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Host Response and Inflammation | Spotlight

Human NOD2 Recognizes Structurally Unique Muramyl Dipeptides from Mycobacterium leprae

Mirjam Schenk, Sebabrata Mahapatra, Phuonganh Le, Hee Jin Kim, Aaron W. Choi, Patrick J. Brennan, John T. Belisle, Robert L. Modlin
S. Ehrt, Editor
Mirjam Schenk
aDivision of Dermatology, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, California, USA
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Sebabrata Mahapatra
cMycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
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Phuonganh Le
aDivision of Dermatology, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, California, USA
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Hee Jin Kim
cMycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
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Aaron W. Choi
aDivision of Dermatology, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, California, USA
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Patrick J. Brennan
cMycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
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John T. Belisle
cMycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
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Robert L. Modlin
aDivision of Dermatology, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, California, USA
bDepartment of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles, California, USA
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S. Ehrt
Weill Cornell Medical College
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DOI: 10.1128/IAI.00334-16
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  • FIG 1
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    FIG 1

    NOD2L is a potent inducer of IL-32 and DC differentiation. siRNA knockdown of NOD2 in purified human monocytes significantly reduced NOD2 expression, shown as arbitrary units (AU) (A), blocked syn-MDP and live M. leprae induction of IL-32 mRNA, shown as mean fold change (FC) (B), and reduced the induction of CD1b+ DC, shown as the percentage of positive cells (C). Data are represented as means ± standard errors of the means (SEM) (n = 4). Statistical significance was calculated by two-tailed Student's t test. Asterisks indicate statistically significant differences: *, P < 0.05; **, P < 0.01.

  • FIG 2
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    FIG 2

    Induction of IL-32 by M. leprae. (A) Purified human monocytes were cultured with live M. leprae (MOI of 10) or 10 μg/ml of either sonicated bacilli (mLEP-son), digested M. leprae peptidoglycan (mLEP-PG), M. leprae mycolyl-arabinogalactan-peptidoglycan (mAGP), synthetic MDP (syn-MDP), or inactive synthetic MDP (LL-syn-MDP), and IL-32 gene expression was measured. (B) Four enriched fractions of muropeptides derived from mLEP-PG (mLEP-PG F-16, -17, -18, and -19) were tested for their ability to induce IL-32 expression and compared to digested M. leprae peptidoglycan (mLEP-PG) and synthetic MDP (syn-MDP). Data are represented as mean fold change (FC) compared to the medium control (ctrl) ± SEM (n = 6). Statistical significance was calculated by two-tailed Student's t test. Asterisks indicate statistically significant differences compared to media control: *, P < 0.05; **, P < 0.01.

  • FIG 3
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    FIG 3

    Chemical structures of synthetic MDP and M. leprae MDP. (A) Structural differences between the different MDPs are highlighted (box). The first amino acid residue of M. leprae MDP is Gly instead of l-Ala, and the d-Glu residue is amidated [mLEP-MDP(NH2)] or nonamidated (mLEP-MDP). (B) mLEP-MDP(NH2) and mLEP-MDP were synthesized enzymatically, and the molecular structures were confirmed by mass spectrometry: [M+H]+ of 493.2168 m/z belongs to syn-MDP, [M+H]+ of 480.1861 m/z has a calculated molecular formula of C18H29N3O12, which is consistent with the molecular formula of mLEP-MDP, and [M+H]+ of 479.2002 m/z has a calculated molecular formula of C18H30N4O11, which is also the molecular formula of mLEP-MDP(NH2).

  • FIG 4
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    FIG 4

    Cytokine response to structurally distinct MDPs. Purified human monocytes were activated by adding 1 μg/ml of syn-MDP, mLEP-MDP(NH2), or mLEP-MDP. After 24 h, IL-32, IL-1β, and IL-6 induction was measured by mRNA expression as fold change (FC) compared to the medium control (A) and protein secretion (B). Data are represented as means ± SEM (n ≥ 6).

  • FIG 5
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    FIG 5

    Induction of DC differentiation by different MDPs. Purified human monocytes were activated using syn-MDP, mLEP-MDP(NH2), and mLEP-MDP, and DC differentiation was measured by flow cytometry for CD1b expression. A representative histogram (A) and the mean percentage of CD1b-positive cells ± SEM (B) are shown (n = 5).

  • FIG 6
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    FIG 6

    Quantification of NOD2 signaling induced by structurally distinct MDPs. HEK-NOD2 reporter cells were used to quantitatively assess NOD2 activation by stimulating cells with structurally different MDP compounds [syn-MDP, mLEP-MDP-(NH2), and mLEP-MDP] over a range of concentrations (0.001 to 1 μg/ml). Data are represented as mean arbitrary units (AU) ± SEM (n = 6).

  • FIG 7
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    FIG 7

    siNOD2 abolished MDP-induced cytokine response. (A) Purified human monocytes were transfected with siNOD2 or siCtrl, and NOD2 gene expression was measured and is shown in arbitrary units (AU). siNOD2 knockdown monocytes were stimulated with 1 μg/ml of syn-MDP, mLEP-MDP(NH2), or mLEP-MDP and induction of IL-32, IL1B, and IL-6 mRNA, shown as fold change (FC) compared to the medium control (B), and protein expression was measured (C). Data are represented as means ± SEM (n = 4). Statistical significance was calculated by two-tailed Student's t test. Asterisks indicate statistically significant differences compared to the medium control: *, P < 0.05; **, P < 0.01.

Tables

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  • TABLE 1

    Muropeptide composition of the fractions testeda

    Fraction no. and predicted muropeptide structureResidue modification(s)Observed massdAbundance (vol)
    SugarbPeptidec
    17
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP)Anhydro muramic acid4 Amidation1,668.7244256,684
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)Anhydro muramic acid4 Amidation1,810.7952272,907
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP)Deacetylated, anhydro muramic acid5 Amidation1,625.7257370,152
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)NA4 Amidation1,757.768448,216
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)NA4 Amidation1,757.7705523,833
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)NA4 Amidation1,828.8073653,560
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)Anhydro muramic acid4 Amidation1,739.7597804,009
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)Anhydro muramic acid4 Amidation1,810.7961888,780
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)NA4 Amidation1,828.80621,293,773
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)Anhydro muramic acid4 Amidation1,739.76011,449,098
    18
        Monomer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)eNA1 Amidation924.392439,795
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP-Gly/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)Deacetylated3 Amidation1,795.736f11,361
        Monomer (GlcNAc-MurNAc-Gly-d-Glu-DAP)eAnhydro muramic acid1 Amidation835.343416,734
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)Deacetylated3 Amidation, Gly1,866.7642f27,999
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)NA4 Amidation1,828.811517,990
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)Anhydro muramic acid4 Amidation1,739.76317,117
        Monomer (GlcNAc-MurNAc-Gly-d-Glu-DAP)eNA1 Amidation, Gly932.3628f15,261
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP)Deacetylated5 Amidation, methylation1,657.769635,943
        Dimer (GlcNAc-MurNAc-Gly-d-Glu-DAP/GlcNAc-MurNAc-Gly-d-Glu-DAP)NA2 Amidation, Gly1,745.72315,281
    19
        Monomer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)eAnhydro muramic acid2 Amidation, Gly, methylation976.433534,655
        Monomer (GlcNAc-MurNAc-Gly-d-Glu-DAP-d-Ala)eAnhydro muramic acid1 Amidation906.3814200,361
        Monomer (GlcNAc-MurNAc-Gly-d-Glu-DAP)eAnhydro muramic acid2 Amidation, Gly, methylation905.381629,404
        Monomer (GlcNAc-MurNAc-Gly-d-Glu-DAP)eNA1 Amidation932.3689f15,237
    • ↵a The muropeptide fraction obtained from M. leprae peptidoglycan was further fractionated by size exclusion chromatography. The dominant muropeptides from each fraction were identified using LC-MS analysis by interrogating the MS data against a database search. The predicted structures, sugar residue modifications, peptide residue modifications, observed mass, and abundance are indicated for fractions 17 to 19.

    • ↵b Anhydro muramic acid is a potential 1,6-anhydromuramic acid. Deacetylated indicates the loss of an N-acetyl group from one of the sugar residues. NA, not applicable.

    • ↵c Amidation, amidation of carboxylic acid of d-Glu and/or DAP; Gly, d-Glu or DAP residues modified by a glycine residue; methylation, methylation of free carboxylic acid groups of d-Glu or DAP. The number (1 to 5) indicates the number of carboxylic acid residues that are amidated.

    • ↵d All observed masses are H+ adducts unless otherwise noted.

    • ↵e Uncross-linked muropeptide.

    • ↵f Observed mass was a Na+ adduct.

Additional Files

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  • Supplemental material

    • Supplemental file 1 -

      Fig. S1. Preparation of natural and synthetic NOD2 ligands of M. leprae. Fig. S2. Titration of structurally different MDP for IL-32 induction.

      PDF, 175K

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Human NOD2 Recognizes Structurally Unique Muramyl Dipeptides from Mycobacterium leprae
Mirjam Schenk, Sebabrata Mahapatra, Phuonganh Le, Hee Jin Kim, Aaron W. Choi, Patrick J. Brennan, John T. Belisle, Robert L. Modlin
Infection and Immunity Aug 2016, 84 (9) 2429-2438; DOI: 10.1128/IAI.00334-16

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Human NOD2 Recognizes Structurally Unique Muramyl Dipeptides from Mycobacterium leprae
Mirjam Schenk, Sebabrata Mahapatra, Phuonganh Le, Hee Jin Kim, Aaron W. Choi, Patrick J. Brennan, John T. Belisle, Robert L. Modlin
Infection and Immunity Aug 2016, 84 (9) 2429-2438; DOI: 10.1128/IAI.00334-16
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