Modification of the structure of peptidoglycan is a strategy to avoid detection by nucleotide-binding oligomerization domain protein 1

Abstract

Nucleotide-binding oligomerization domain (NOD) protein 1 (NOD1) and NOD2 are pathogen recognition receptors that sense breakdown products of peptidoglycan (PGN) (muropeptides). It is shown that a number of these muropeptides can induce tumor necrosis factor alpha (TNF-alpha) gene expression without significant TNF-alpha translation. This translation block is lifted when the muropeptides are coincubated with lipopolysaccharide (LPS), thereby accounting for an apparently synergistic effect of the muropeptides with LPS on TNF-alpha protein production. The compounds that induced synergistic effects were also able to activate NF-kappaB in a NOD1- or NOD2-dependent manner, implicating these proteins in synergistic TNF-alpha secretion. It was found that a diaminopimelic acid (DAP)-containing muramyl tetrapeptide could activate NF-kappaB in a NOD1-dependent manner, demonstrating that an exposed DAP is not essential for NOD1 sensing. The activity was lost when the alpha-carboxylic acid of iso-glutamic acid was modified as an amide. However, agonists of NOD2, such as muramyl dipeptide and lysine-containing muramyl tripeptides, were not affected by amidation of the alpha-carboxylic acid of iso-glutamic acid. Many pathogens modify the alpha-carboxylic acid of iso-glutamic acid of PGN, and thus it appears this is a strategy to avoid recognition by the host innate immune system. This type of immune evasion is in particular relevant for NOD1.

FIG. 1.

Structure of PGN. (a) Common primary structure of PGN. (b) Variations in the peptide chain of PGN. Residues in parentheses may replace corresponding amino acids. The α-carboxylic acid of iso-d-Glu may be modified by an amide, Gly, or d-Ser. Dab, 2,4-diaminobutyric acid; DAHP, 2,6-diamino-3-hydroxypimelic acid; Hyg, threo-3-hydroxyglutamic acid; Hyl, hydroxylysine; Lan, lanthionine; Orn, ornithine.

FIG. 2.

Structures of synthetic muropeptides.

FIG. 3.

Effect of MDP (muropeptide 2) on dose response of LPS. Mono Mac 6 cells were preincubated with muropeptide 2 (100 μM) (•) or medium (▴) as control for 30 min at 37°C. Increasing concentrations of LPS were added, and after an incubation of 5 h, TNF-α protein was measured by ELISA. TNF-α values were normalized for the E_max of LPS (2,305 pg/ml; 100%). Stimulation with muropeptide 2 alone (100 μM) resulted in a TNF-α concentration of 69 ± 12 pg/ml (3% of control).

FIG. 4.

Induction of TNF-α production by MM6 cells treated with muropeptides 1 to 5 and LPS. Mono Mac 6 cells were preincubated with medium as control, peptides 1 and 2, or lysine-containing muropeptides 3 to 5 (100 μM each) for 30 min at 37°C. After 5 h of stimulation with LPS (EC₅₀; 0.5 ng/ml) TNF-α was determined. TNF-α values were normalized for the EC₅₀ of LPS (100%). Asterisks indicate significant differences from cells incubated with LPS alone (P < 0.05).

FIG. 5.

Induction of TNF-α mRNA by MM6 cells incubated with muropeptides 1 to 5. Mono Mac 6 cells were stimulated with medium alone (control), medium containing LPS (10 and 0.1 ng/ml), peptides 1 and 2, or lysine-containing muropeptides 3 to 5 (100 μM each) for 1.5 h before RNA was isolated for RT-PCR analysis of TNF-α message (40 PCR cycles). mRNA of the 18S ribosomal gene amplified under the same conditions was used as an internal control. Asterisks indicate significant differences from untreated (control) cells (P < 0.05).

FIG. 6.

Response of HEK293T cells expressing NOD2 to muropeptides 1 to 5. Induction of NF-κB activation was determined in triplicate cultures of HEK293T cells transiently transfected with the pcDNA-NOD2 expression vector in the presence of pELAM-Luc, pRL-TK, and pcDNA3 plasmids. Forty-four hours posttransfection, cells were treated with synthetic compounds 1 to 5 (5 μM each) or were left untreated (control). Forty-eight hours posttransfection, NF-κB activation, as firefly luciferase activity relative to Renilla luciferase activity, was determined. In the transfection experiment shown, human TNF-α (10 ng/ml) induced 14.0-fold ± 1.7-fold activation of NF-κB. Asterisks indicate significant differences from untreated (control) cells (P < 0.05).

FIG. 7.

Induction of TNF-α production by MM6 cells treated with muropeptides 6 to 9 and LPS. Mono Mac 6 cells were preincubated with medium as control or DAP-containing muropeptides 6 to 9 (100 μM each) for 30 min at 37°C. After 5 h of stimulation with LPS (EC₅₀; 0.5 ng/ml) TNF-α was determined. TNF-α values were normalized for the EC₅₀ of LPS (100%). Asterisks indicate significant differences from cells incubated with LPS alone (P < 0.05).

FIG. 8.

Induction of TNF-α mRNA by MM6 cells incubated with muropeptides 6 to 9. Mono Mac 6 cells were stimulated with medium alone (control), medium containing LPS (10 and 0.1 ng/ml), or DAP-containing muropeptides 6 to 9 (100 μM each) for 1.5 h before RNA was isolated for RT-PCR analysis of TNF-α message (40 PCR cycles). mRNA of the 18S ribosomal gene amplified under the same conditions was used as an internal control. Asterisks indicate significant differences from untreated (control) cells (P < 0.05).

FIG. 9.

Response of HEK293T cells expressing NOD1 to muropeptides 6 to 9. Induction of NF-κB activation was determined in triplicate cultures of HEK293T cells transiently transfected with the pcDNA-NOD1 expression vector in the presence of pELAM-Luc, pRL-TK, and pcDNA3 plasmids. Forty-four hours posttransfection, cells were treated with the synthetic compounds 6 to 9 (5 μM each) or were left untreated (control). Forty-eight hours posttransfection, NF-κB activation, as firefly luciferase activity relative to Renilla luciferase activity, was determined. In the transfection experiment shown, human TNF-α (10 ng/ml) induced 10.8-fold ± 0.9-fold activation of NF-κB. Asterisks indicate significant differences from untreated (control) cells (P < 0.05).