24 h and TLR4 dependent NFκB activation was measured. (C–D) Human THP-1 macrophages were preincubated for 3 h with purified lipid A, LA-core or LPS samples at the concentration indicated. Then the cells were stimulated with 1 ng/ml E. coli O111 LPS for further 20 h and TNFα release was measured. Data were combined from n = 3 independent experiments, error bars indicated are standard error of the mean.

(A–B) Dose-response curve of purified lipid A, LA-core or LPS. Samples were assayed for TLR4 dependent NFκB activation with HEKBlue human TLR4 cells. (C–D) Purified lipid A, LA-core or LPS samples were assayed for induction of TNFα release by human THP-1 macrophages. (E) Purified LPS samples or lipid IV_A were assayed for induction of IL-6 release by canine DH82 macrophages. (F) Dose-response curve of NFκB activation by lipid A or LPS. Purified lipid A or LPS samples were assayed for TLR4-dependent NFκB activation with HEKBlue human TLR4 cells. The C. canimorsus lipid A stock solution was pretreated with either 0.1% TEN or 50% DMSO and sonication to increase its solubility in water/buffer. Identical concentrations of DMSO or TEN were added to E. coli O111 LPS as a control. Data were combined from n = 3 independent experiments, error bars indicated are standard error of the mean.

(A) C. canimorsus lipid A consists of a β-(1′→6)-linked GlcN3N′-GlcN disaccharide, to which 3-hydroxy-15-methylhexadecanoic acid, 3-hydroxy-13-methyltetradecanoic acid, 3-O-(13-methyltetradecanoyl)-15-methylhexadecanoic acid, and 3-hydroxyhexadecanoic acid are attached at positions 2, 3, 2′, and 3′, respectively. The disscharide carries a positively charged ethanolamine at the 1 phosphate and lacks a 4′ phosphate. (B) Structure of E. coli hexa-acylated lipid A. (C) C. canimorsus LPS core features only one Kdo, to which a phosphoethanolamine (P-Etn) is attached. The only net negative charge present is from the carboxy group of the Kdo. The inner core continues with Man to which another a P-Etn is attached. The outer core consists of Gal and l-Rhamnose (l-Rha), to which the O-antigen is attached (U. Zähringer, unpublished results).

Soluble human MD-2 from cell culture supernatant was combined with the indicated mixture of E. coli LPS-Biotin and a competitor (either C. canimorsus lipid A, lipid A-core, E. coli O111 LPS, penta-acyl E. coli lipid A or lipid IV_A). Biotinylated E. coli LPS-MD-2 complexes were purified and analyzed by non-reducing, denaturing Western blotting for presence of MD-2. (A) Untreated human MD-2 did not bind to the Strep-column (lane 1), addition of E. coli LPS-biotin lead to co-purification of human MD-2 (lane 2). Results shown are representative of three independent determinations. (B) Quantification of Western-blots as shown in A. Values are shown as percentage of the corresponding positive control. Data were combined from n = 3 independent experiments, error bars indicated are standard error of the mean. (C) as in (A) but the C. canimorsus lipid A stock solution was pretreated with either 0.1% TEN or 50% DMSO and sonication in both cases to increase its solubility in water/buffer.