Identification of Key Residues That Confer <i>Rhodobacter sphaeroides</i> LPS Activity at Horse TLR4/MD-2

<div><p>The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from <i>Rhodobacter sphaeroides</i> (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.</p></div

RSLPS requires specific residues within horse MD-2 and TLR4, yet is independent of CD14.

<p>HEK293 cells were transiently transfected with combinations of human and horse TLR4 and MD-2, with or without horse CD14, and reporter constructs NF-κB-luc and phRG-TK. Cells were stimulated 48 hours later for 6 hours with 100 ng/ml RSLPS, 10 ng/ml ECLPS, 100 ng/ml RSLPS+10 ng/ml ECLPS, or medium alone. Data are from a representative experiment (n = 3 experiments) and expressed as triplicate mean ±SEM for that experiment, relative to the maximum ECLPS response. A) Cells were transfected with different combinations of human and horse TLR4 and MD-2. B) Horse TLR4/MD-2 was transfected with and without CD14. C) MD-2 mutants were transfected with horse TLR4/CD14. D) TLR4 mutants were transfected with horse MD-2/CD14.</p

Chemical structures of lipid A derivatives.

<p>A) Lipid A from <i>E. coli</i>. B) Lipid A synthesis intermediate lipid IVa. C) Lipid A from <i>Rhodobacter sphaeroides</i>.</p

RSLPS activity requires the presence of both R385 and P442 in horse TLR4.

<p>HEK293 cells were transiently transfected with combinations of human and horse TLR4 and MD-2, together with horse CD14 and reporter constructs NF-κB-luc and phRG-TK. Cells were stimulated 48 hours later for 6 hours. Data are from a representative experiment (n = 3 experiments) and expressed as triplicate mean ±SEM for that experiment, relative to the maximum ECLPS response. A) TLR4 point mutants were transfected with horse MD-2/CD14 and stimulated with 100 ng/ml RSLPS, 10 ng/ml ECLPS, 100 ng/ml RSLPS+10 ng/ml ECLPS or medium alone. B) TLR4 point mutants were transfected with horse MD-2/CD14 and stimulated with 1 µg/ml lipid IVa, 1 µg/ml lipid IVa+10 ng/ml ECLPS, or medium alone.</p

RSLA and lipid IVa sit differently within the MD-2 pocket.

<p>A) Docking models of RSLA and lipid IVa bound to horse TLR4/MD-2 were overlaid to assess ligand and receptor positioning. The acyl chains of RSLA (blue) sit more deeply in the MD-2 (pink) pocket than lipid IVa (green), and the R2 chain of RSLA protrudes from the MD-2 pocket to contact TLR4* (grey). The 1-PO₄ is also moved away from TLR4 due to lowering of the diglucosamine backbone. B) Overlay of RSLA (blue; horse model), lipid IVa (green; horse model) and lipid A (red; human crystal) in situ in the MD-2 pocket. TLR4 and MD-2 have been removed for clarity. The PO₄ groups and acyl chains of all three ligands sit somewhat differently to one another within the pocket. Lipid A and RSLA appear to occupy a similar volume within the pocket.</p

The absence of either CD14 or CD36 does not affect the TLR activation triggered by rPCN.

<p>HEK293T were cotransfected with CD14 and/or CD36 along with TLR2/1 (A) or TLR2/6 (B). The total amount of DNA in each transfection was kept constant by adding empty expression vector. The HEK293T cells were stimulated with rPCN (1.25 µg/mL), which was previously incubated with polymyxin to neutralize LPS. The positive controls were Pam3CysSK4 (P3C) for TLR2/1 and FSL-1 for TLR2/6. Medium was used as negative control for cell stimulation (white bars). Results are representative of three independent experiments. Statistical differences were assessed by comparing the response of cells lacking one of the co-receptors to the response of cells expressing both co-receptors, under similar stimuli. Values are the mean ± S.D. *** p<0.001.</p

TLR2 heterodimerization is not critical for the cell activation triggered by rPCN.

<p>HEK293T cells were transfected with CD14 and CD36 along with TLR2, TLR2/TLR1 or TLR2/TLR6. The total amount of DNA in each transfection was kept constant by adding empty expression vector. After 48 h of transfection, cells were stimulated with the agonists: Pam3CysSK4 (P3C) for TLR2/1 and FSL-1 for TLR2/6. Medium was used as negative control for cell stimulation (white bars). rPCN (1.25 µg/mL), previously incubated with polymyxin to neutralize LPS, was assayed. The cell supernatants were analyzed for IL-8 by ELISA. Panel A: Cells transfected with TLR2 and TLR1. Panel B: Cells transfected with TLR2 and TLR6. Results are representative of five independent experiments. Statistical differences were assessed by comparing the response of cells expressing TLR2 to the response of cells expressing TLR2/TLR1 or TLR2/TLR6. Values are the mean ± S.D. * p<0.05; ** p<0.01; *** p<0.001.</p

Therapeutic groups.

a<p>PBS alone.</p><p>Therapeutic groups.</p

Therapeutic administration of rPCN increases proinflammatory cytokine and NO production.

<p>Lung homogenates were analyzed for IL-12p40 (A), IFN-γ (B), TNF-α (C), IL-10 (D), IL-4 (E), and NO (F) concentrations. Data represent the mean and SD of five mice per group; the experiments were performed in triplicate. * p<0.05; ** p<0.01; *** p<0.001 <i>vs.</i> the PBS group.</p

Possible mechanism of the protection against murine paracoccidioidomycosis conferred by paracoccin administration, as suggested by <i>in vivo</i> and <i>in vitro</i> studies.

<p>Once administered to BALB/c mice, before or after inoculation of <i>P. brasiliensis</i> yeasts, rPCN interacts with N-glycans of TLRs on antigen-presenting cells (APC). It triggers IL-12 production, which drives immunity to the Th1 axis. Production of IL-10 is also induced. The consequent balanced Th1 immunity that is developed protects mice against PCM, as manifested by lower incidence of granulomatous lesions and more efficient fungal clearance in the lungs, at day 30 post-infection.</p