The Rockefeller University Press $30.00
J. Exp. Med. Vol. 206 No. 8 1709-1716
BRIEF DEFINITIVE REPORT
In seminal studies, Jules T. Freund reported that
the presence of mycobacteria in a water-in-oil
emulsion containing protein antigen stimulated a
particularly strong delayed hypersensitivity reac-
tion in guinea pigs (Freund et al., 1950; Freund,
1951). Later, it was shown that mycobacterial
peptidoglycan (PGN) could replace whole my-
cobacterial cells in CFA (Lederer et al., 1975)
and that synthetic N-acetylmuramyl-l-alanyl-d-
isoglutamine PGN moiety, usually referred to as
N-acetyl muramyl dipeptide (MDP), presented
adjuvant activity (Ellouz et al., 1974; Kotani
et al., 1975). However, mycobacterial PGN is
unique in that the N-acetyl group at carbon 2
of muramic acid is preferentially hydroxylated
to an N-glycolyl group (Adam et al., 1969) via
the action of N-acetyl muramic acid hydroxy-
lase (NamH), an enzyme present only in certain
aerobic Actinomycetes (Raymond et al., 2005).
Notably, these are the very organisms to which
Freund attributed increased immunogenic activ-
ity (Freund, 1956).
Adjuvants act by stimulating the innate
immune response of the host. The response
to bacterial PGN is mediated largely by NOD1
and NOD2, two members of the cytosolic-
localized Nod-like receptor family, in coopera-
tion with other pattern recognition molecules
such as Toll-like receptors (Kufer and Sansonetti,
2007). NOD2 has been implicated in sens-
ing PGN-derived N-acetyl MDP and shown
to activate the NF-B and mitogen-activated
protein kinase (MAPK) pathways via polyubiq-
uitination of the RIP2 kinase (Girardin et al.,
2003; Yang et al., 2007; Hasegawa et al., 2008).
However, the role of NOD2 in recognition of
the unique mycobacterial N-glycolyl MDP has
not been studied.
Using both bacterial genetics and chemical
synthesis, we have determined the importance
Marcel A. Behr:
Abbreviations used: CD,
Crohn’s disease; JNK, c-Jun
N-terminal kinase; MAPK,
mitogen-activated protein ki-
nase; MDP, muramyl dipeptide;
NamH, N-acetyl muramic acid
hydroxylase; PGN, peptidogly-
can; TDM, trehalose
Increased NOD2-mediated recognition
of N-glycolyl muramyl dipeptide
François Coulombe,1 Maziar Divangahi,1 Frédéric Veyrier,1 Louis de Léséleuc,1
James L. Gleason,2 Yibin Yang,3 Michelle A. Kelliher,3 Amit K. Pandey,4
Christopher M. Sassetti,4 Michael B. Reed,1 and Marcel A. Behr1
1Department of Medicine, McGill University Health Centre, Montreal, Quebec H3G 1A4, Canada
2Department of Chemistry, McGill University, Montreal, Quebec H3A 2K6, Canada
3Department of Cancer Biology and 4Department of Molecular Genetics and Microbiology, University of Massachusetts
Medical School, Worcester MA 01655
Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor
NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomy-
cetes 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 infect-
ing macrophages with 12 bacteria, tumor necrosis factor (TNF) secretion was NOD2 depen-
dent 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 B, 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 ovalbu-
min-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 histori-
cal observation attributing exceptional immunogenic activity to the mycobacterial cell wall.
© 2009 Coulombe et al. This article is distributed under the terms of an Attribu-
tion–Noncommercial–Share Alike–No Mirror Sites license for the first six months
after the publication date (see http://www.jem.org/misc/terms.shtml). After six
months it is available under a Creative Commons License (Attribution–Noncom-
mercial–Share Alike 3.0 Unported license, as described at http://creativecommons
The Journal of Experimental Medicine
N-GLYCOLYL MDP AND NOD2 | Coulombe et al.
shown (Raymond et al., 2005), the Mycobacterium smegmatis
namH mutant was more susceptible to ampicillin than WT M.
smegmatis and the mutant complemented with namH (Fig. 1 B,
bottom). After ex vivo infection of WT and Nod2-deficient
macrophages, NOD2-dependent TNF- production was
abrogated in the absence of namH and restored with gene
complementation (Fig. 1 B).
To verify that NOD2 recognition was sufficient for namH-
dependent TNF- production, we stimulated HEK293 cells
transiently expressing NOD2 and a NF-B luciferase reporter
vector with purified PGN derived from the Actinomycetes
M. tuberculosis (namH positive) and Streptomyces sp. (namH
negative). In this assay, M. tuberculosis–derived PGN, but not
Streptomyces sp.–derived PGN, induced NF-B activation
(Fig. 1 C). Therefore, namH is critical for optimal NOD2-
mediated recognition of mycobacterial PGN.
N-glycolyl MDP is sensed by NOD2 and is more potent
than N-acetyl MDP at activating RIP2, NF-B,
and c-Jun N-terminal kinase (JNK) but not p38 MAPK
Because mycobacterial PGN contains a mixture of N-gly-
colylated 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-B 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 un-
able to activate NF-B via the mutated NOD2 (Fig. 2 A).
The potency of a compound is a measure of its activity,
as expressed by the concentration of compound required to
produce a defined response. To determine the relative po-
tency of the two forms of MDP at activating intracellular
signaling downstream of NOD2, we stimulated macrophages
with increasing concentrations of these compounds ranging
from 0.1 to 10 µg/ml. N-glycolyl MDP was 30-fold more
potent than N-acetyl MDP at inducing polyubiquitination
of RIP2 (Fig. 2 B), and 100-fold more potent than N-
acetyl MDP at inducing phospho-IB (Fig. 2 C). Only
N-glycolyl MDP could induce detectable phospho-JNK
under current experimental conditions (Fig. 2 D). Both
forms of MDP induced similar phosphorylation of p38
MAPK (Fig. 2 E). Collectively, these data indicate that N-
glycolyl MDP is more potent than N-acetyl MDP at acti-
vating NOD2-mediated RIP2 polyubiquitination and
selective downstream pathways.
N-glycolyl MDP is more active than N-acetyl MDP
at inducing synergistic proinflammatory cytokine production
It is known that MDP alone is a potent inducer of TNF-
mRNA, which remains untranslated, and that presence of
LPS abrogates this translation block (Wolfert et al., 2002).
To compare N-glycolyl and N-acetyl MDP, we stimulated
of the bacterial NamH enzyme for NOD2-mediated sensing
and compared the activity of N-acetyl and N-glycolyl MDP
in vitro and in vivo. Our findings identify N-glycolyl MDP as
more stimulatory than N-acetyl MDP at eliciting NOD2-me-
diated immune responses in the context of an intact bacterium
and as a pure compound. Together, these findings indicate
that the NOD2 pathway may be exquisitely tuned to detect
mycobacterial infections, and suggest a likely mechanism to
explain the remarkable adjuvancy of mycobacterial cell walls.
RESULTS AND DISCUSSION
Macrophage recognition of selected Actinomycetes
is NOD2 dependent
To address the effect of NOD2 on recognition of diverse bac-
teria, 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- secretion.
As shown by others, naive macrophages produced undetect-
able levels of TNF- in response to N-acetyl MDP alone, and
synergistic NOD2-dependent TNF- after co-stimulation
with MDP and LPS (Fig. 1 A). After infection with Gram-
negative Escherichia coli, Salmonella typhimurium, and Pseudomo-
nas aeruginosa, and Gram-positive Bacillus cereus, Staphylococcus
aureus, and Listeria monocytogenes, TNF- 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- production was significantly reduced in
Nod2-deficient cells after infection with different mycobacte-
rial 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).
As a trend, Nod2-deficient cells produced less TNF- af-
ter infection with Gram-positive but not Gram-negative or-
ganisms, consistent with the greater quantity of PGN in the
cell wall of Gram-positive bacteria (Yang et al., 2001). How-
ever, the observation that significant NOD2-dependent rec-
ognition was restricted to a subset of aerobic Actinomycetes
led us to ask whether these organisms in particular may share
a common difference in their PGN.
Bacterial NamH is required for optimal
Several differences in PGN amount, location, and structure
exist among bacterial species and have in some instances been
described to alter immunological activity (Stewart-Tull,
1980). A common feature shared by NOD2-stimulatory Ac-
tinomycetes is the elaboration of N-glycolylated PGN via the
action of the NamH hydroxylase that converts the UDP-N-
acetylmuramic acid PGN precursor moiety to UDP-N-
glycolylmuramic acid (UDP-MurNAc + O2 + NADPH +
H+ → UDP-MurNGlyc + NADP+ + H2O; Raymond et al.,
2005). Because hydroxylation of UDP-N-acetylmuramic
acid ultimately affects the structure of MDP (Fig. 1 A, right),
we tested the importance of namH for NOD2-dependent
bacterial recognition through gene disruption. As previously
JEM VOL. 206, August 3, 2009
BRIEF DEFINITIVE REPORT
10- to 20-fold lower concentration. (Fig. 3 A, bottom) The
synergistic response to MDP plus TDM in Nod2+/+ cells was
abrogated in Nod2/ cells (Fig. 3 B). Thus, N-glycolyl
MDP is more stimulatory and more potent than N-acetyl
MDP at inducing proinflammatory cytokine production
upon co-stimulation with nonmycobacteria- and myco-
WT M. smegmatis induces increased NOD2-dependent
macrophage activation compared with namH-deficient
M. smegmatis after i.p. challenge in mice
To investigate the effect of N-glycolyl MDP on in vivo in-
nate inflammatory response in the context of a live bacterial
mouse peritoneal macrophages with various concentrations
of MDP along with a fixed concentration of LPS, and mea-
sured TNF- and IL-6 production (Fig. 3 A, top). At all
concentrations ranging from 0.5 to 10 µg/ml, N-glycolyl
MDP was more stimulatory than N-acetyl MDP. In terms of
potency, N-glycolyl MDP was active at a 10-fold lower
concentration than N-acetyl MDP (0.5 vs. 5 µg/ml). Be-
cause Actinomycetes do not produce LPS, we repeated
this experiment using M. tuberculosis–derived trehalose 6,6-
dimycolate (TDM), a molecule that has been shown to have
synergistic immunological activity in combination with
MDP (Masihi et al., 1985). Again, N-glycolyl MDP was
more stimulatory than N-acetyl MDP and was active at a
Figure 1. NOD2-mediated recognition of selected Actinomycetes by macrophages depends on bacterial NamH. (A, left) Naive peritoneal macro-
phages 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. smegmatisnamH), and namH-disrupted M. smegmatis complemented with namH (M. smegmatisnamH::namH). (bottom) Ampicillin zone
of inhibition assay on WT M. smegmatis and namH variants. In A and B, the amount of TNF- 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-B luciferase reporter in the presence of PGN derived from indicated bacteria. The fold increase in NF-B activation compared with
transfected but unstimulated cells was assessed. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05.
N-GLYCOLYL MDP AND NOD2 | Coulombe et al.
infection, we harvested and cultured peritoneal macrophages
from Nod2+/+ and Nod2/ mice after i.p. stimulation with
WT M. smegmatis, namH-deficient M. smegmatis, and namH-
complemented mutant. 3 d after infection, there was no sig-
nificant difference in the number of colony-forming units in
the spleens of mice infected with the different organisms
(unpublished data). Cells harvested from Nod2+/+ mice that
had been infected with different strains of M. smegmatis spon-
taneously released low levels of TNF- (Fig. 4 A, left).
Nonetheless, cells from mice infected with namH-deficient
M. smegmatis produced significantly less TNF- than cells
from mice infected by either the WT or the complemented
strain. Upon ex vivo restimulation of these cells with live
mycobacteria, TNF- levels were increased, again in a namH-
dependent manner (Fig. 4 A, left). The namH dependence
for both spontaneous cytokine production and restimulated
cells was abrogated in Nod2/ mice. The same pattern was
observed with IL-6 (Fig. 4 A, right).
Because the M. smegmatis namH mutant is more sensitive
to lysozyme than WT M. smegmatis (Raymond et al., 2005),
it is possible that infection with the former organism leads to
increased PGN shedding and macrophage exhaustion rather
than decreased stimulation. To control for this possibility, we
digested the PGN from heat-killed M. smegmatis and namH
variants, and performed a short-term i.p. stimulation of WT
and Nod2-deficient mice. After 2 h, KC levels were signifi-
cantly increased in the peritoneum of WT mice stimulated
with N-glycolyl MDP–containing organisms (Fig. 4 B, left).
This increase was abrogated in Nod2-deficient mice. Further-
more, akin to what was observed after 72 h, macrophages re-
cruited to the peritoneum of WT mice after a 2-h challenge
with M. smegmatis and the complemented namH mutant were
more responsive to Toll-like receptor 4 stimulation than
macrophages from namH-deficient M. smegmatis–treated mice
(Fig. 4 B, right). This effect was again dependent on the pres-
ence of NOD2 because it was abrogated in Nod2-deficient
mice (Fig. 4 B, right). Thus, optimal NOD2-dependent macro-
phage activation by mycobacteria in vivo requires expression
of the bacterial NamH.
N-glycolyl MDP confers increased immunological activity
to live mycobacteria and is a critical active component of CFA
Our group recently showed that activation of the IFN-–IL-
12 axis during mycobacterial infection is impaired in Nod2-
deficient mice (Divangahi et al., 2008). To investigate the
NOD2-dependent contribution of mycobacterial NamH to
T cell activation, we performed a short-term immunization
experiment using live WT M. smegmatis and namH-deficient
Figure 2. N-glycolyl MDP is more potent than N-acetyl MDP at
inducing RIP2 polyubiquitination and at activating NF-B and
JNK. (A) HEK293 cells were transfected with indicated vectors and a
NF-B luciferase reporter in the presence of various concentrations of
N-glycolyl or N-acetyl MDP. Fold increase in NF-B activation com-
pared with transfected but unstimulated cells was assessed. Represen-
tative data from three independent replicates are shown (means ±
SEM). *, P < 0.05 compared with unstimulated cells; **, P < 0.05 be-
tween 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 polyu-
biquitinated RIP2 proteins were detected by immunoblotting (IB) with
the indicated antibody. Total immunoprecipitated RIP2 protein was
measured as a control. (C–E) The NF-B (C), JNK (D), and p38 MAPK (E)
activities were measured by immunoblotting with the indicated anti-
phospho antibodies. Total IB, JNK, p38 MAPK, and -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.
JEM VOL. 206, August 3, 2009
BRIEF DEFINITIVE REPORT
To specifically test the relative adjuvant activity of the two
forms of MDP present in CFA, we immunized mice s.c. with
preparations of OVA emulsified in (a) IFA alone, (b) CFA, (c)
N-glycolyl MDP with IFA, or (d) N-acetyl MDP with IFA.
After 7 d, OVA-specific IFN-–producing cells from the
draining lymph nodes were enumerated by ELISPOT. IFA
alone was not sufficient to generate antigen-specific immu-
nity, whereas CFA induced a high frequency of OVA-specific
IFN-–producing T cells (Fig. 5 B). Strikingly, N-glycolyl
MDP plus IFA induced a comparable response to CFA, which
M. smegmatis. 14 d after immunization, no bacteria could be
detected in the mouse spleens (unpublished data). As shown
in Fig. 5 A, the number of IFN-–producing splenocytes
(top) and the level of IFN- production by these cells (mid-
dle) were significantly increased in Nod2+/+ compared with
Nod2/ cells after immunization with WT M. smegmatis.
This NOD2 dependence was lost after immunization with
namH-deficient M. smegmatis. Similar results were obtained
when measuring IL-12p40 production by splenic antigen-
presenting cells (Fig. 5, bottom).
Figure 3. N-glycolyl MDP is more potent than N-acetyl MDP at inducing proinflammatory cytokine production in macrophages. (A) Naive
peritoneal macrophages from Nod2+/+ mice were stimulated for 6 h with LPS (top) or for 12 h with TDM (bottom) in combination with various concentra-
tions of N-glycolyl or N-acetyl MDP. (B) Naive peritoneal macrophages from Nod2+/+ and Nod2/ mice were either left unstimulated or were stimulated
for 12 h with 10 µg/ml N-acetyl MDP alone, 10 µg/ml N-glycolyl MDP alone, TDM alone and a combination of N-acetyl MDP and TDM, or N-glycolyl MDP
and TDM. The amount of TNF- and IL-6 released in the supernatant was quantified by ELISA. Representative data from three independent replicates are
shown (means ± SEM). *, P < 0.05 compared with no MDP added; **, P < 0.05 between N-glycolyl versus N-acetyl MDP.
N-GLYCOLYL MDP AND NOD2 | Coulombe et al.
NOD2 is a susceptibility gene for CD, a polygenic sys-
temic inflammatory disease featuring recurring lesions in the
gastrointestinal tract (Hugot et al., 2001). Cells from humans
with CD-associated NOD2 polymorphisms manifest a re-
duced response to MDP (Inohara et al., 2003), which could
increase susceptibility to intracellular bacterial infection.
Because only a small minority of all individuals with CD-asso-
ciated NOD2 polymorphisms will develop CD in their
lifetime, it is possible that decreased resistance to specific
bacteria will play an important role in disease pathogenesis.
Given the importance of NOD2 as a key modulator of the
host response to N-glycolyl MDP–containing bacteria, it
will be a priority to investigate the role of these organisms in
the pathogenesis of CD.
MATERIALS AND METHODS
Mice. Nod2+/ males backcrossed six generations onto a C57BL/6 back-
ground were obtained from the Congenics Facility at Yale University. They
were bred with C57BL/6 mice purchased from Harlan Laboratories to estab-
lish a Nod2/ breeding colony at the McGill University Health Centre.
was significantly greater than N-acetyl MDP plus IFA. As a
control, there was no response in nondraining lymph nodes
(unpublished data). These results point to a key role of N-gly-
colylated MDP in the adjuvant activity of CFA.
The major findings of this work are that the disruption of
namH leads to impaired NOD2-mediated recognition of my-
cobacterial PGN, and that N-glycolyl MDP is more potent at
modulating host response compared with N-acetyl MDP.
Thus, it appears that the mammalian NOD2 pathway is ex-
quisitely sensitive to PGN from mycobacteria and related or-
ganisms, and hence, that NOD2 serves as a major receptor
for CFA. Efforts are currently underway to address the role of
NamH in relationship to NOD2 using more virulent bacte-
rial organisms. Determining whether increased potency of
N-glycolyl MDP is a result of altered affinity, stability, or cy-
tosolic delivery is expected to lead to a better understanding
of the mechanism of action of CFA.
Figure 4. Increased NOD2-dependent immunogenicity of WT M. smegmatis compared with M. smegmatisnamH during short-term mouse
i.p. challenge. (A) Naive Nod2+/+ and Nod2/ mice (n = 3 per genotype per infection) were injected i.p. with live preparations of either M. smegmatis,
M. smegmatisnamH, or M. smegmatisnamH::namH. After 72 h, peritoneal macrophages were harvested and either left unstimulated or stimulated with
M. smegmatisnamH. (B) Naive Nod2+/+ and Nod2/ mice (n = 3 per genotype per stimulation) were injected i.p. with mutanolysin-treated killed prepa-
rations of either M. smegmatis, M. smegmatisnamH, or M. smegmatisnamH::namH. After 2 h, peritoneal lavage was performed, and peritoneal macro-
phages were harvested and either left unstimulated or stimulated with LPS. The amount of KC in the peritoneal lavage as well as TNF- and/or IL-6
released in the culture supernatant was quantified by ELISA. Representative data from two independent replicates are shown (means ± SEM). *, P < 0.05.
JEM VOL. 206, August 3, 2009
BRIEF DEFINITIVE REPORT
A WT breeding colony was established from the Nod2+/+ littermates from
this breeding, and these animals were used for experiments in Figs. 1, 3, 4, and
5 A. For Fig. 5 B, C57BL/6 mice were purchased from the Jackson Labora-
tory. All study mice were 8–12 wk old, and experiments were conducted in
accordance with the guidelines of animal research ethics boards of McGill
and Harvard Universities.
Bacterial strains, growth conditions, and bacteria-derived reagents.
M. bovis BCG Russia, M. smegmatis mc2155, and M. avium ssp. paratuberculo-
sis (MAP) K10 were grown as previously described (Divangahi et al., 2008).
For MAP K10, 1 µg/ml mycobactin J (Allied Monitor Inc.) was added to
the culture medium. S. aureus (American Type Culture Collection), E. coli
(American Type Culture Collection), S. typhimurium (American Type Cul-
ture Collection), P. aeruginosa (American Type Culture Collection), and
recent clinical isolates of L. monocytogenes, B. cereus, R. equi, N. asteroides, and
Streptomyces sp., as well as E. coli DH5- used for cloning purposes, were
cultured at 37°C in Luria broth (Difco) at 250 rpm. Kanamycin (50 µg/ml
for E. coli and mycobacteria) and hygromycin (100 µg/ml for E. coli and
50 µg/ml for mycobacteria) were used when needed.
Pure LPS from E. coli 055:B5, N-acetyl MDP (98% purity), Streptomyces
sp.–derived PGN, TDM from M. tuberculosis (99% purity), CFA, and IFA
were purchased from Sigma-Aldrich. M. tuberculosis–derived PGN was a gift
from P. Brennan and J. Spencer (Colorado State University, Fort Collins,
CO). N-glycolyl MDP was custom synthesized (Carbohydrate Synthesis;
Kobayashi et al., 1980) and shown to be >95% pure by nuclear magnetic
resonance spectrometry. N-acetyl and N-glycolyl MDP were free of endo-
toxin contamination, as confirmed by the Limulus amebocyte lysate assay
(Pyrotell; Associates of Cape Cod, Inc.).
Construction and complementation of the M. smegmatis namH mu-
tant. The hygromycin resistance cassette from PSC301 was digested by
XmnI and inserted into XbaI-digested and T4 polymerase-treated pUC19 to
generate the pUC19::Hyg suicide vector. Subsequently, a 1,070-bp fragment
derived from the M. smegmatis namH gene (amplified using KONamHF [5-
CCGCATATGTCGCCTCGTGGTTC-3] and KONamHR [5-AGCG-
GATCCTCTCGTCGGGGATC-3]) was inserted into pUC19::Hyg using
NdeI and BamHI. This plasmid was electroporated in M. smegmatis, and the
hygromycin-resistant clones were confirmed by Southern blotting to have
the plasmid inserted in namH. To complement the ∆namH mutant, the full-
length M. smegmatis namH gene was amplified along with its putative pro-
moter using MsNamHF (5-CGAGCTAGCTGGTGTGGTTGATCG-3)
and MsNamHR (5-GATAAGCTTCGATGTGCCCGACG-3). This
fragment was inserted in pSUM37 using NheI and HindIII to generate the
pSUMnamH complementation vector.
Ex vivo macrophage culture, infection, and stimulation. Peritoneal
macrophages were harvested from naive Nod2+/+ and Nod2/ mice with-
out prestimulation. 105 cells per well were purified by adherence and cul-
tured in 96-well plates with or without live bacterial infection (2 CFU/cell)
or stimulation with 10 ng/ml LPS, 10 µg/ml TDM, and N-acetyl and N-
glycolyl MDP. TDM was dissolved in petroleum ether, coated to the wells
of a 96-well plate, and allowed to evaporate before macrophage addition
with and without MDP. The culture supernatants were collected at desig-
nated intervals and stored at 20°C until cytokine measurements. Cytokine
production by macrophages was assayed using ELISA (R&D Systems) to
measure TNF- and IL-6 in culture supernatants.
Antibiotic sensitivity assay. 107 cells from log-phase M. smegmatis strains
were resuspended in 200 µl 7H9 and spread onto 25 ml 7H10 in standard
15-cm-diameter Petri dishes. 10-µg ampicillin disks (Oxoid Ltd.) were
placed on inoculated plates. After a 48-h incubation at 37°C, the diameter of
the zone of inhibition was measured.
HEK293 cell transfection and stimulation. Plasmids containing cDNA
for the WT human NOD2 or the CD-associated human NOD2 3020insC
Figure 5. N-glycolyl MDP is a critical active constituent of the
adjuvant-active mycobacterial cell wall. (A) Naive Nod2+/+ and
Nod2/ mice (n = 4 per genotype per immunization) were immunized
i.p. with either M. smegmatis or M. smegmatisnamH and rechallenged
14 d later with the same organism. Saline-injected mice were used as a
control. The frequency of IFN-–producing splenocytes (top) as well as
total IFN- (middle) and IL-12p40 (bottom) production by these cells
was quantified using ELISPOT and ELISA, respectively. (B) C57BL/6 mice
(n = 3–4 per immunization) were immunized s.c. with the indicated
emulsified preparations for 7 d. The frequency of OVA-specific IFN-–
producing T cells in the draining lymph nodes was analyzed by ELISPOT
of unstimulated and OVA-stimulated cells. Representative data from two
independent replicates are shown (means ± SEM). *, P < 0.05.
N-GLYCOLYL MDP AND NOD2 | Coulombe et al.
2007. Mycobacterium paratuberculosis is recognized by Toll-like receptors and
NOD2. J. Leukoc. Biol. 82:1011–1018.
Freund, J. 1951. The effect of paraffin oil and mycobacteria on antibody forma-
tion and sensitization; a review. Am. J. Clin. Pathol. 21:645–656.
Freund, J. 1956. The mode of action of immunologic adjuvants. Bibl. Tuberc.
Freund, J., M.M. Lipton, and L.R. Morrison. 1950. Demyelination in the guinea
pig in chronic allergic encephalomyelitis produced by injecting guinea pig
brain in oil emulsion containing a variant of Mycobacterium butyricum. Arch.
Pathol. (Chic). 50:108–121.
Gandotra, S., S. Jang, P.J. Murray, P. Salgame, and S. Ehrt. 2007. Nucleotide-
binding oligomerization domain protein 2-deficient mice control infection
with Mycobacterium tuberculosis. Infect. Immun. 75:5127–5134.
Girardin, S.E., I.G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas,
D.J. Philpott, and P.J. Sansonetti. 2003. Nod2 is a general sensor of pep-
tidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem.
Hasegawa, M., Y. Fujimoto, P.C. Lucas, H. Nakano, K. Fukase, G. Nunez,
and N. Inohara. 2008. A critical role of RICK/RIP2 polyubiquitination in
Nod-induced NF-kappaB activation. EMBO J. 27:373–383.
Hugot, J.P., M. Chamaillard, H. Zouali, S. Lesage, J.P. Cezard, J. Belaiche, S.
Almer, C. Tysk, C.A. O’Morain, M. Gassull, et al. 2001. Association of
NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.
Inohara, N., Y. Ogura, A. Fontalba, O. Gutierrez, F. Pons, J. Crespo, K. Fukase,
S. Inamura, S. Kusumoto, M. Hashimoto, et al. 2003. Host recognition
of bacterial muramyl dipeptide mediated through NOD2. Implications for
Crohn’s disease. J. Biol. Chem. 278:5509–5512.
Kobayashi, S., T. Fukuda, H. Yukimasa, M. Fujino, I. Azuma, and
Y. Yamamura. 1980. Synthesis of muramyl dipeptide analogs with en-
hanced adjuvant activity. Bull. Chem. Soc. Jpn. 53:2570–2577.
Kotani, S., Y. Watanabe, F. Kinoshita, T. Shimono, and I. Morisaki. 1975.
Immunoadjuvant activities of synthetic N-acetyl-muramyl-peptides or -
amino acids. Biken J. 18:105–111.
Kufer, T.A., and P.J. Sansonetti. 2007. Sensing of bacteria: NOD a lonely job.
Curr. Opin. Microbiol. 10:62–69.
Leber, J.H., G.T. Crimmins, S. Raghavan, N.P. Meyer-Morse, J.S. Cox, and
D.A. Portnoy. 2008. Distinct TLR- and NLR-mediated transcriptional
responses to an intracellular pathogen. PLoS Pathog. 4:e6.
Lederer, E., A. Adam, R. Ciorbaru, J.F. Petit, and J. Wietzerbin. 1975. Cell
walls of Mycobacteria and related organisms; chemistry and immunostimu-
lant properties. Mol. Cell. Biochem. 7:87–104.
Mahapatra, S., H. Scherman, P.J. Brennan, and D.C. Crick. 2005. N glycolylation
of the nucleotide precursors of peptidoglycan biosynthesis of Mycobacterium
spp. is altered by drug treatment. J. Bacteriol. 187:2341–2347.
Masihi, K.N., W. Brehmer, W. Lange, H. Werner, and E. Ribi. 1985. Trehalose
dimycolate from various mycobacterial species induces differing anti-in-
fectious activities in combination with muramyl dipeptide. Infect. Immun.
Raymond, J.B., S. Mahapatra, D.C. Crick, and M.S. Pavelka Jr. 2005.
Identification of the namH gene, encoding the hydroxylase responsible
for the N-glycolylation of the mycobacterial peptidoglycan. J. Biol. Chem.
Stewart-Tull, D.E. 1980. The immunological activities of bacterial peptidogly-
cans. Annu. Rev. Microbiol. 34:311–340.
Wolfert, M.A., T.F. Murray, G.J. Boons, and J.N. Moore. 2002. The origin
of the synergistic effect of muramyl dipeptide with endotoxin and pep-
tidoglycan. J. Biol. Chem. 277:39179–39186.
Yang, S., R. Tamai, S. Akashi, O. Takeuchi, S. Akira, S. Sugawara, and H.
Takada. 2001. Synergistic effect of muramyldipeptide with lipopolysac-
charide or lipoteichoic acid to induce inflammatory cytokines in human
monocytic cells in culture. Infect. Immun. 69:2045–2053.
Yang, Y., C. Yin, A. Pandey, D. Abbott, C. Sassetti, and M.A. Kelliher.
2007. NOD2 pathway activation by MDP or Mycobacterium tuberculosis
infection involves the stable polyubiquitination of Rip2. J. Biol. Chem.
(NOD2fs) were gifts from S. Girardin (University of Toronto, Toronto,
Canada). 3xFLAG-tagged NOD2wt and NOD2fs were PCR amplified and
subcloned into the pIRES-puro3 expression vector (Clontech Laboratories,
Inc.). For NF-B activation assays, 105 HEK293 cells per well were seeded
into 24-well plates and transfected overnight using FuGene6 (Roche) with
1 ng of NOD2, NOD2fs, or empty vector, 75 ng pTAL-NF-B-luc (Clon-
tech Laboratories, Inc.), and 7.5 ng pGL4-RL (Promega), along with de-
fined concentrations of PGN or MDP. After 16 h, a dual-luciferase reporter
assay (Promega) was performed on cell lysates.
Immunoprecipitation and Western blot analysis. Immunoprecipita-
tion and immunoblotting were performed as previously described (Yang
et al., 2007). Experiments were performed using the RAW 264.7 macro-
phage cell line stimulated with MDP for 1 h.
i.p. M. smegmatis challenges. Nod2+/+ and Nod2/ mice were injected i.p.
with 0.5 ml PBS containing 107 live or heat-killed, mutanolysin-treated M.
smegmatis (Sigma-Aldrich) or modified strains for 72 and 2 h, respectively.
Peritoneal lavage was performed at 2 h with 3 ml PBS, and peritoneal macro-
phages from infected mice were harvested and cultured as described with or
without stimulation. KC production in the peritoneal lavage as well as TNF-
and IL-6 production in culture supernatants was assayed using ELISA.
Immunizations and analysis of T cell response. Nod2+/+ and Nod2/
mice were immunized i.p. with 0.5 ml PBS alone or containing 106 live
M. smegmatis or M. smegmatisnamH. After 14 d, each mouse was given a 5-d
boost with the same preparation used for immunization. The number of
IFN-–producing splenocytes (0.5 × 106/well) as well as total IL-12p40 and
IFN- production by unstimulated cells was measured using ELISPOT
(R&D Systems) and ELISA, respectively.
C57BL/6 mice received s.c. injections (two injection sites, 100 µl per site)
of 200 µg OVA (Sigma-Aldrich) mixed with IFA, CFA, 30 µg N-glycolyl MDP
plus IFA, or 30 µg N-acetyl MDP plus IFA. 7 d after immunization, a single-cell
suspension was prepared from draining lymph nodes (inguinal) and nondraining
lymph nodes (axillary). Cells were incubated for 24 h with medium alone or
containing 100 µg OVA, and then subjected to IFN- ELISPOT.
This work was funded by an operating grant (MOP-86536) from the Canadian
Institutes for Health Research (CIHR) to M.A. Behr. F. Coulombe is supported by a
studentship award from the Fonds de la Recherche en Santé du Québec (FRSQ),
M.B. Reed is a New Investigator of the CIHR, and M.A. Behr is a Chercheur-Boursier
Senior of the FRSQ and a William Dawson Scholar of McGill University.
The authors have no conflicting financial interests.
Submitted: 11 August 2008
Accepted: 16 June 2009
Adam, A., J.F. Petit, J. Wietzerbin-Falszpan, P. Sinay, D.W. Thomas, and E.
Lederer. 1969. FEBS Lett. 4:87–92.
Divangahi, M., S. Mostowy, F. Coulombe, R. Kozak, L. Guillot, F. Veyrier, K.S.
Kobayashi, R.A. Flavell, P. Gros, and M.A. Behr. 2008. NOD2-deficient
mice have impaired resistance to Mycobacterium tuberculosis infection through
defective innate and adaptive immunity. J. Immunol. 181:7157–7165.
Ellouz, F., A. Adam, R. Ciorbaru, and E. Lederer. 1974. Minimal structural
requirements for adjuvant activity of bacterial peptidoglycan derivatives.
Biochem. Biophys. Res. Commun. 59:1317–1325.
Ferwerda, G., S.E. Girardin, B.J. Kullberg, L. Le Bourhis, D.J. De Jong,
D.M. Langenberg, R. Van Crevel, G.J. Adema, T.H. Ottenhoff, J.W.
Van Der Meer, and M.G. Netea. 2005. NOD2 and toll-like receptors
are nonredundant recognition systems of Mycobacterium tuberculosis. PLoS
Ferwerda, G., B.J. Kullberg, D.J. De Jong, S.E. Girardin, D.M. Langenberg,
R. Van Crevel, T.H. Ottenhoff, J.W. Van Der Meer, and M.G. Netea.