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J. Biol. Chem., Vol. 278, Issue 32, 29880-29889, August 8, 2003
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From the
Institut de Pharmacologie et de Biologie
Structurale du CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex and
¶FRE 2358, Experimental and Molecular Genetics,
3B rue de la Ferrolerie, 45071 Orleans, Cedex 2, France
Received for publication, April 3, 2003 , and in revised form, May 20, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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through TLR2, irrespective of their acylation pattern, and that
they signal through the adaptor MyD88. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Mycobacterium bovis BCG 1173P2 (the Pasteur strain) (1), Mycobacterium smegmatis ATCC-607 (1), and Mycobacterium tuberculosis H37Rv ATCC-27294 were found to mainly contain two PIM families, the dimmanosylated (PIM2) and the hexamannosylated (PIM6) ones. PIM1, PIM3, PIM4, and PIM5 were observed in very small amounts, suggesting that they are biosynthetic intermediates.
PIM are known from the 1940s and have been structurally investigated by
Ballou and co-workers in the 1960s
(9). By 1965, studies of
deacylated PIM from M. tuberculosis and Mycobacterium phlei
revealed the structure of the saccharidic part. PIM6 were the
highest PIM which were fully characterized from M. phlei
(10) and were shown to contain
a pentamannoside of sequence
Manp1
2Manp12Manp16Manp16Manp1attached
to position 6 of the myo-inositol, whereas a Manp unit is
linked to the position 2 of the myo-inositol. Recently, the complete
structure of native PIM2 has been achieved
(1,
2). These last studies focused
on the characterization of their lipidic part and unambiguously established
the existence of a tetra-acylated form that was thus far suggested
(4).
Several biological functions have been recently attributed to PIM. PIM2 were shown to recruit natural killer T cells, which have a primary role in the local granulomatous response (1, 11). Moreover, a role for surface-exposed PIM as M. tuberculosis adhesins that mediate attachment to non-phagocytic cells has also been established (12, 13). Analysis of infected macrophages revealed that PIM, among other mycobacterial lipids, are actively trafficked out of the mycobacterial phagosome (14). This could be of particular importance relating to the potential role played by these constituents in extending the influence of the bacterium over its surroundings. An unfractionated preparation of PIM, as well as phosphoinositol capped LAM (PILAM), was recently shown to activate cells via Toll-like receptor-2 (TLR-2) (15). Activation of TLR-dependent signaling pathways leads to the activation of genes that participate in innate immune responses, such as expression of cytokines, coactivation molecules, and nitric oxide (16, 17). Finally, PIM6 as well as ManLAM from Mycobacterium leprae and M. tuberculosis are presented by antigen-presenting cells in the context of CD1b (18). The high affinity interaction of CD1b molecules with the PIM2 acyl side chains was then established (19). The phosphatidylinositol moiety plays a central role in the process of PIM and ManLAM binding to CD1b proteins.
Here we investigated the structure of the most polar PIM isolated from M. bovis BCG, PIM6. The earlier NMR studies conducted on PIM2 and PIM6 by Severn et al. (20) focused on the deacylated molecules, thus excluding the study of the lipidic moieties. In this study, we investigated the chemical structure of native PIM6, focusing on the characterization of the different "acyl forms," using sophisticated analytical tools such as MALDI-MS and two-dimensional NMR. Then we demonstrated the capacity of the PIM2 and PIM6 acyl forms to stimulate macrophages to produce cytokines, and we investigated the implication of the different TLR in this process.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Purification of the PIM Acyl Forms—Fraction C (20 mg) was loaded in 0.1 M ammonium acetate solution containing 15% (v/v) propanol-1 to octyl-Sepharose CL-4B (Amersham Biosciences) column (20 x 1.5 cm) pre-equilibrated with the same buffer. The column was first eluted with 50 ml of equilibration buffer and then with a linear propanol-1 gradient from 15 to 65% (v/v) (250 ml each) in 0.1 M ammonium acetate solution at a flow rate of 5 ml/h. The fractions were collected every 30 min. 20 µl of each fraction was dried and submitted to acidic hydrolysis (100 µl of trifluoroacetic acid, 2 M, 2 h at 110 °C). The hydrolysates were dried, reconstituted in water, and then analyzed by high pH anion exchange chromatography for mannose content giving the presented chromatogram (Fig. 2A). Fractions were pooled according to the purification profile, and repeated lyophilizations were performed to eliminate ammonium acetate salts. An acetone precipitation step was done on each fraction in order to eliminate contaminants issued from the propanol-1. Finally, 1.2 (fraction I), 1 (fraction II), 7.5 (fraction III), and 3 mg (fraction IV) were obtained.
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Purification was checked by TLC on aluminum-backed plates of silica gel (Alugram Sil G, Macherey-Nagel, Duren, Germany), using chloroform/methanol/water, 60:35:8 (v/v/v), as migration solvent. A sulfuric anthron spray and a Dittmer-Lester spray were used to detect carbohydrates containing lipids and phosphorus-containing lipids, respectively.
Acetolysis Procedure—200 µg of PIM were treated with 200 µl of anhydrous acetic acid-d4/acetic anhydride-d6, 1:1 (v/v), at 110 °C for 12 h. The reaction mixture was dried under stream of nitrogen and was submitted to acetylation. The mixture was dissolved in acetic anhydride/anhydrous pyridine, 1:1 (v/v), at 80 °C for 2 h. The reaction mixture was dried under stream of nitrogen. 20 µl of chloroform/methanol, 9:1 (v/v), was added and analyzed in MALDI-Tof-MS in positive and negative modes.
Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry
(MALDI-Tof-MS)—Analysis by MALDI-Tof-MS was carried out on
a Voyager DE-STR (PerSeptive Biosystems, Framingham, MA) using the reflectron
mode. Ionization was effected by irradiation with pulsed UV light (337 nm)
from an N2 laser. PIM were analyzed by the instrument operating at
20 kV in the negative ion mode using an extraction delay time set at 200 ns.
Typically, spectra from 100 to 250 laser shots were summed to obtain the final
spectrum. All of the samples were prepared for MALDI analysis using the
on-probe sample cleanup procedure with cation-exchange resin
(22). The HABA matrix (from
Sigma) was used at a concentration of 
10 mg/ml in ethanol/water (1:1,
v/v). Typically, 0.5 µl of PIM sample (10 µg) in a
CHCl3/CH3OH/H2O solution and 0.5 µl of the
matrix solution, containing 5–10 cation exchange beads, were
deposited on the target, mixed with a micropipet, and dried under a gentle
stream of warm air. The measurements were externally calibrated at two points
with PIM.
NMR Analysis—NMR spectra were recorded with an Avance DMX500
spectrometer (Bruker GmbH, Karlsruhe, Germany) equipped with an Origin 200 SGI
using Xwinnmr 2.6. Samples were dissolved in
CDCl3/CD3OD/D2O, 60:35:8 (v/v/v), and
analyzed in 200 x 5-mm 535-PP NMR tubes at 308 K. Proton chemical shifts
are expressed in ppm downfield from the signal of the chloroform
(
H/TMS 7.27 and C/TMS 77.7). The
one-dimensional phosphorus (31P) spectra were measured at 202 MHz
with phosphoric acid (85%) as external standard (p 0.0). All the
details concerning NMR sequences used and experimental procedures were
detailed in previous study on PIM2
(1).
Primary Macrophage Cultures—TLR2- and/or TLR4-deficient mice obtained by inter-cross from TLR4-deficient mice (23) and TLR2-deficient mice (24), TLR6-deficient mice (25) and MyD88-deficient mice (26), and their control littermates were bred under specific-pathogen-free conditions in the Transgenose Institute animal breeding facility (Orléans, France). Murine bone marrow cells were isolated from femurs and cultivated (106/ml) for 7 days in Dulbecco's minimal essential medium supplemented with 20% horse serum and 30% L929 cell-conditioned medium (as source of M-CSF, as described in Ref. 27). Three days after washing and re-culturing in fresh medium, the cell preparation contained a homogenous population of macrophages. The bone marrow-derived macrophages were plated in 96-well microculture plates (at 105 cells/well) and stimulated with LPS (Escherichia coli, serotype O111:B4, Sigma, at 100 ng/ml), bacterial lipopeptide (Pam3-Cys-Ser-Lys4, EMC microcollections; at 0.5 µg/ml), or PIM preparations at the indicated concentration. Lyophilized PIM preparations were solubilized in Me2SO and added in the cultures at a non-cytotoxic final concentration of 1–1.5% Me2SO (cell viability monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay).
Alternatively the macrophages were infected with M. bovis BCG
(Pasteur strain 1173P2; kind gift from G. Marchal, Pasteur Institute, Paris,
France; at a multiplicity of infection of 2 bacteria per cell). After 18 h of
stimulation, the supernatants were harvested and analyzed for cytokine content
using commercially available enzyme-linked immunosorbent assay reagents for
TNF- and IL-12p40 (Duoset R & D Systems, Abingdon, UK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To proceed in the separation of the acyl forms, 20 mg of fraction C were applied on an octyl-Sepharose column, using propanol-1 as eluent (Fig. 2A). The different acyl forms were eluted at concentrations of propanol-1 ranging from 25 to 50% and separated into 4 sub-fractions according to the profile elution determined by the mannose content. Each sub-fraction (I to IV) was collected and analyzed by negative MALDI-Tof-MS.
MALDI-Tof-MS Characterization of the Octyl-Sepharose Column Fractions—The mass spectra of the fraction I (Fig. 2, B–C) revealed that it contained mono-acylated forms of the molecules (1Ac). Indeed, the analysis of fraction Ia revealed deprotonated molecular ions at m/z 1543.6 characterizing mono-acylated forms with C16 (Fig. 2B), whereas mono-acylated forms with C19 appeared more abundant in fraction Ib (m/z 1585.7) (Fig. 2C). The mass spectrum corresponding to fraction II showed three peaks with an intensity above 10% and were assigned to (M – H)– ions of di-acylated forms: two major peaks at m/z 1781.8 and 1823.9 and one minor one at m/z 1795.8 (Fig. 2D). The two major peaks were characterized as (M – H)– ions of di-acylated forms containing 2C16 and 1C16,1C19, respectively, and the minor peak corresponded to (M – H)– ions of the molecules acylated by 1C16,1C17. The mass spectrum of fraction III (Fig. 2E) revealed two peaks of intensity superior to 10% assigned to tri-acylated forms of the molecules. The major peak at m/z 2062.1 was attributed to (M – H)– ions corresponding to tri-acylated forms containing 2C16,1C19, whereas the minor one at m/z 2090.1 contains 1C16,1C18, and 1C19. The mass spectrum of fraction IV appeared more complex, constituted by a series of peaks (Fig. 2F) between m/z 2230.2 and 2356.4 and assigned to (M – H)– ions of different tetra-acylated forms. Indeed, the most abundant (M – H)– ions at m/z 2300.3 and 2342.4 characterized tetra-acylated forms containing 3C16,1C19 and 2C16,2C19, whereas the ions at m/z 2314.3 corresponded to tetra-acylated forms containing 2C16,2C18. Minor ions at m/z 2272.3, 2286.3, 2328.4, and 2356.4 were attributed to the molecules esterified by 3C16,1C17, 3C16,1C18, 2C16,1C18,1C19, and 1C16,2C18,1C19 or 1C16 and 1C17,2C19, respectively. In addition, species esterified by unsaturated fatty acids were also present as each peak consisted of a –2 analog.
Therefore, we have developed a powerful preparative method of fractionation, leading to the purification of four purified PIM6, corresponding to mono-, di-, tri-, and tetra-acyl forms. The structural analysis is further detailed below for the most complex entities, tri- and tetra-acyl forms.
Glycosidic Analysis of Native Tetra-acyl Forms—The sequence
of glycosyl residues in PIM6 was established using a range of high
resolution NMR techniques applied to the tetra-acylated molecules. The
1H NMR spectrum of the native molecules in
CDCl3/CD3OD/D2O, 60:35:8 (v/v/v), at 500 MHz
showed a complex anomeric proton region (between 4.4 and 5.1 ppm)
(Fig. 3). Anomeric signals were
investigated thanks to the 1H-13C HMQC spectrum
(Fig. 3). Indeed, from the
protons resonating between 4.4 and 5.1 ppm, two of them correlated with
carbons out of the anomeric zone: proton at 5.02 correlated with a
carbon at 70.6 ppm and proton at 4.53 correlated with a carbon at 71.7
ppm. From previous studies (1,
2), they were respectively
assigned to H2 of Gro and H3 of myo-Ins. These protons are deshielded
due to the presence of gem acyl group. Moreover, the coupling constants
measured on the proton at 4.53 (J2,3 2.4 Hz and
J3,4 10.6 Hz) confirmed its attribution to
myo-Ins H3. The other proton resonances correlating with carbons
resonating around 100 ppm accounted for the six mannose units labeled from I
to VI in decreasing order of their chemical shifts. Indeed, integration of the
anomeric signals (H1) proved that the signal resonating at 4.88 ppm
corresponded to two protons (II1 and III1). The
-anomeric configuration of the mannoses was deduced from the values of
the one bound coupling constant (1JC1,H1) above
170 Hz, measured on non-decoupled 1H-13C HMQC spectrum
(not shown).
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The H2 protons of each -Manp unit was determined using the
COSY spectrum (Fig.
4A) although H2 of system I was partly hidden by the
H3/H3' of glycerol (Gro). The entire spin system of Gro can be analyzed
from the COSY spectrum (Fig.
4A) and those of the myo-Ins from the HOHAHA
spectrum (Fig. 4B).
The six mannose spin systems could be completely assigned
(Table I) using
1H-1H HOHAHA (Fig.
4B) and 1H-13C HMQC
(Fig. 3). The deshielded values
of the chemical shifts of the C2 of units I ( 78.5) and IV (
79.1) and of C6 of units II ( 65.8) and VI ( 66.3) revealed the
sites of glycosylation of the concerned units. The absence of deshielding
concerning the carbons of systems III and V indicated that these units III and
V were terminal. The chemical shift of the C6 of unit III ( 63.9)
(Table I) was intermediate
between those of the C6 of units I, IV, and V (around 61.5 ppm) that were not
glycosylated in C6 and those of the C6 of units II and VI (around 66.0) that
were glycosylated in C6. Moreover, H6/H6' protons of unit III were
deshielded (4.02:4.15) (Table
I). Thus, these data demonstrated that this unit is acylated in
position 6.
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The glycosidic sequence was next deduced from the inter-residual nuclear
Overhauser effect contacts observed in the 1H-1H ROESY
spectrum (Fig. 4C).
This sequence was used here to observe short through space connectivities
between the anomeric proton of each mannose and the proton of the adjacent
glycosidically linked residue. The anomeric proton of spin system I (
5.02) correlated with H2 of system IV, indicating that -Manp
unit I is linked to the C2 position of -Manp unit IV. In the
same way, the occurrence of cross-peaks relating H1 (IV)/H6 (VI), H1 (V)/H2
(I), and H1 (VI)/H6 (II) established the partial linear sequence
V-(12)-I-(12)-IV-(16)-VI-(16)-II. Anomeric protons of
-Manp units II and III were then separated showing both
correlations with protons belonging to the Ins unit.
The H1 of system III showed a nuclear Overhauser effect contact with proton 2 of myo-Ins, revealing that this unit is linked in position 2 of the myo-Ins. The H1 of system II showed two cross-peaks with protons 1 and 6 of myo-Ins. As explained below, two-dimensional 1H-31P HMQC analysis allowed us to define one of the positions of substitution of the phosphorus as position 1 of the myo-Ins. Thus, system II was deduced to be linked in position 6 of the myo-Ins.
Therefore, the glycosidic analysis of the tetra-acylated form of the
molecules using a combination of scalar and dipolar homonuclear and
heteronuclear NMR sequences demonstrated the structure
t--Manp-(12)--Manp-(12)--Manp-(16)--Manp-(16)--Manp-(16)-myo-Ins-(2
1)-t--Manp,
as depicted in Fig. 5.
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Anchor Structure of Native Tetra-acyl Forms—The NMR strategy
developed to study PIM2 containing a total of 4 fatty acids
(1) was used here. From the
1H-31P HMQC experiment, the prochiral H-3 and H-3'
Gro protons and the myo-Ins H-1 were easily assigned (not shown). The
remaining myo-Ins and Gro protons were then observed on the
two-dimensional 1H-31P HMQC-HOHAHA spectrum
(Fig. 6B) and were
assigned from their multiplicity and chemical shifts
(29) and with the help of the
1H-1H HOHAHA spectrum
(Fig. 6, C–D).
The different chemical shifts typified the presence or absence of an acyl
appendage. The chemical shifts of the H-1 (4.19/3.95 ppm) and H-2 (5.02 ppm)
revealed a di-acyl-Gro (29).
The downfield shift of the myo-Ins H-3 resonating at 4.53 ppm then
signed the C3 acyl appendage. A fourth position of acylation, the C6 of the
Manp, could be assigned by analysis of the two-dimensional
1H-13C HMQC spectrum. Indeed, the chemical shifts of
H-6/H-6' of -Manp unit III (4.02/4.15 ppm) proved that
this position is also acylated. The slight deshielding of C6 resonance (from
61.5 to 63.9 ppm) confirmed this assignment.
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Therefore, the analytical approach applied to this acyl form of PIM6 (containing four fatty acids in total) revealed that the four positions of acylation were the same as the ones described in case of the corresponding acyl form of PIM2 (1): the positions C1 and C2 of the Gro, position C3 of the myo-Ins unit, and position C6 of the Manp unit linked to C2 of the myo-Ins.
Acyl Distribution of Native Tetra-acyl Forms—The nature of the fatty acids esterifying the different sites was investigated by mass spectrometry using MALDI-Tof-MS analysis of the acetolysis reaction products of the native tetra-acyl forms of the molecules (not shown). Acetolysis cleaves the phosphoglycerol linkage without altering the fatty acid esters, leading to two entities: the hexamannosyl-inositol phosphate moiety (Man6-Ins-P) and the acyl-glycerol residue, as already described in Ref. 1. The hexamannosyl-inositol phosphate moiety was observed in negative mode as [M – H]– ions, whereas the acyl-glycerol part was analyzed in positive mode as [M + Na]+ ions. As described previously, the acetolysis reaction produces two populations of Man6-Ins-P moieties that differ by the presence or absence of an acetyl group on the phosphate (1). But as this acetate present on the phosphate is very labile (mixed anhydride), it is partially hydrolyzed when the sample is mixed with the matrix (HABA diluted in EtOH/H2O). To discriminate between acetate groups and changes of C19 by C16, the reaction was made with perdeuterated acetic acid and acetic anhydride.
The positive MALDI-Tof-MS spectrum of the perdeutero-acetolyzed of the tetra-acylated molecules showed an intense peak at m/z 678.6 assigned to sodium adduct of the di-acylated C16/C19 Gro (not shown). The negative mass spectrum mainly showed two peaks at m/z 2653.7 and 2695.8 corresponding to the Man6-Ins-P moiety acylated with 2C16 and C16,C19, respectively. Less intense peaks were also observed corresponding to the Man6-Ins-P moiety acylated with other combinations of fatty acids. Taken together, these results indicate that the glycerol is always acylated by C16,C19, and we can postulate that the mannose is acylated by a C16, whereas the nature of the fatty acid present on the inositol is variable, being predominantly C16 or C19.
Analysis of Native Tri-acyl
Forms—1H-13C HMQC spectrum of the
tri-acylated molecules exhibited the same cross-peaks as for the
tetra-acylated one, except for the inositol spin system defined as
H1/C1 3.93/70.64, H2/C2 4.04/78.68, H3/C3 3.27/70.63,
H4/C4 3.36/67.63, H5/C5 3.10/73.68, and H6/C6 3.60/79.03,
indicating that there is no fatty acyl appendage on C3. The three fatty acids
were found on both positions of Gro and on the -Manp unit on
the C2 of myo-Ins. The positive MALDI-Tof-MS spectrum of
deutero-acetolyzed molecules showed a similar intense peak at
m/z 678.6 assigned to the sodium adduct of the di-acylated
C16/C19 Gro as the one observed in the case of the
tetra-acylated molecules (not shown). The negative mass spectrum mainly showed
one peak at m/z 2460.5 corresponding to the
Man6-Ins-P moiety acylated with one 1C16. Thus, the
results indicate that the tri-acylated molecules predominantly exist with
2C16 and 1C19, in agreement with the negative
MALDI-Tof analysis of the native molecules
(Fig. 2E), the
glycerol being di-acylated by C16/C19 and the mannose
bearing a C16.
Macrophage Stimulation by PIM2 and
PIM6—Unfractionated PIM have been
shown to stimulate the murine RAW 264.7 monocytic cell line to produce
TNF- (15), and we asked
whether purified PIM2 and PIM6 were equally
pro-inflammatory. First, unfractionated PIM2 preparation was tested
and shown to stimulate TNF- and IL-12 p40 secretion by primary murine
macrophages (not shown). Then two different well defined acyl forms of
PIM2 were assayed as follows: a fraction (F6) containing lyso-PI
and lyso-PIM2 (both containing C16) and a fraction (F7)
containing more acylated forms of PIM2 (tri-acylated and
tetra-acylated molecules). Both PIM2 fractions induced TNF-
secretion irrespective of their acyl forms
(Fig. 7). The concentration of
TNF- secreted was clearly sub-maximal (1 ng/ml) as compared with
those achieved by stimulation with strong stimuli such as LPS or BLP or after
infection with live BCG (15 ng/ml;
Fig. 7A). We next
asked whether PIM6 exhibited a similar function. The unfractionated
PIM6 preparation (F1) also stimulated TNF- secretion by
macrophages (Fig. 7B).
To determine whether the number of fatty acids played a role in the
stimulation of TNF- secretion, the purified acyl forms of
PIM6 (F2 to F5) were tested for their capacity to stimulate
macrophages to produce TNF-. No clear enrichment in stimulatory
activity was observed with the purified PIM6 acylated forms (mono-
to tetra-acylated molecules) as compared with the unfractionated
PIM6 (Fig.
7B). Only marginal levels of IL-12p40 were detected after
incubation of macrophages with PIM2 or PIM6 (data not
shown).
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Thus, both PIM2 and PIM6 stimulated TNF-
secretion by primary macrophages, and this activity seemed independent from
the number and the nature of the PIM2 and PIM6 acyl
moieties.
TLR Dependence of PIM Activity—The unfractionated PIM
preparation was shown to be a TLR2 agonist, based on reporter assay with
Chinese hamster ovary cell lines transfected with the tlr2 gene
(15). Here bone marrow-derived
macrophages prepared from mice rendered deficient for TLR2 and/or TLR4, for
TLR6, or for MyD88, the adaptor common to the different TLR, were used to
investigate the TLR dependence of the PIM2 and PIM6
responses. The macrophages were stimulated with the fraction (F6) containing
lyso-PI and lyso-PIM2 (both containing C16), and the
fraction (F7) containing more acylated forms of PIM2 (tri-acylated
and tetra-acylated molecules) or with the PIM6 fraction (F1) and
TNF- (Fig. 8) and IL-12
p40 (not shown) secretions were assessed. The production of TNF- by
primary macrophages in response to the PIM2 and PIM6
fractions is dependent on TLR2, as no TNF- could be detected in the
supernatant of macrophages deficient for TLR2, although cells deficient for
TLR4 were efficiently stimulated by these fractions
(Fig. 8, B and
C). As expected, no cytokine production was detected in
the supernatants of macrophages isolated from the double knock-out mice
(TLR2–/– and TLR4–/–)
(Fig. 8, B and
C). Thus, PIM6 are capable of inducing
TNF- secretion via a TLR2-dependent pathway. TLR2 has been shown to
heterodimerize with TLR1 or TLR6
(30), and we next looked at
the implication of TLR6 in the PIM-TLR2 interaction. TLR6 deficiency did not
impair the ability of the macrophages to respond to PIM2 or
PIM6 (Fig. 8,
D–F). MyD88 is involved in most of the TLR-mediated
signals (31), including TLR2
as shown here for the TLR2 agonist BLP and for the best of the response to the
TLR4 agonist LPS (Fig.
8D), and we showed that macrophages deficient for MyD88
did not respond to the PIM2 or PIM6 stimulation
(Fig. 8, E and
F). Thus, both PIM2 and PIM6
stimulate TNF- secretion by macrophages through TLR2 and not TLR4. TLR6
is not essential in this response which involves signaling through the adaptor
MyD88.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The structural strategy used to characterize PIM6 acyl forms was similar to the one successfully used to define PIM2 acyl forms (1) and combines the potency of complementary analytical techniques, NMR and mass spectrometry. NMR was used to characterize the acylation positions, whereas mass spectrometry gave information concerning the number of fatty acids and the nature of the fatty acids present at each position. MALDI or ESI modes were chosen rather than fast atom bombardment, because the molecules could be analyzed without derivatization preventing the loss of any labile substituents. In this study, MALDI-MS was chosen rather than ESI-MS as a same deposit could be analyzed in positive or negative mode. Indeed, concerning the analysis of the acetolysis products, the hexamannosyl-inositol phosphate moiety (Man6-Ins-P) was observed in negative mode, whereas the acyl-glycerol residue was analyzed in positive mode. The complete NMR study required some milligrams of purified product. This was detailed for the more complex acyl forms, the tri-acylated and the tetra-acylated ones.
The results obtained are summarized in Table II. The mono-acylated forms are identified for lyso-PIM6 with C16 or C19 in position 1 of the glycerol. The di-acylated forms appear as two populations almost equally represented as one with 2C16 and one with 1C16 and 1C19, both fatty acids being on the glycerol and structurally corresponding to "true" PIM6. In contrast, concerning tri-acylated forms, a major acyl form was observed, containing 2C16 and 1C19, the glycerol being di-acylated by C16,C19 and the mannose bearing a C16. They should then correspond to mono-acylated-PIM6 (or Ac1PIM6). Interestingly, the acyl form containing 3C16 was not observed, indicating that the tri-acylated forms arose from the di-acylated forms containing C16,C19 as fatty acyl appendages. The tetra-acylated forms were present as essentially two populations, 3C16,1C19 or 2C16,2C19. The acylation positions were here clearly elucidated as being both positions of Gro, the C3 of the myo-Ins unit and the C6 of the Manp linked to the C2 of the myo-Ins unit. Taken together, the same conclusions as for the acyl forms of PIM2 could be made. Indeed, the results indicate that the glycerol is preferentially acylated by C16,C19. However, the nature of the fatty acid present on the myo-inositol appears highly variable, being essentially C16 or C19.
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As mentioned previously (1),
the biosynthetic linkage between PIM2, LM, and LAM appears now to
be more and more evident, as the same anchor structures were found for all
these lipoglycans. However, even if the acyl forms found for PIM6
were exactly the same as the ones found for PIM2, LM, and LAM,
PIM6 are not part of the LM/LAM biosynthetic pathway. Indeed, the
linear mannan backbone found in LM and LAM is constituted by a linear
-(16)-linked Manp, whereas PIM6 exhibit at
its extremity -(12) links. PIM6 appears to be an end
product and may have a specific role.
Mammalian TLR proteins are pattern recognition receptors for a wide array
of bacterial and viral products
(17). Gram-negative bacterial
lipopolysaccharide (LPS) activates cells through TLR4, whereas the
mycobacterial cell wall lipoglycans, AraLAM, activate cells through TLR2.
Recently, Jones et al.
(15) identified a secreted
TLR2 agonist activity in short term culture filtrates of M.
tuberculosis, which they called STF, for "soluble tuberculosis
factor." To determine the identity of the TLR2 agonist present in
soluble tuberculosis factor, they used preparative SDS-PAGE. The TLR2 agonist
activity was present in one fraction, with an apparent molecular size of 6
kDa, raising the possibility that the TLR2 agonist was PIM2. By
using TLC, the authors reported the presence of two major species,
PIM1 and PIM2. In that case, the precise structure of
the TLR2 agonist was not determined. We confirm here that structurally defined
acyl forms of PIM2 stimulated TNF- secretion by primary
macrophages in a TLR2-dependent fashion. We then demonstrated that the major
polar PIM from M. bovis BCG and M. tuberculosis,
PIM6, also stimulated TNF- secretion and that this secretion
was mediated by TLR2 but not TLR4. TLR2 has been shown to heterodimerize with
TLR1 or TLR6 (30). We show
here that the TLR2-mediated stimulation of macrophages by PIM2 and
PIM6 was independent of TLR6 but that both PIM2 and
PIM6 signaled via the adaptor molecule MyD88.
It has been demonstrated in the case of LPS that saturated fatty acids
acylated in lipid A moiety are essential for its biological activities.
Saturated fatty acids, but not unsaturated fatty acids, induce NF-
B
activation and expression of inflammatory markers in macrophages
(37). In addition, it has been
proposed that the shape of lipid A, influenced by the length and number of
acyl chains, asymmetry of acyl groups, and distribution of negative charges,
determine the interaction of LPS with different TLR
(38). This would explain how
E. coli LPS, with a strong conical shape lipid A, interact with TLR4,
whereas Porphyromonas gingivalis or Rhodobacter sphaeroides
LPS, with a more cylindrical shape lipid A, are TLR2 agonists
(38). Here we asked whether
different PIM2 and PIM6 acyl forms, bearing 1–4
acyl residues, were equally potent in stimulating macrophages to produce
TNF-. We show that the TNF- stimulating activity of
PIM6 is independent from the number and the nature of the acyl
moieties present on the molecules. This seemed to be also the case for
PIM2 as both mono- (F6) and tri- and tetra-acylated (F7) forms
studied here had similar activity.
In infected macrophages, PIM were shown to traffic out of the mycobacterial phagosome among other mycobacterial lipids, such as LAM, and are released to the medium and bystander uninfected cells (14). We show here that both major PIM species from M. bovis BCG, M. smegmatis 607, and M. tuberculosis H37Rv, PIM2 and PIM6, are agonists of TLR2, irrespective of their acylation state. ManLAM from slow-growing mycobacteria such as M. bovis or M. tuberculosis do not show such pro-inflammatory effects or TLR2-dependent activity (39).2 TLR2 may be present intracellularly, recruited to macrophage phagosomes, where they sample the content of the vacuole, and contribute to elaborate an inflammatory response appropriate for defense against a specific pathogen (40). The trafficking and export of PIM2 and the more polar PIM6 from M. bovis or M. tuberculosis could thus contribute to maintaining the smoldering activation state of the infected macrophage and of the neighboring cells in the tuberculous granuloma through TLR2 activation, and contribute to the innate immunity necessary to contain latent infection.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 33 5 61 17 55 57; Fax: 33 5
61 17 59 94, E-mail:
Martine.Gilleron{at}ipbs.fr.
1 The abbreviations used are: PIM, phosphatidyl-myo-inositol
mannosides; BCG, bacillus Calmette Guérin; BLP, bacterial lipopeptide;
C16, palmitate; C18, stearate; C19,
tuberculostearate (10-methyloctadecanoate); COSY, correlation spectroscopy;
ESI-MS, electrospray ionization-mass spectrometry; Gro, glycerol; HABA,
2-(4-hydroxyphenylazo)-benzoic acid; HMQC, heteronuclear multiple quantum
correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; LAM,
lipoarabinomannans; LM, lipomannans; ManLAM, LAM with mannosyl extensions;
MALDI-Tof-MS, matrix-assisted laser desorption/ionization-time of
flight-mass spectrometry; myo-Ins, myo-inositol; MyD88,
myeloid differentiation factor; p, pyranosyl; PI,
phosphatidyl-myo-inositol; QMA, quaternary methyl ammonium; ROESY,
rotating frame nuclear Overhauser effect spectroscopy; t, terminal; TLR,
Toll-like receptor; TMS, trimethylsilyl; TNF-, tumor necrosis
factor-; IL, interleukin; LPS, lipopolysaccharide.
2 M. Gilleron, V. F. J. Quesniaux, and G. Puzo, unpublished data.
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ACKNOWLEDGMENTS |
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