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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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NOD2-mediated recognition of selected Actinomycetes by macrophages depends on bacterial NamH. (A, left) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were left unstimulated; stimulated with 10 µg/ml N-acetyl MDP alone, LPS alone, or a combination of N-acetyl MDP and LPS; or were infected with various live Gram-negative and -positive organisms. (right) Schematic representation of the effect of NamH on MDP. (B, top) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were either left uninfected or were infected with WT M. smegmatis, namH-disrupted M. smegmatis (M. smegmatisΔnamH), and namH-disrupted M. smegmatis complemented with namH (M. smegmatisΔnamH::namH). (bottom) Ampicillin zone of inhibition assay on WT M. smegmatis and namH variants. In A and B, the amount of TNF-α released in the supernatant after 16 h was quantified by ELISA. Results represent averaged data from two independent replicates (A) or one representative experiment out of three (B). (C) HEK293 cells were transfected with NOD2 and a NF-κB luciferase reporter in the presence of PGN derived from indicated bacteria. The 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.
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fig1: NOD2-mediated recognition of selected Actinomycetes by macrophages depends on bacterial NamH. (A, left) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were left unstimulated; stimulated with 10 µg/ml N-acetyl MDP alone, LPS alone, or a combination of N-acetyl MDP and LPS; or were infected with various live Gram-negative and -positive organisms. (right) Schematic representation of the effect of NamH on MDP. (B, top) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were either left uninfected or were infected with WT M. smegmatis, namH-disrupted M. smegmatis (M. smegmatisΔnamH), and namH-disrupted M. smegmatis complemented with namH (M. smegmatisΔnamH::namH). (bottom) Ampicillin zone of inhibition assay on WT M. smegmatis and namH variants. In A and B, the amount of TNF-α released in the supernatant after 16 h was quantified by ELISA. Results represent averaged data from two independent replicates (A) or one representative experiment out of three (B). (C) HEK293 cells were transfected with NOD2 and a NF-κB luciferase reporter in the presence of PGN derived from indicated bacteria. The 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.

Mentions: To address the effect of NOD2 on recognition of diverse bacteria, peritoneal macrophages derived from naive WT or Nod2-deficient mice were infected with a panel of live Gram-negative and -positive organisms to measure TNF-α secretion. As shown by others, naive macrophages produced undetectable levels of TNF-α in response to N-acetyl MDP alone, and synergistic NOD2-dependent TNF-α after co-stimulation with MDP and LPS (Fig. 1 A). After infection with Gram-negative Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa, and Gram-positive Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes, TNF-α levels did not depend on NOD2 (Fig. 1 A). Conversely, as previously described for mycobacterial infection (Ferwerda et al., 2005; Ferwerda et al., 2007; Gandotra et al., 2007; Divangahi et al., 2008; Leber et al., 2008), TNF-α production was significantly reduced in Nod2-deficient cells after infection with different mycobacterial species (Fig. 1 A). NOD2-dependent recognition extended to other Actinomycetales class members (Nocardia asteroides and Rhodococcus equi) but not Streptomyces sp. (Fig. 1 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)

NOD2-mediated recognition of selected Actinomycetes by macrophages depends on bacterial NamH. (A, left) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were left unstimulated; stimulated with 10 µg/ml N-acetyl MDP alone, LPS alone, or a combination of N-acetyl MDP and LPS; or were infected with various live Gram-negative and -positive organisms. (right) Schematic representation of the effect of NamH on MDP. (B, top) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were either left uninfected or were infected with WT M. smegmatis, namH-disrupted M. smegmatis (M. smegmatisΔnamH), and namH-disrupted M. smegmatis complemented with namH (M. smegmatisΔnamH::namH). (bottom) Ampicillin zone of inhibition assay on WT M. smegmatis and namH variants. In A and B, the amount of TNF-α released in the supernatant after 16 h was quantified by ELISA. Results represent averaged data from two independent replicates (A) or one representative experiment out of three (B). (C) HEK293 cells were transfected with NOD2 and a NF-κB luciferase reporter in the presence of PGN derived from indicated bacteria. The 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: NOD2-mediated recognition of selected Actinomycetes by macrophages depends on bacterial NamH. (A, left) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were left unstimulated; stimulated with 10 µg/ml N-acetyl MDP alone, LPS alone, or a combination of N-acetyl MDP and LPS; or were infected with various live Gram-negative and -positive organisms. (right) Schematic representation of the effect of NamH on MDP. (B, top) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were either left uninfected or were infected with WT M. smegmatis, namH-disrupted M. smegmatis (M. smegmatisΔnamH), and namH-disrupted M. smegmatis complemented with namH (M. smegmatisΔnamH::namH). (bottom) Ampicillin zone of inhibition assay on WT M. smegmatis and namH variants. In A and B, the amount of TNF-α released in the supernatant after 16 h was quantified by ELISA. Results represent averaged data from two independent replicates (A) or one representative experiment out of three (B). (C) HEK293 cells were transfected with NOD2 and a NF-κB luciferase reporter in the presence of PGN derived from indicated bacteria. The 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.
Mentions: To address the effect of NOD2 on recognition of diverse bacteria, peritoneal macrophages derived from naive WT or Nod2-deficient mice were infected with a panel of live Gram-negative and -positive organisms to measure TNF-α secretion. As shown by others, naive macrophages produced undetectable levels of TNF-α in response to N-acetyl MDP alone, and synergistic NOD2-dependent TNF-α after co-stimulation with MDP and LPS (Fig. 1 A). After infection with Gram-negative Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa, and Gram-positive Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes, TNF-α levels did not depend on NOD2 (Fig. 1 A). Conversely, as previously described for mycobacterial infection (Ferwerda et al., 2005; Ferwerda et al., 2007; Gandotra et al., 2007; Divangahi et al., 2008; Leber et al., 2008), TNF-α production was significantly reduced in Nod2-deficient cells after infection with different mycobacterial species (Fig. 1 A). NOD2-dependent recognition extended to other Actinomycetales class members (Nocardia asteroides and Rhodococcus equi) but not Streptomyces sp. (Fig. 1 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