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Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide.

Coulombe F, Divangahi M, Veyrier F, de Léséleuc L, Gleason JL, Yang Y, Kelliher MA, Pandey AK, Sassetti CM, Reed MB, Behr MA - J. Exp. Med. (2009)

Bottom Line: In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion.Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy.Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, McGill University Health Centre, Montreal, Quebec H3G 1A4, Canada.

ABSTRACT
Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) alpha secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

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N-glycolyl MDP is more potent than N-acetyl MDP at inducing RIP2 polyubiquitination and at activating NF-κB and JNK. (A) HEK293 cells were transfected with indicated vectors and a NF-κB luciferase reporter in the presence of various concentrations of N-glycolyl or N-acetyl MDP. Fold increase in NF-κB activation compared with transfected but unstimulated cells was assessed. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05 compared with unstimulated cells; **, P < 0.05 between N-glycolyl versus N-acetyl MDP. (B–E) RAW 264.7 cells were left untreated or treated with various concentrations of N-acetyl or N-glycolyl MDP. (B) Cell lysates were immunoprecipitated (IP) and polyubiquitinated RIP2 proteins were detected by immunoblotting (IB) with the indicated antibody. Total immunoprecipitated RIP2 protein was measured as a control. (C–E) The NF-κB (C), JNK (D), and p38 MAPK (E) activities were measured by immunoblotting with the indicated anti-phospho antibodies. Total IκBα, JNK, p38 MAPK, and α-tubulin protein levels were measured with the indicated antibodies. Representative data from two independent replicates are shown in B–E. Black lines indicate that intervening lanes have been spliced out.
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fig2: N-glycolyl MDP is more potent than N-acetyl MDP at inducing RIP2 polyubiquitination and at activating NF-κB and JNK. (A) HEK293 cells were transfected with indicated vectors and a NF-κB luciferase reporter in the presence of various concentrations of N-glycolyl or N-acetyl MDP. Fold increase in NF-κB activation compared with transfected but unstimulated cells was assessed. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05 compared with unstimulated cells; **, P < 0.05 between N-glycolyl versus N-acetyl MDP. (B–E) RAW 264.7 cells were left untreated or treated with various concentrations of N-acetyl or N-glycolyl MDP. (B) Cell lysates were immunoprecipitated (IP) and polyubiquitinated RIP2 proteins were detected by immunoblotting (IB) with the indicated antibody. Total immunoprecipitated RIP2 protein was measured as a control. (C–E) The NF-κB (C), JNK (D), and p38 MAPK (E) activities were measured by immunoblotting with the indicated anti-phospho antibodies. Total IκBα, JNK, p38 MAPK, and α-tubulin protein levels were measured with the indicated antibodies. Representative data from two independent replicates are shown in B–E. Black lines indicate that intervening lanes have been spliced out.

Mentions: Because mycobacterial PGN contains a mixture of N-glycolylated and N-acetylated muramic acid (Mahapatra et al., 2005), we determined the relative capacity of N-acetyl and N-glycolyl MDP to stimulate NOD2-dependent responses. As shown in Fig. 2 A, both forms of MDP led to activation of NF-κB in HEK293 cells expressing WT NOD2, but N-glycolyl MDP was more stimulatory than N-acetyl MDP at 1 and 0.1 µg/ml. The Crohn's disease (CD)–associated NOD2 variant containing a frame-shift mutation at position 3,020 (NOD2fs) is unresponsive to N-acetyl MDP (Girardin et al., 2003). Likewise, the glycolylated variant was also unable to activate NF-κB via the mutated NOD2 (Fig. 2 A).


Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide.

Coulombe F, Divangahi M, Veyrier F, de Léséleuc L, Gleason JL, Yang Y, Kelliher MA, Pandey AK, Sassetti CM, Reed MB, Behr MA - J. Exp. Med. (2009)

N-glycolyl MDP is more potent than N-acetyl MDP at inducing RIP2 polyubiquitination and at activating NF-κB and JNK. (A) HEK293 cells were transfected with indicated vectors and a NF-κB luciferase reporter in the presence of various concentrations of N-glycolyl or N-acetyl MDP. Fold increase in NF-κB activation compared with transfected but unstimulated cells was assessed. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05 compared with unstimulated cells; **, P < 0.05 between N-glycolyl versus N-acetyl MDP. (B–E) RAW 264.7 cells were left untreated or treated with various concentrations of N-acetyl or N-glycolyl MDP. (B) Cell lysates were immunoprecipitated (IP) and polyubiquitinated RIP2 proteins were detected by immunoblotting (IB) with the indicated antibody. Total immunoprecipitated RIP2 protein was measured as a control. (C–E) The NF-κB (C), JNK (D), and p38 MAPK (E) activities were measured by immunoblotting with the indicated anti-phospho antibodies. Total IκBα, JNK, p38 MAPK, and α-tubulin protein levels were measured with the indicated antibodies. Representative data from two independent replicates are shown in B–E. Black lines indicate that intervening lanes have been spliced out.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2722178&req=5

fig2: N-glycolyl MDP is more potent than N-acetyl MDP at inducing RIP2 polyubiquitination and at activating NF-κB and JNK. (A) HEK293 cells were transfected with indicated vectors and a NF-κB luciferase reporter in the presence of various concentrations of N-glycolyl or N-acetyl MDP. Fold increase in NF-κB activation compared with transfected but unstimulated cells was assessed. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05 compared with unstimulated cells; **, P < 0.05 between N-glycolyl versus N-acetyl MDP. (B–E) RAW 264.7 cells were left untreated or treated with various concentrations of N-acetyl or N-glycolyl MDP. (B) Cell lysates were immunoprecipitated (IP) and polyubiquitinated RIP2 proteins were detected by immunoblotting (IB) with the indicated antibody. Total immunoprecipitated RIP2 protein was measured as a control. (C–E) The NF-κB (C), JNK (D), and p38 MAPK (E) activities were measured by immunoblotting with the indicated anti-phospho antibodies. Total IκBα, JNK, p38 MAPK, and α-tubulin protein levels were measured with the indicated antibodies. Representative data from two independent replicates are shown in B–E. Black lines indicate that intervening lanes have been spliced out.
Mentions: Because mycobacterial PGN contains a mixture of N-glycolylated and N-acetylated muramic acid (Mahapatra et al., 2005), we determined the relative capacity of N-acetyl and N-glycolyl MDP to stimulate NOD2-dependent responses. As shown in Fig. 2 A, both forms of MDP led to activation of NF-κB in HEK293 cells expressing WT NOD2, but N-glycolyl MDP was more stimulatory than N-acetyl MDP at 1 and 0.1 µg/ml. The Crohn's disease (CD)–associated NOD2 variant containing a frame-shift mutation at position 3,020 (NOD2fs) is unresponsive to N-acetyl MDP (Girardin et al., 2003). Likewise, the glycolylated variant was also unable to activate NF-κB via the mutated NOD2 (Fig. 2 A).

Bottom Line: In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion.Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy.Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, McGill University Health Centre, Montreal, Quebec H3G 1A4, Canada.

ABSTRACT
Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) alpha secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

Show MeSH
Related in: MedlinePlus