THE JOURNAL OF BIOLOGICAL CHEMISTRY
Accelerated Publication
The Cell Surface Receptor
DC-SIGN Discriminates between
Mycobacterium Species through
Selective Recognition of the
Mannose Caps on
Lipoarabinomannan*
Received for publication, October 23, 2002,
and in revised form, December 12, 2002
Published, JBC Papers in Press, December 20, 2002,
DOI 10.1074/jbc.C200586200
Norihiro Maeda‡§¶, Jérôme Nigou¶储,
Jean-Louis Herrmann**, Mary Jackson‡,
Ali Amara‡‡, Philippe Henri Lagrange**,
Germain Puzo储, Brigitte Gicquel‡,
and Olivier Neyrollesत
From the ‡Institut Pasteur, Unité de Génétique
Mycobactérienne, 28 rue du Dr Roux, 75724 Paris Cedex
15, 储Institut de Pharmacologie et de Biologie Structurale
du CNRS, UMR 5089, 205 route de Narbonne, 31077
Toulouse Cedex, **Hôpital Saint-Louis, Service de
Microbiologie, 1 avenue Claude Vellefaux, 75010 Paris,
and ‡‡Institut Pasteur, Unité d’Immunologie Virale,
28 rue du Dr Roux, 75724 Paris Cedex 15, France
Interactions between dendritic cells (DCs) and
Mycobacterium tuberculosis, the etiological agent of tu-
berculosis, most likely play a key role in anti-mycobac-
terial immunity. We have recently shown that M. tuber-
culosis binds to and infects DCs through ligation of the
DC-specific intercellular adhesion molecule-3-grabbing
nonintegrin (DC-SIGN) and that M. tuberculosis man-
nose-capped lipoarabinomannan (ManLAM) inhibits
binding of the bacilli to the lectin, suggesting that Man-
LAM might be a key DC-SIGN ligand. In the present
study, we investigated the molecular basis of DC-SIGN
ligation by LAM. Contrary to what was found for slow
growing mycobacteria, such as M. tuberculosis and the
vaccine strain Mycobacterium bovis bacillus Calmette-
Guérin, our data demonstrate that the fast growing sa-
prophytic species Mycobacterium smegmatis hardly
binds to DC-SIGN. Consistent with the former finding,
we show that M. smegmatis-derived lipoarabinoman-
nan, which is capped by phosphoinositide residues (PI-
LAM), exhibits a limited ability to inhibit M. tuberculosis
binding to DC-SIGN. Moreover, using enzymatically de-
mannosylated and chemically deacylated ManLAM mol-
ecules, we demonstrate that both the acyl chains on the
ManLAM mannosylphosphatidylinositol anchor and the
mannooligosaccharide caps play a critical role in DC-
SIGN-ManLAM interaction. Finally, we report that DC-
* This work was supported by grants from the European Community
“Cluster for Tuberculosis Vaccine Development,” the Institut Pasteur,
and the National Institutes of Health (NIAID Contract NO1 AI-75320,
“Tuberculosis Research Materials and Vaccine Testing”). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A JSPS overseas research fellow.
¶ These authors contributed equally to the work.
§§ To whom correspondence should be addressed. Tel.: 33-1-45-68-88-
40; Fax: 33-1-45-68-88-43; E-mail: neyrolle@pasteur.fr.
This paper is available on line at http://www.jbc.org 5513
Vol. 278, No. 8, Issue of February 21, pp. 5513–5516, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
SIGN binds poorly to the PILAM and uncapped AraLAM-
containing species Mycobacterium fortuitum and
Mycobacterium chelonae, respectively. Interestingly,
smooth colony-forming Mycobacterium avium, in which
ManLAM is capped with single mannose residues, was
also poorly recognized by the lectin. Altogether, our re-
sults provide molecular insight into the mechanisms of
mycobacteria-DC-SIGN interaction, and suggest that
DC-SIGN may act as a pattern recognition receptor and
discriminate between Mycobacterium species through
selective recognition of the mannose caps on LAM
molecules.
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The interaction between Mycobacterium tuberculosis and
host dendritic cells (DCs)1 is thought to be critical for mounting
a protective anti-mycobacterial immune response and for de-
termining the outcome of infection (1– 4). However, the molec-
ular bases of DC infection by mycobacteria remain poorly un-
derstood. We have recently shown that M. tuberculosis, as well
as the vaccine strain Mycobacterium bovis bacillus Calmette-
Guérin (BCG), use the DC-specific intercellular adhesion mol-
ecule-3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) to bind to
and enter human DCs (5), a feature that may allow the bacillus
to persist within a unique immature compartment of the cells
(6). DC-SIGN/CD209 is a calcium-dependent (C-type) trans-
membrane lectin that contains a single carbohydrate recogni-
tion domain at its extracellular C-terminal end. It is expressed
on DCs as well as on some macrophage (M␾) subsets, including
alveolar M␾s (7, 8). DC-SIGN has been described initially as a
receptor for ICAM-3 and ICAM-2, as well as for human and
simian immunodeficiency viruses, enabling the trans infection
of susceptible CD4⫹ T lymphocytes by these viruses (9 –12).
Thereafter, it was shown to bind to other microbes, namely
Ebola virus and Leishmania pifanoi (13, 14).
The DC-SIGN carbohydrate recognition domain binds to
mannose-rich glycoconjugates (15), a feature that is consistent
with our finding that M. tuberculosis lipoarabinomannan
(termed ManLAM; see below), a highly mannosylated surface
lipoglycan, might be a key mycobacterial ligand for DC-SIGN
(5). Indeed, purified M. tuberculosis-derived ManLAM was
found to inhibit the binding of M. tuberculosis to human mono-
cyte-derived DCs, as well as to recombinant HeLa-derived cells
expressing DC-SIGN. LAM is a major component of the myco-
bacterial cell wall. It contains a carbohydrate backbone com-
posed of D-mannan and D-arabinan (Fig. 1). The mannan core is
attached to an acylated mannosylphosphatidylinositol (MPI)
anchor at its reducing end; the arabinan domain is capped with
either mannose residues in so-called ManLAMs or with phos-
phoinositide motifs in so-called PILAMs (16, 17), or uncapped
in so-called AraLAM (40). The caps of ManLAMs consist of
mono-, ␣(132)-di-, and ␣(132)-tri-mannopyranosides, with di-
1
The abbreviations used are: DC, dendritic cell; DC-SIGN, DC-spe-
cific intercellular adhesion molecule-3-grabbing nonintegrin; BCG, ba-
cillus Calmette-Guérin; ICAM, intercellular adhesion molecule; M␾,
macrophage; ManLAM, mannose-capped lipoarabinomannan; ␣Man-
LAM, ␣-exomannosidase-treated ManLAM; dManLAM, deacylated
ManLAM; PILAM, phosphoinositide-capped lipoarabinomannan;
AraLAM, uncapped LAM; MPI, mannosylphosphatidylinositol; CE-LIF,
capillary electrophoresis coupled to laser-induced fluorescence; APTS,
1-aminopyrene-3,6,8-trisulfonate; IL, interleukin; MR, mannose recep-
tor; TLR, toll-like receptor.
5514 Mycobacterial Binding to DC-SIGN
FIG. 1. Structural organization of ManLAM versus PILAM.
Manp, mannopyranose; Ins, myo-inositol; P, phosphate; R1–R4,
acyl chains.
mannopyranosides being the most abundant motif (18 –20). So
far, ManLAMs have been detected in slow growing mycobacte-
ria only. These include various strains of M. tuberculosis,
M. bovis BCG, the leprosy agent Mycobacterium leprae, and the
opportunistic species Mycobacterium avium (16, 17, 21, 22). By
contrast, PILAM or AraLAM expression seems to be fairly
limited to fast growing mycobacteria, including nonpathogenic
Mycobacterium smegmatis (16, 17, 21, 23), Mycobacterium che-
lonae (40), and Mycobacterium fortuitum.2 In addition to their
structural role in organizing of the cell wall, LAMs are known
to be potent inducers of various cytokines when in contact with
mammalian phagocytic cells (24).
We have shown that the slow growing mycobacteria
M. tuberculosis and M. bovis BCG, which express ManLAM,
interact with human DCs through DC-SIGN and that purified
M. tuberculosis-derived ManLAM inhibits M. tuberculosis
binding to recombinant HeLa-derived cells expressing DC-
SIGN and to monocyte-derived DCs. The goal of the present
report was to obtain a better understanding of the molecular
determinants of the LAM molecule involved in binding to DC-
SIGN. Using a DC-SIGN-expressing recombinant cell line as a
readout, we first report that DC-SIGN binds poorly to the fast
growing species M. smegmatis and that M. smegmatis-derived
PILAM, which lacks mannose caps, exhibits a very limited
ability to inhibit M. tuberculosis binding to the lectin. Using
various chemically or enzymatically generated variants of the
ManLAM molecule, we further demonstrate that both the acyl
chains on the MPI anchor and the mannose-capping residues
play a key role in the ManLAM-DC-SIGN ligation process.
Moreover, we show that DC-SIGN does not bind to the PILAM-
and AraLAM-containing species M. fortuitum and M. chelonae,
respectively. Altogether, our findings provide evidences that
DC-SIGN may discriminate between ManLAM-containing slow
growers, such as M. tuberculosis, and nonpathogenic PILAM-
containing fast growers, such as M. smegmatis, through a high
affinity for mannose-capping residues on ManLAM.
EXPERIMENTAL PROCEDURES
Cells and Bacteria—DC-SIGN⫹ HeLa (HeLa-DC) cells were obtained
by transducing HeLa cells with the retroviral vector TRIP-⌬U3 encod-
ing for human DC-SIGN (25). HeLa and HeLa-DC cells were propa-
gated in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf
serum (Dutscher, Brumath, France). M. tuberculosis H37Rv,
M. smegmatis mc2155, and clinical isolates of M. fortuitum, M. chelonae,
and M. avium (smooth colony-forming) were propagated in 7H9 me-
dium containing 10% albumin-dextrose-catalase supplement.
Mycobacterial Glycoconjugate Purification and Chemical Degrada-
tion—ManLAM from M. bovis BCG Pasteur and PILAM from M. smeg-
matis were purified as described previously (19, 23, 26). M. tuberculosis
H37Rv-purified ManLAM was a kind gift from the Colorado State
University. dManLAM was prepared by incubating ManLAM (200 ␮g)
in 200 ␮l of NaOH 0.1 M for 2 h at 37 °C. After neutralization with 200
␮l of HCl, 0.1 M, the reaction products were dialyzed against water and
2
L. Bala, M. Gilleron, M. Rivière, and G. Puzo, unpublished data.
freeze-dried. ␣ManLAM was prepared by incubating ManLAM (200 ␮g)
for 6 h at 37 °C in 30 ␮l of a jack beans ␣-mannosidase (Sigma) solution
(2 mg/ml, 0.1 M sodium acetate buffer, pH 4.5, 1 mM ZnSO4). After a
second addition of 50 ␮l of enzyme solution, the reaction was continued
overnight. The reaction products were then dialyzed against 50 mM
NH4CO3, pH 7.6. Elimination of ␣-mannosidase was achieved by dena-
turation (2 min at 110 °C) followed by overnight tryptic digestion
(37 °C, 3.2 ␮g of trypsin). ␣ManLAM was recovered after dialysis
against water, freeze-dried, and analyzed for cap contents by CE-LIF as
previously described (27). Briefly, ManLAM or ␣ManLAM (1 ␮g) was
submitted to mild acidic hydrolysis (15 ␮l of HCl, 0.1 M, for 20 min at
110 °C) in the presence of mannoheptose (100 pmol) as the internal
standard. The reaction products were then submitted to 1-aminopy-
rene-3,6,8-trisulfonate (APTS) tagging and subjected to CE-LIF analy-
sis. Separations were performed using an uncoated, fused silica capil-
lary column (50 ␮m internal diameter; 40 cm effective length; 47 cm
total length; Sigma), and analyses were carried out at a temperature of
25 °C with an applied voltage of 20 kV using acetic acid 1% (w/v),
triethylamine 30 mM in water, pH 3.5, as a running electrolyte. The
amount of each cap motif was determined relative to the
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internal standard.
Binding and Inhibition Assay—Cells were infected at a multiplicity
of infection of 1 bacterium/cell for 4 h at 4 °C in RPMI 1640, washed
extensively in RPMI 1640, and analyzed by scoring colony-forming
units after plating on agar and incubation at 37 °C. In binding inhibi-
tion experiments, cells were preincubated for 1 h at 4 °C with 10 ␮g/ml
of the indicated components. These components were left in the culture
medium during the infection process.
RESULTS AND DISCUSSION
Mycobacterial species can be divided into slow and fast grow-
ers. To gain a better understanding of the molecular basis of
their ligation to DC-SIGN, we first compared the relative abil-
ity of the slow growing pathogenic species M. tuberculosis ver-
sus the fast growing saprophytic species M. smegmatis to bind
to the lectin. A binding assay was performed on HeLa-derived
cells expressing or not DC-SIGN (HeLa-DC and HeLa, respec-
tively). As we reported previously, M. tuberculosis was found to
bind to HeLa-DC ⬃15 times more than to HeLa cells (Fig. 2A),
and in a multiplicity of infection-dependent manner (data not
shown). Conversely, M. smegmatis binding to HeLa-DC was
found to be only ⬃2 times more than to HeLa cells (Fig. 2A).
Our previous finding that M. tuberculosis ManLAM can inhibit
M. tuberculosis binding to DC-SIGN suggests that the reduced
ability of M. smegmatis to bind to HeLa-DC cells may be due to
the inability of M. smegmatis PILAM to bind to the lectin. To
test this hypothesis, we performed a M. tuberculosis binding
assay on HeLa-DC cells that had been preincubated or not with
LAM molecules from various mycobacterial species. As re-
ported previously, yeast mannan and M. tuberculosis- as well
as M. bovis BCG-derived ManLAMs were found to inhibit my-
cobacterial binding to HeLa-DC cells by as much as ⬃90% (Fig.
2B). By contrast, M. smegmatis-derived PILAM was found to
inhibit poorly (⬃25% inhibition) the binding of M. tuberculosis
to HeLa-DC cells. Interestingly, PILAM was found able to fully
inhibit M. smegmatis binding to HeLa-DC cells (data not
shown). The fact that BCG-derived ManLAM inhibits M. tuber-
culosis binding to DC-SIGN as well as M. tuberculosis-derived
ManLAM does is consistent with our previous result showing
that M. bovis BCG binds to DC-SIGN to the same extent as
M. tuberculosis and with the known structural similarity be-
tween ManLAMs from M. bovis BCG and M. tuberculosis (18).
Because DC-SIGN is a mannose-binding lectin and because
PILAMs are devoid of mannose caps (Fig. 1), we next reasoned
that the results described above might indicate that ManLAM
capping residues may be the ManLAM subdomains preferen-
tially recognized by the lectin. To test this hypothesis, we
treated M. bovis BCG-derived ManLAM with ␣-exomannosi-
dase to obtain ManLAM devoid of mannose caps (␣ManLAM).
The reaction was assessed by CE-LIF analysis as previously
described (18, 27). A typical electropherogram obtained for
 Mycobacterial Binding to DC-SIGN
FIG. 2. DC-SIGN has high affinity for slow versus fast growing
Mycobacterium species. A, epithelial HeLa-derived cells expressing
or not DC-SIGN (HeLa-DC and HeLa, respectively) were infected with
M. smegmatis or M. tuberculosis H37Rv at a multiplicity of infection of
1 bacterium/cell. Bacterial binding was evaluated after 4 h at 4 °C by
counting colony-forming units (CFUs). Data represent the means
(⫾S.D.) of three separate experiments. B, cells were preincubated with
10 ␮g/ml yeast-derived mannan (MAN), M. tuberculosis H37Rv- or
M. bovis BCG-derived ManLAM, M. smegmatis-derived PILAM, or sa-
line (control, Ø) for 1 h at 4 °C and infected as described in A. Bacteria
binding was measured as described in A. Data are expressed as per-
centages of binding relative to control values (100%, preincubation of
HeLa-DC cells with saline), and the means (⫾S.D.) of three independ-
ent experiments are shown.
␣ManLAM is presented in Fig. 3A. Peaks corresponding to
mannooligosaccharide caps, i.e. mono-, ␣ (132)-di-, and
␣(132)-tri-mannopyranosides, were almost undetectable.
Quantification indicated that more than 95% of cap demanno-
sylation was achieved. The ability of ␣ManLAM to inhibit
M. tuberculosis binding to DC-SIGN was evaluated in binding
experiments. In contrast to native ManLAM, ␣ManLAM failed
to inhibit mycobacterial binding to the lectin (⬃10% binding
inhibition, Fig. 3B). Similar results were obtained when cells
were treated with M. tuberculosis-derived ␣ManLAM prior to
the binding assay (data not shown). These results indicate that
mannooligosaccharide caps are critical structural features for
ManLAM-mediated inhibition of M. tuberculosis binding
to DC-SIGN.
Because MPI anchor has been shown previously to be in-
volved in some of the biological activities of ManLAM, partic-
ularly their binding to C-type lectins (28, 29), we then evalu-
ated the role of the acyl part of the MPI anchor in ManLAM-
DC-SIGN interaction. To this end, M. bovis BCG-derived
dManLAM was prepared by alkali treatment. As shown on Fig.
3B, dManLAM failed to inhibit M. tuberculosis binding to
HeLa-DC cells, revealing that a native acylated MPI anchor is
required for ManLAM-mediated inhibition of mycobacterial
binding to DC-SIGN.
Finally, we wished to know whether our finding was still
valid in other Mycobacterium species for which the LAM struc-
5515
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FIG. 3. Mannooligosaccharide caps and the acyl groups on the
MPI anchor play a critical role in ManLAM binding to DC-SIGN.
A, CE-LIF analysis of mannooligosaccharide caps from M. bovis BCG-
derived ManLAM (upper electropherogram, dotted line) and ␣ManLAM
(lower electropherogram, solid line). ManLAM or ␣ManLAM was sub-
mitted to mild acid hydrolysis (0.1 M HCl for 30 min at 110 °C) in the
presence of mannoheptose as the internal standard. The liberated oli-
gosaccharide was derivatized by APTS before CE-LIF analysis (18, 27).
A, Ara-APTS; M, Man-APTS; S, internal standard, mannoheptose-
APTS; AM, Manp-(␣135)-Ara-APTS; AMM, Manp-(␣132)-Manp-
( ␣ 135)-Ara-APTS; AMMM, Manp-( ␣ 132)-Manp-( ␣ 132)-Manp-
(␣135)-Ara-APTS. B, cells were infected as described in the legend for
Fig. 2B. HeLa-DC cells were treated with 10 ␮g/ml M. bovis BCG-
derived ManLAM, ␣ManLAM, dManLAM, or saline (control, Ø) prior to
the assay. Data are expressed as described in the legend for Fig. 2B.
ture was known. In agreement with what we reported above,
the PILAM- and AraLAM-containing species M. fortuitum and
M. chelonae were poorly recognized by DC-SIGN. Indeed, in a
representative binding assay done in triplicate, M. fortuitum
and M. chelonae bound to HeLa-DC cells 2.3 and 1.5 times more
than to HeLa cells, respectively (data not shown). Interest-
ingly, the ManLAM-containing slow grower M. avium was also
found to bind poorly to DC-SIGN-expressing HeLa cells (⬃1.7
times more than to HeLa cells; data not shown). This is not
surprising because ManLAM from smooth colony-forming
M. avium, which is the one used in our assay, has been reported
to be capped mainly with single mannose residues instead of
the di- and tri-mannopyranoside motifs found in M. tuberculo-
sis- and M. bovis BCG-derived ManLAM (22). Because DC-
SIGN does not bind to single mannose molecules but to more
complex mannosylated structures (15), it is likely that such
mono-mannosylated ManLAM is not recognized by the lectin.
These results raise the possibility, currently under investiga-
tion, that DC-SIGN may recognize mycobacteria from the tu-
berculosis complex only.
Altogether, our results demonstrate that the DC-SIGN-Man-
LAM interaction involves both the MPI anchor acyl chains and
the mannose residues from caps of the ManLAM molecule. As
established recently for the binding of ManLAM to the human
surfactant pulmonary protein A C-type lectin (29, 30), the MPI
fatty acids are most likely involved in the supermolecular or-
ganization of the ManLAM molecules in aggregates, allowing
macromolecular clustering in aqueous solution. Micelle forma-
tion probably results in a huge increase in ManLAM valence
and increases ManLAM avidity to DC-SIGN. This is likely to
explain the poor ability of dManLAM to inhibit M. tuberculosis
5516 Mycobacterial Binding to DC-SIGN
binding to HeLa-DC cells but does not indicate whether LAM
acyl chains, which are likely to be buried within the bacterial
cell wall, can interact with the lectin in vivo. However, the
latter definitely should be investigated, as our result is remi-
niscent of the involvement of the acyl chains of the
M. tuberculosis 19-kDa lipoprotein antigen in binding to toll-
like receptor-2 (TLR2) on phagocytic cells (31).
Selective recognition of the ManLAM mannose-capping res-
idues by DC-SIGN on the surface of DCs is likely to have
important consequences for both the pathogenesis and immu-
nology of tuberculosis and other mycobacterial diseases. LAMs
have various effects on phagocytic cells, including M␾s and
DCs (24). PILAMs induce the secretion of proinflammatory
cytokines, such as tumor necrosis factor-␣, interleukin-1 (IL-1),
IL-12, and IL-6, and the production of microbicidal radicals,
such as NO2⫺, in a much more potent way than do ManLAMs.
In addition, ManLAMs, but not PILAMs, inhibit the M␾ acti-
vation effect of interferon-␥ produced by effector T lympho-
cytes. PILAMs are thus now considered as proinflammatory
molecules, whereas ManLAMs are viewed rather as anti-in-
flammatory components (17, 24), which is consistent with the
known ability of ManLAM-containing slow growing mycobac-
teria to resist immune defense mechanisms of their susceptible
host (32). In particular, our recent results demonstrate that
ManLAM inhibits the secretion of IL-12 by human DCs, a
process that, like DC-SIGN ligation, requires both the MPI
anchor acyl chains and the mannose caps (27). In this previous
study (27), based on experiments using monoclonal antibodies,
we suggested that ManLAM was acting through ligation of the
mannose receptor (MR). Although MR is also involved in LAM
mannose caps recognition (33), one cannot rule out the possi-
bility that ManLAM could act also through the ligation of
DC-SIGN, which is currently under investigation. Indeed, DC-
SIGN ligation by ManLAM, either attached to the bacilli or
released in the milieu through exocytosis (34), is likely to
induce major signaling events, possibly including cell deactiva-
tion and/or secretion of anti-inflammatory cytokines such as
transforming growth factor-␤ or IL-10 (35). Interestingly, PIL-
AMs but not ManLAMs can activate cells in a TLR2-dependent
manner (36). It will be of interest to study the cross-talk be-
tween phagocytic cell surface lectins, such as MR and DC-
SIGN, and TLRs in response to mycobacterial ligands, includ-
ing LAMs from various mycobacterial species.
From an evolutionary perspective, it is interesting that DC-
SIGN can discriminate between fast growing saprophytic and
slow growing virulent or potentially virulent mycobacteria.
Until recently, it was proposed that mannose capping of the
LAM molecules was a unique feature of virulent mycobacteria.
This is unlikely to be the case because the attenuated species
M. bovis BCG also contains mannose-capped LAM (21). Even if
not virulent stricto sensu, M. bovis BCG can be pathogenic
under certain conditions, especially in children and immuno-
compromised patients, in whom it may cause a variety of ef-
fects ranging from local adenitis to disseminated disease (37).
Moreover, M. bovis BCG is derived from virulent M. bovis,
which shares a common ancestor with M. tuberculosis (38). One
cannot exclude that mannose capping of the ManLAM molecule
is a feature of virulent mycobacteria that has been conserved
during the recent evolution of M. bovis BCG from M. bovis. In
this context, DC-SIGN could be viewed as a pattern recognition
receptor (39) that has evolved to recognize potentially harmful
mycobacteria through their specific surface glycosylated moi-
eties. In parallel, mycobacteria could have evolved mechanisms
(LAM capping) to resist host immunity (deactivation of the
inflammatory response) in contrast with their fast growing,
soil-living, harmless ancestors.
Acknowledgments—We thank L. Tailleux, V. Abadie, O. Schwartz
(Institut Pasteur, Paris), and C. Petit (Institut Cochin, Paris) for careful
reading of the manuscript and helpful discussions. We thank P. Char-
neau (Institut Pasteur, Paris) for providing TRIP-⌬U3. We acknowl-
edge Colorado State University for the gift of purified M. tuberculosis
H37Rv-derived ManLAM.
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