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Originally published In Press as doi:10.1074/jbc.M310906200 on March 18, 2004

J. Biol. Chem., Vol. 279, Issue 22, 22973-22982, May 28, 2004
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Tsukamurella paurometabola Lipoglycan, a New Lipoarabinomannan Variant with Pro-inflammatory Activity*

Kevin J. C. Gibson{ddagger}, Martine Gilleron§, Patricia Constant§, Thérèse Brando§, Germain Puzo§, Gurdyal S. Besra{ddagger}, and Jérôme Nigou§

From the {ddagger}School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom and the §Department of Molecular Mechanisms of Mycobacterial Infections, Institut de Pharmacologie et de Biologie Structurale, CNRS, Unité Mixte de Recherche 5089, 205 Route de Narbonne, 31077 Toulouse, cedex 4, France

Received for publication, October 3, 2003 , and in revised form, February 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan, one of the most potent mycobacterial immunomodulatory molecules. We have investigated the presence of lipoglycans in Tsukamurella paurometabola and report here the isolation and structural characterization of a new lipoarabinomannan variant, designated TpaLAM. Matrix-assisted laser desorption ionization-mass spectrometric analysis revealed that TpaLAM had an average molecular mass of 12.5 kDa and consequently was slightly smaller than Mycobacterium tuberculosis lipoarabinomannan. Using a range of chemical degradations, NMR experiments, capillary electrophoresis, and mass spectrometry analyses, TpaLAM revealed an original carbohydrate structure. Indeed, TpaLAM contained a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by a linear ({alpha}1->6)-Manp mannan domain, which is further substituted by an ({alpha}1->5)-Araf chain. Half of the Araf units are further substituted at the O-2 position by a Manp-({alpha}1->2)-Manp-({alpha}1-> dimannoside motif. Altogether, TpaLAM appears to be the most elaborated non-mycobacterial LAM molecule identified to date. TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-{alpha} when tested with either human or murine monocyte/macrophage cell lines. This induction was completely abrogated in the presence of an anti-toll-like receptor-2 (TLR-2) antibody, suggesting that TLR-2 participates in the mediation of TNF-{alpha} production in response to TpaLAM. Moreover, we established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-{alpha}-inducing activity. This conclusively demonstrates that a linear ({alpha}1->6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is a bewildering range of aerobic actinomycetes, found in almost any environment imaginable, with some pathogenic to humans and others that are not (1). The aerobic actinomycetes may be further subdivided into the "nocardioform actinomycetes" (2). This informal terminology is now widely used to describe a number of organisms with similar characteristics, with key members including mycobacteria, nocardia, rhodococcus, and corynebacteria (3). Unlike the previously mentioned members the genus Tsukamurella is in its infancy, whereas the type strain, Tsukamurella paurometabola, has had a long taxonomical history, with names including Corynebacterium paurometabolum (4), Gordona aurantiaca (5), and Rhodococcus aurantiacus (6). This taxonomical puzzle was finally resolved, when in 1988 Collins et al. (7) showed that the 16 S RNAs of R. aurantiacus and C. paurometabolum were 99% homologous. And so they proposed reclassifying and renaming this organism T. paurometabola, after the microbiologist M. Tsukamura, who first isolated the species (7).

Cases of human infection with T. paurometabola are infrequent, nevertheless diagnosis rates are increasing, typically in patients with underlying predisposing factors, including immunosuppression (8, 9), chronic pathology (6), and indwelling foreign bodies (10). However, there are a number of reported cases in which infected patients had no underlying factors, with Granel et al. (11) describing an inflammatory cutaneous infection in an otherwise healthy individual.

All members of the nocardioform actinomycetes possess a similar whole cell carbohydrate composition, whereas the majority also contain long-chain branched fatty acids, termed mycolic acids (12). The majority of our current knowledge about actinomycetes cell wall architecture comes from pioneering studies on mycobacterial strains (13). Such work led to the identification, within the cell envelope, of a biosynthetically related family of glycolipids, phosphatidyl-myo-inositol mannosides (PIMs),1 and lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (13). Mycobacterial LAM is a large heterogeneous macroamphiphile that possesses three distinct domains, a mannosylphosphatidyl-myo-inositol (MPI) anchor, a carbohydrate backbone, and various capping motifs (14, 15). The carbohydrate backbone is composed of two homopolysaccharides, D-mannan and D-arabinan. In all species described to date the D-mannan domain exists as a linear {alpha}(1->6)-Manp backbone substituted according to the species at the O-2 or O-3 positions by single Manp residues. The D-arabinan domain consists of a linear {alpha}(1->5)-Araf backbone punctuated by branching fashioned from 3,5-O-linked {alpha}-D-Araf residues (14, 15).

In slow growing mycobacteria, such as Mycobacterium tuberculosis, the capping motifs consist of Manp residues (16, 17); whereas fast growing mycobacteria, such as Mycobacterium smegmatis, possess phosphoinositol residues (18, 19), resulting in LAM being termed either ManLAM or PILAM, respectively. In addition, a new class of LAMs has been described that lacks any capping motifs (20), termed AraLAM. These subtle differences in the capping motifs are thought to explain the different immunomodulatory functions of ManLAM and PILAM. Indeed, a paradigm is emerging whereby ManLAMs possess the ability to inhibit the production of pro-inflammatory cytokines, such as interleukin-12 and TNF-{alpha} (21, 22); conversely, PILAM stimulates the production of such cytokines (18, 23).

Lipoglycans from other nocardioform actinomycetes have been identified and have been shown to have related, but distinct structures to that of mycobacterial LAM. In particular, they are smaller in size and do not necessarily possess distinct mannan and arabinan domains (24–30). Few of these studies examined whether these lipoglycans possessed any in vitro biological activity; nevertheless, in a recent publication we have demonstrated that a LAM-like molecule from Amycolatopsis sulphurea, designated AsuLAM, which possessed mannose capping motifs, failed to induce a pro-inflammatory cytokine pattern (27), in agreement with the findings that ManLAMs can modulate the immune response by inhibiting the induction of pro-inflammatory cytokines.

We report here the isolation and structural characterization of a lipoglycan originating from T. paurometabola. Furthermore, we provide evidence for the molecular motifs underlying bacterial lipoglycan mediated pro-inflammatory cytokine responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria and Growth Conditions—T. paurometabola, type strain DSM 20162, was purchased from Deutsche Sammlung van Mikroorganismen and Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures), Germany. It was routinely grown at 30 °C in GYM streptomyces medium, which contained 4 g of glucose, 4 g of yeast, and 10 g of maltose extract per liter of deionized water supplemented with 0.05% (w/v) Tween 80. Cells were grown to late log phase, harvested by centrifugation, washed, and lyophilized.

Purification of TpaLAM—Purification procedures were adapted from protocols established for the extraction and purification of mycobacterial lipoglycans (31, 32). Briefly, the cells were delipidated at 60 °C by mixing in CHCl3/CH3OH (1:1, v/v) overnight. The organic extract was removed by filtration, and the delipidated biomass was resuspended in deionized water and disrupted by sonication (MSE Soniprep, 12 micro amplitude, 60 s on, 90 s off for 10 cycles, on ice). The cellular glycans and lipoglycans were further extracted by refluxing the broken cells in 50% ethanol at 65 °C overnight. Contaminating proteins and glucans were removed by enzymatic degradation using protease and {alpha}-amylase treatments followed by dialysis. The resulting extract was resuspended in buffer A, 15% propan-1-ol in 50 mM ammonium acetate, and loaded onto an octyl-Sepharose CL-4B column (50 x 2.5 cm) and eluted with 400 ml of buffer A at 5 ml/h, enabling the removal of non-lipidic moieties (22). The retained lipoglycans were eluted with 400 ml of buffer B, 50% propan-1-ol in 50 mM ammonium acetate. The resulting lipoglycans were resuspended in buffer C, 0.2 M NaCl, 0.25% sodium deoxycholate (w/v), 1 mM EDTA, and 10 mM Tris, pH 8, to a final concentration of 200 mg/ml and loaded onto a Sephacryl S-200 HR column (50 x 2.5 cm) and eluted with buffer C at a flow rate of 5 ml/h. Fractions (1.25 ml) were collected and analyzed by SDS-PAGE followed by periodic acid-silver nitrate staining. The resulting lipoglycan fractions were pooled, dialyzed extensively against water, lyophilized, and stored at –20 °C.

Preparation of Chemically or Enzymatically Modified TpaLAM— Deacylated TpaLAM (dTpaLAM) was obtained by incubating 100 µg of TpaLAM with 200 µl of 0.1 N NaOH for 2 h at 37 °C. The reaction was stopped by extensive dialysis against water. TpaLAM lipomannan core (i.e. mild acid hydrolyzed TpaLAM, mahTpaLAM) was prepared by mild acid hydrolysis (0.1 M HCl at 110 °C for 25 min) of TpaLAM. TpaLAM lipomannan core was recovered after dialysis against water and analyzed for carbohydrate content via capillary electrophoresis coupled to laser-induced fluorescence (CE-LIF) as described below. {alpha}-Exomannosidase-treated TpaLAM ({alpha}TpaLAM) was prepared by incubating TpaLAM (100 µg) for 6 h at 37 °C in 30 µl of a jack bean {alpha}-mannosidase (Sigma) solution (2 mg/ml, 0.1 M sodium acetate buffer, pH 4.5, 1 mM ZnSO4). After a second addition of 50 µl of enzyme solution, the reaction was continued overnight. The reaction products were then dialyzed against 50 mM NH4CO3, pH 7.6. Elimination of {alpha}-mannosidase was achieved by denaturation (2 min at 110 °C) followed by overnight tryptic digestion (37 °C, 3.2 µg of trypsin). {alpha}TpaLAM was recovered after dialysis against water and analyzed for cap contents by CE-LIF (22).

MALDI-TOF/MS—The matrix used was 2,5-dihydroxybenzoic acid at a concentration of 10 µg/µl in a mixture of water/ethanol (1:1, v/v). 0.5 µl of TpaLAM, at a concentration of 10 µg/µl, was mixed with 0.5 µl of the matrix solution. Analyses were performed on a Voyager DE-STR MALDI-TOF instrument (PerSeptive Biosystems, Framingham, MA) using linear mode detection. Mass spectra were recorded in the negative mode using a 300-ns time delay with a grid voltage of 94% of full accelerating voltage (20 kV) and a guide wire voltage of 0.1%. The mass spectra were mass assigned using external calibration.

Fatty Acid Analysis—200 µg of TpaLAM was deacylated using strong alkaline hydrolysis (200 µlof1 M NaOH at 110 °C for 2 h). The reaction mixture was neutralized with HCl, and the liberated fatty acids were extracted three times with chloroform and, after drying under nitrogen, were methylated using three drops of 10% (w/w) BF3 in methanol (Fluka) at 60 °C for 5 min. The reaction was stopped by the addition of water and the fatty acid methyl esters were extracted three times with chloroform. After drying, the fatty acid methyl esters were solubilized in 10 µl of pyridine and trimethylsilylated using 10 µl of hexamethyldisilazane and 5 µl of trimethylchlorosilane at room temperature for 15 min. After drying under a stream of nitrogen, the fatty acid derivatives were solubilized in cyclohexane before analysis by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS).

Glycosidic Linkage Analysis—Glycosyl linkage composition was performed according to the modified procedure of Ciucanu and Kerek (33). The per-O-methylated TpaLAM was hydrolyzed using 500 µl of 2 M trifluoroacetic acid at 110 °C for 2 h, reduced using 350 µl of a 10 mg/ml solution of NaBD4 (1 M NH4OH/C2H5OH, 1:1, v/v) and per-O-acetylated using 300 µl of acetic anhydride for 1 h at 110 °C. The resulting alditol acetates were solubilized in cyclohexane before analysis by GC and GC/MS.

APTS Derivatization—1–5 µg of lipoglycans, in the presence of mannoheptose as an internal standard, were hydrolyzed using either strong acid hydrolysis (30 µl of 2 M trifluoroacetic acid at 110 °C for 2 h) (total carbohydrate analysis) or mild acid hydrolysis (30 µl of 0.1 M HCl at 110 °C for 20 or 30 min) (caps analysis). The samples were dried and mixed with 0.3 µl of 0.2 M 1-aminopyrene-3,6,8-trisulfonate (APTS) (Interchim, Montluçon, France) in 15% acetic acid and 0.3 µl of a 1 M sodium cyanoborohydride solution dissolved in tetrahydrofuran. The reaction mixture was heated at 55 °C for 90 min and subsequently quenched by the addition of 20 µl of water. A 2-µl solution of the APTS derivatized solution was diluted 10-fold before being subjected to capillary electrophoresis.

CE-LIF—Analyses were performed on a P/ACE capillary electrophoresis system (Beckman Instruments, Inc.) with the cathode on the injection side and the anode on the detection side (reverse polarity). The electropherograms were acquired and stored on a Dell XPS P60 computer using the System Gold software package (Beckman Instruments, Inc.). APTS derivatives were loaded by applying 0.5 p.s.i. (3.45 kPa) vacuum for 5 s (6.5 nl injected). Separations were performed using an uncoated fused-silica capillary column (Sigma, Division Supelco, Saint-Quentin-Fallavier, France) of 50-µm internal diameter with 40 cm of effective length (47-cm total length). 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 running electrolyte. The detection system consisted of a Beckman laser-induced fluorescence (LIF) equipped with a 4-milliwatt argon-ion laser with the excitation wavelength of 488 nm and emission wavelength filter of 520 nm.

CE-ESI/MS—Analyses were performed on a CE system P/ACETM MDQ (Beckman Coulter, Inc) with a 75-µm x 80-cm fused-silica capillary. The outlet was integrated into the electrospray ionization (ESI) needle that was directly coupled to an ion trap MS system (LCQTM DUO, ThermoFinnigan, Inc.). Separations were carried out with an electrolyte composed of acid acetic (1%, v/v), triethylamine (30 mM), pH 3.5, and an applied voltage of –20 kV. Migration was monitored by the total ion current. During analysis, temperature was constantly maintained (25 °C) along the capillary, and the outlet end of the capillary was at a spray voltage of 4 kV. The sheath liquid, consisting of water: isopropanol (20:80), was infused to the ESI needle through a syringe pump at a flow rate of 3 µl min–1 using nitrogen as a nebulizing gas. For measurements, negative mode was used and all data were collected and analyzed on Xcalibur software.

NMR Spectroscopy—NMR spectra were recorded on a Bruker DMX-500 spectrometer equipped with a double resonance (1H/X)-BBi z-gradient probe head. TpaLAM (10 mg) was exchanged in D2O (D, 99.97% from Euriso-top, Saint-Aubin, France), with intermediate lyophilization, then re-dissolved in 0.4 ml of Me2SO-d6 (D, 99.8% from Euriso-top, Saint-Aubin, France) and analyzed in 200 x 5 mm 535-PP NMR tubes at 343 K. Data were processed on a Bruker-X32 workstation using the xwinnmr program. Proton and carbon chemical shifts are expressed in ppm and referenced relative to internal Me2SO signals at {delta}H 2.52 and {delta}C 40.98. The one dimensional (1D) proton (1H) spectrum was recorded using a 90° tipping angle for the pulse and 1 s as recycle delay between each of the 128 acquisitions of 1.64 s. The spectral width of 2948 Hz was collected in 16,000 complex points that were multiplied by a Gaussian function (LB = –1, GB = 0.2) prior to processing to 32,000 real points in the frequency domain. After Fourier transformation, the spectra were baseline-corrected with a fourth order polynomial function. Two-dimensional (2D) spectra were recorded without sample spinning, and data were acquired in the phase-sensitive mode using the time-proportional phase increment (TPPI) method. The 2D 1H-13C heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) were recorded in the proton-detected mode with a Bruker 5-mm 1H broad band tunable probe with reverse geometry. The Globally optimized Alternating-phase Rectangular Pulses sequence (34) at the carbon frequency was used as a composite pulse decoupling during acquisition. The 1H-13C HMQC spectrum was obtained according to Bax and Subramanian pulse sequence (35). Spectral widths of 25,154 Hz in 13C and 2,948 Hz in 1H dimensions were used to collect a 2,048 x 413 (TPPI) point data matrix with 80 scans/t1 value expanded to 4,096 x 1,024 by zero filling. The relaxation delay was 1 s. A sine bell window shifted by {pi} /2 was applied in both dimensions. The 1H-13C HMBC spectrum was obtained using the Bax and Summers pulse sequence (36). Spectral widths of 25,154 Hz in 13C and 2,948 Hz in 1H dimensions were used to collect a 2,048 x 512 (TPPI) point data matrix with 96 scans/t1 value expanded to 4,096 x 1,024 by zero filling. The relaxation delay was 1 s. A sine bell window shifted by {pi} /2 was applied in both dimensions. The 2D 1H-1H HOHAHA spectrum was recorded using a MLEV-17 mixing sequence of 112 ms (37). The spectral width was 2,948 Hz in both F2 and F1 dimensions. 512 spectra of 4,096 data points with 24 scans/t1 increment were recorded.

TNF-{alpha} Production by Macrophages—THP-1 and J774 monocyte/macrophage human and murine cell lines, respectively, were maintained in continuous culture with RPMI 1640 medium (Invitrogen), 10% fetal calf serum (Invitrogen) in an atmosphere of 5% CO2 at 37 °C, THP-1 as non-adherent and J774 as adherent cells. Purified native or modified TpaLAM were added in duplicate or triplicate to monocyte/macrophage cells (5 x 105 cells/well) in 24-well culture plates and then incubated for 20 h at 37 °C. Stimuli were previously incubated for 1 h at 37 °C in the presence or absence of 10 µg/ml polymyxin B (Sigma) known to inhibit the effect of (contaminating) LPS (23). To investigate the TLR dependence of TNF-{alpha}-inducing TpaLAM activity, monoclonal anti-TLR-2 (clone TL2.1, eBioscience) or anti-TLR-4 (clone HTA125, Serotec) antibodies or an IgG2a isotype control (clone eBM2a, eBioscience) at a concentration of 10 µg/ml were added together with TpaLAM to THP-1 cells. Supernatants from THP-1 cells were assayed for TNF-{alpha} by sandwich enzyme-linked immunosorbent assay using commercially available kits and according to manufacturer's instructions (R&D Systems). Supernatants from J774 cells were assayed for TNF-{alpha} using the previously described cytotoxic assay against WEHI164 clone 13 cells (38). Basically, 50 µl of supernatant was added to 50 µlof WEHI cells (5 x 105 cells/ml) in flat-bottom 96-well plates and incubated for 20 h at 37 °C, then 50 µl of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 1 mg/ml in phosphate-buffered saline) were added to each well and incubated for 4 h. Formazan crystals were solubilized with 100 µl of lysis buffer (N,N-dimethyl formamide, 30% SDS (1:2) adjusted to pH 4.7 with acetic acid), and the optical density was read at 570 nm with an enzyme-linked immunosorbent assay plate reader (Bio-Tek Instruments). The TNF-{alpha} content of supernatants was determined by comparing to a reference curve obtained using serial dilutions of human recombinant TNF-{alpha} (Invitrogen). LPS was from Escherichia coli 055:B5 (Sigma) and Man-LAM from Mycobacterium bovis BCG.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
General Features
A lipoglycan with a SDS-PAGE migration similar to that of M. tuberculosis LAM was purified from T. paurometabola (Fig. 1A). The negative MALDI mass spectrum of the lipoglycan exhibited a broad unresolved peak centered at m/z 12500, indicating a molecular mass around 12.5 kDa for the major molecular species of this lipoglycan (Fig. 1B). CE-LIF analysis of the total acid hydrolyzed lipoglycan showed that it contained Ara and Man, in a ratio of 1:1.7. In addition, myo-inositol, glycerol, and fatty acids were also detected by GC analysis. The predominant fatty acids identified were palmitic (C16:0, 50%) and octadecenoic (C18:1, 20%), with smaller amounts of stearic (C18:0, 15%) and tuberculostearic (methyl-10-methyloctadecanoic, C19:0, 15%) acids. Altogether, the lipoglycan exhibited the basic components of a structure related to mycobacterial LAM and was subsequently termed TpaLAM.



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  		FIG. 1.
SDS-PAGE (A) and MALDI/MS (B) analyses of T. paurometabola lipoglycan. In A: lane 1, M. tuberculosis ManLAM, LM (top and bottom bands, respectively); lane 2, T. paurometabola lipoglycan. B, 0.5 µlofa T. paurometabola lipoglycan solution at 10 µg/µl were mixed with 0.5 µl of the matrix solution (10 µg/µl of 2,5-dihydroxybenzoic acid in ethanol/water, 1:1, v/v) and analyzed by MALDI-TOF in the negative mode.

 
Structural Characterization
NMR Signal Assignment—Per-O-methylation analysis of TpaLAM indicated the presence of 5-Araf, t-Manp, 2,5-Araf, and 2-Manp residues in similar ratios, with slightly less 6-Manp detectable. Accordingly, the 1H-NMR anomeric region of TpaLAM exhibited five anomeric signals at {delta} 5.07 (I1), {delta} 5.02 (II1), {delta} 4.94 (III1), {delta} 4.85 (IV1), and {delta} 4.71 (V1), in a ratio 1.4/1.5/1.8/1.5/1 (Fig. 2A). As revealed by the 1H-13C HMQC spectrum, their corresponding anomeric carbons resonate at {delta} 99.5 (I1), {delta} 107.1 (II1), {delta} 103.1 (III1), {delta} 109.3 (IV1), and {delta} 100.7 (V1), respectively (Fig. 2D). Proton and carbon resonances of the different spin systems were assigned from 1H-13C HMQC and 1H-1H HOHAHA experiments (partially shown in Figs. 2C, 2D, and Fig. 3, respectively) and were based on our previous studies with mycobacterial LAMs (17, 39) and LAM-related structures (25, 40, 41). The assignments are summarized in Table I. Spin systems I, II, III, IV, and V were unambiguously assigned to 2-{alpha}-Manp, 2,5-{alpha}-Araf, t-{alpha}-Manp, 5-{alpha}-Araf, and 6-{alpha}-Manp, respectively, according to the following lines of evidence.



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  		FIG. 2.
1D 1H (A), and 2D 1H-13C HMBC (B) and HMQC (C, D) NMR spectra of TpaLAM in Me SO-d6 at 343K. Expanded regions ({delta} 1H: 24.62–5.15) (A), ({delta} 1H: 4.62–5.15, {delta} 13C: 60–90) (B), ({delta}1H: 3.30–4.10, {delta} 13C: 60–90) (C), and ({delta} 1H: 4.62–5.15, {delta} 13C: 97–112) (D) are shown. Glycosyl residues are labeled in roman numerals, and their carbons and protons are in Arabic numbers. I, 2-{alpha}-Manp; II, 2,5-{alpha}-Araf; III, t-{alpha}-Manp; IV, 5-{alpha}-Araf; and V, 6-{alpha}-Manp.

 



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  		FIG. 3.
2D 1H-1H HOHAHA {tau} 112 ms spectrum of TpaLAM in Me2SO-d6 m at 343 K. Expanded region ({delta} 1H: 2 4.62–5.18, {delta} 1H: 3.35–4.10) is shown. Glycosyl residues are labeled in roman numerals, and their carbons and protons are in Arabic numbers. I, 2-{alpha}-Manp; II, 2,5-{alpha}-Araf; III, t-{alpha}-Manp; IV, 5-{alpha}-Araf; and V, 6-{alpha}-Manp.

 


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  		TABLE I
Proton and carbon chemical shifts of TpaLAM

Chemical shifts were measured at 343 K in Me2SO-d6 and are referenced relative to internal Me2SO signals at {delta}H 2.52 and {delta}C 40.98.

 
The {alpha}-anomeric configuration of 2,5-{alpha}-Araf and 5-{alpha}-Araf (II and IV) was based on their C-1 chemical shift at {delta} 107.1 and {delta} 109.3, respectively, compared with {alpha}/{beta}-Araf units in mycobacterial LAM ({alpha}-Araf: {delta}C-1 108–110; {beta}-Araf: {delta}C-1 103) (17, 18). Moreover, this was in agreement with the magnitude of the 3JH1,H2-coupling constant determined as 1.9 Hz for spin system IV ({alpha}-Araf: 3JH1,H2 1.7 Hz; {beta}-Araf: 3JH1,H2 4.6 Hz (42)) (Table I). The C-4 carbon chemical shift at {delta} 82.9 (II4) and {delta} 83.0 (IV4), respectively (Table I and Fig. 2C) confirmed a furanose ring form ({alpha}-Araf: {delta}C-4 84.9; {alpha}-Arap: {delta}C-4 69.4 (43)). Glycosylation at position 5 was shown through the deshielding of the C-5 resonances at {delta} 68.1 (II5 and IV5) (Table I and Fig. 2C) as compared with the unsubstituted t-{alpha}-Araf units present in LAM from Rhodococcus equi (ReqLAM), with C-5 resonances at {delta} 64.2 ({Delta}{delta} 3.9 ppm) (25). Glycosylation at position 2 of 2,5-{alpha}-Araf (II2) was determined by the deshielding of its C-2 resonance at {delta} 88.1 (Table I and Fig. 2C) as compared with the C-2 resonance of 5-{alpha}-Araf (IV2) at {delta} 82.9 ({Delta}{delta} 5.2 ppm). Glycosylation at O-2 was in agreement with the shielding observed for the C-1 of 2,5-{alpha}-Araf (II) at {delta} 107.1 compared with C-1 to 5-{alpha}-Araf (IV) at {delta} 109.3, as previously reported (17). The {alpha}-anomeric configuration of Manp units was determined through the magnitude of their 1JH1,C1 coupling constants determined as 172, 172, and 170 Hz for spin systems I, III, and V, respectively ({alpha}-O-Me-Manp: 1JH1,C1 170 Hz; {beta}-O-Me-Manp: 1JH1,C1 161 Hz (44)) (Table I). Glycosylation at position 2 of 2-{alpha}-Manp (I) was determined by the deshielding of its C-2 resonance at {delta} 78.5 (I2) (Table I and Fig. 2C) as compared with the C-2 resonance of t-{alpha}-Manp in M. tuberculosis LAM at {delta} 70.8 ({Delta}{delta} 7.7 ppm) (39). Spin systems III and V showed proton and carbon chemical shifts (Table I) typical of those of t-{alpha}-Manp and 6-{alpha}-Manp units found in mycobacterial LAM (39). Moreover, the pyranose ring of systems I and III was confirmed by the cross-peaks observed on the 1H-13C HMBC experiment (Fig. 2B) between their H-1 at {delta} 5.07 and {delta} 4.94, respectively, and their respective C-5 at {delta} 75.1 and {delta} 75.0, respectively.

Arabinan Domain and Mannooligosaccharide Caps—The sequence of the different units was investigated by 1H-13C HMBC NMR experimentation (Fig. 2B). H-1 of 2-{alpha}-Manp (I1) at {delta} 5.07 showed intracyclic connectivities with their C-2 at {delta} 78.5, C-3 at {delta} 71.6, and C-5 at {delta} 75.1. An additional intercyclic connectivity with C-2 of 2,5-{alpha}-Araf (II2) at {delta} 88.1 indicated that 2-{alpha}-Manp were linked at O-2 of 2,5-{alpha}-Araf. H-1 of t-{alpha}-Manp (III1) at {delta} 4.94 showed intracyclic connectivities with their C-2 at {delta} 71.3, C-3 at {delta} 72.2, and C-5 at {delta} 75.0. An additional intercyclic connectivity with C-2 of 2-{alpha}-Manp (I2) at {delta} 78.5 indicated that t-{alpha}-Manp were linked at O-2 of 2-{alpha}-Manp. H-1 of 2,5-{alpha}-Araf (II1) showed connectivities with their own C-3, C-4, and C-5 at {delta} 77.4, 82.9, and 68.1, respectively. Because it was confirmed that the 2,5-{alpha}-Ara units were in the furanose form, the cross-peak between the H-1 and C-5 resonances could only be assigned to an intercyclic connectivity, establishing that 2,5-{alpha}-Araf are interconnected by ({alpha}1->5) linkages. Nevertheless, as C-5 resonances of 2,5-{alpha}-Araf and 5-{alpha}-Araf units cannot be distinguished (Fig. 2C and Table I), one cannot exclude the possibility of an interconnection of 2,5-{alpha}-Araf and 5-{alpha}-Araf units by an ({alpha}1->5) linkage. No correlation was observed between H-1 of t-{alpha}-Manp (III1) and C-2 of 2,5-{alpha}-Araf (II2) on the HMBC spectrum (Fig. 2B), indicating that single mannose units do not substitute the arabinan chain. Altogether the data indicated that 2,5-{alpha}-Araf units were substituted at O-2 by the Manp-({alpha}1->[2)-Manp-({alpha}1]n-> oligomannoside motif. Indeed, the correlation observed between H-1 of 2-{alpha}-Manp with their own C-2 could be due to intracyclic connectivity, intercyclic connectivity, or both. Consequently, an interconnection of 2-{alpha}-Manp by an ({alpha}1->2) linkage cannot be excluded from NMR data. Nevertheless, peak integration of the 1D 1H spectrum showed that t-{alpha}-Manp,2-{alpha}-Manp, and 2,5-{alpha}-Araf are present in similar amounts, suggesting that the dimannoside, Manp-({alpha}1->2)-Manp-({alpha}1->, is the most abundant motif.

To investigate the degree of mannosylation present in the side chain caps, TpaLAM was partially hydrolyzed using mild acid conditions (0.1 M HCl for 20 min at 110 °C), APTS-derivatized, and subjected to CE-LIF analysis (45). The electropherogram (Fig. 4) exhibited, beside the peaks assigned to Ara-APTS (I), Man-APTS (II), and mannoheptose-APTS (III, internal standard), two main peaks (IV and V) that were likely to correspond to tri- and tetrasaccharide-APTS compounds, based on their retention times. But, compounds IV and V did not co-inject with the corresponding structural elements obtained by mild acid hydrolysis of M. bovis BCG or M. tuberculosis ManLAM, i.e. Manp-({alpha}1->2)-Manp-({alpha}1->5)-Ara-APTS and Manp-({alpha}1->2)-Manp-({alpha}1->2)-Manp-({alpha}1->5)-Ara-APTS (data not shown). Because CE-LIF is a high resolution technique that allows the separation of oligosaccharide fragments differing only by their glycosidic linkage (45), this finding indicated that the glycosidic links present in the TpaLAM side chains are different to those in mycobacterial LAM. Indeed, this observation was in agreement with the fact that the mannose side chains present in TpaLAM are linked through the O-2 position of the arabinan chain and not at O-5 as in mycobacterial LAM. Consequently, the structure of compounds IV and V was investigated by CE/ESI-MS. The negative ESI-mass spectra were dominated by peaks assigned to mono- and double-charged deprotonated molecular ions, [M-H]– and [M-2H]2–, respectively. Peak IV was attributed to Man-Man-Ara-APTS due to the ions (M-H)– at m/z 914 and (M-2H)2– at m/z 456 (Fig. 4C) and peak V to Man-Man-Ara-Ara-APTS due to the ions (M-H)– at m/z 1045 and (M-2H)2– at m/z 522 (Fig. 4D). Furthermore, MS analysis also permitted the attribution of peak VI to Man-Man-Man-Ara-APTS due to the ions (M-H)– at m/z 1075 and (M-2H)2– at m/z 537 (Fig. 4E), with this data indicating that the dimannoside units were the main motif substituting the arabinan chain. To further confirm these attributions, TpaLAM was submitted to a prolonged mild acid hydrolysis to tentatively obtain dimannoside motifs with a single Ara unit at the reducing end (45). As expected, after 30 min of hydrolysis, the resulting electropherogram in the oligosaccharide region was dominated by a single peak (IV), attributed to Man-Man-Ara-APTS, definitively demonstrating that when the TpaLAM arabinan chain is substituted at the O-2 position it is predominately (90%) by the ({alpha}1->2)-dimannoside units.



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  		FIG. 4.
Partial electropherograms of oligosaccharide derivatives obtained after mild acid hydrolysis of TpaLAM followed by APTS derivatization (A and B) and ESI negative mass spectra of compounds IV (C), V(D), and VI (E). A and B, 1 µg of TpaLAM and 0.1 nmol of mannoheptose were hydrolyzed with 15 µl of HCl 0.1 M for 20 min (A) or 30 min (B) at 110 °C, dried, APTS-derivatized, and subjected to CE-LIF. CE analysis was carried out with a 470-mm x 50-µm capillary, at a temperature of 25 °C, with an applied voltage of 20 kV and monitored by LIF. Acetic acid 1% (w/v), triethylamine 30 mM in water, pH 3.5, was used as running electrolyte. I, Ara-APTS; II, Man-APTS; III, internal standard, mannoheptose-APTS; IV, Manp-{alpha}(1->2)-Manp-{alpha}(1->2)-Ara-APTS; V, Manp-{alpha}(1->2)-Manp-{alpha}(1->2)-Araf-{alpha}(1->5)-Ara-APTS; VI, Manp-{alpha}(1->2)-Manp-{alpha}(1->2)-Manp-{alpha}(1->2)-Ara-APTS. M1->2M1->5A, Manp-{alpha}(1->2)-Manp-{alpha}(1->5)-Ara-APTS standard, corresponding to the dimannoside motif found in mycobacterial ManLAM. C–E, CE conditions were the same as in A with a 75-µm x 80-cm fused-silica capillary, with the outlet end of the capillary at a spray voltage of 4 kV. The sheath liquid, consisted of water:isopropanol (20:80, v/v) and was infused to the ESI needle at a flow rate of 3 µl·min–1 using nitrogen as nebulizing gas.

 
The relative abundance of 2,5-{alpha}-Araf, 2-{alpha}-Manp, and t-{alpha}-Manp units, determined by integration of the anomeric proton signals, was in the ratio 1.5/1.4/1.8. Indicating that most, if not all the 2,5-{alpha}-Araf units were linked to the dimannoside units, and consequently were unlikely to be substituted by lateral ({alpha}1->5)-Araf chains. Moreover, methylation analysis showed only trace amounts of t-Ara units (t-Ara/2,5-Ara ratio of 1.5/100), confirming that the occurrence of lateral Araf chains, which would be terminated by t-Ara units, is statistically very weak. Taken together, one may conclude that the arabinan domain is likely to be arranged as a linear ({alpha}1->5)-Araf chain, with half of the units being substituted at O-2 by the oligomannoside motifs. Indeed, integration of the anomeric proton signals provided a ratio 5-{alpha}-Araf/2,5-{alpha}-Araf of 1/1, indicating a branching degree of 50% for the arabinan chain.

Mannan Domain—Per-O-methylation analysis indicated the presence of 6-Manp units, but almost no 2,6-Manp units were detected in TpaLAM, indicating that the TpaLAM mannan domain was likely to consist of a linear ({alpha}1->6)-Manp polymer. This assumption is contrary to all other LAM-like molecules analyzed so far, in that these mannan domains are composed from branched chains. To confirm this assumption, TpaLAM lipomannan core was obtained after mild acid hydrolysis and dialysis to separate the released mono- and oligosaccharides (those previously analyzed by CE in Fig. 4) from the nonhydrolyzed lipomannan core (41). CE-LIF (Fig. 5A) and GC (not shown) analyses of the totally hydrolyzed recovered lipomannan core fraction showed that it contained essentially mannosyl residues, as compared with the native TpaLAM molecule. In addition, the anomeric zone of the 1D 1H NMR spectrum of TpaLAM lipomannan core (Fig. 5B) was dominated by a single resonance at {delta} 4.71 attributed to the H-1 of the 6-{alpha}-Manp units. Moreover, GC analyses (not shown) demonstrated that the TpaLAM lipomannan core fraction contained inositol and fatty acids as opposed to the mono- and oligosaccharide fraction. Taken together, these data demonstrate that, in contrast to mycobacterial LAM and other LAM-like molecules, the mannan domain in TpaLAM is a linear non-branching chain composed entirely from 6-{alpha}-Manp residues; however, similarly to mycobacterial LAM and other LAM-like molecules, the mannan domain is linked to the MPI anchor.



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  		FIG. 5.
CE-LIF analysis (A) and 1D 1H NMR spectrum in Me2SO-d6 at 343K (B) of TpaLAM lipomannan core. A, native TpaLAM (upper electropherogram) or TpaLAM lipomannan core (lower electropherogram) in the presence of mannoheptose as internal standard were hydrolyzed with 2 M trifluoroacetic acid at 110 °C for 2 h, APTS-derivatized, and submitted to CE-LIF migration (same conditions as in Fig. 4A). I, Ara-APTS; II, Man-APTS; III, internal standard, mannoheptose-APTS. B, expanded regions ({delta}1H: 4.62–5.15) of 1D 1H NMR spectra of native TpaLAM (upper spectrum) or TpaLAM lipomannan core (lower spectrum) are shown. I, 2-{alpha}-Manp; II, 2,5-{alpha}-Araf; III, t-{alpha}-Manp; IV, 5-{alpha}-Araf; and V, 6-{alpha}-Manp.

 
MPI Anchor—Per-O-methylation analysis showed the presence of a product that afforded in CI mode (M+H)+ and (M+NH4)+ ions at m/z 321 and 338, respectively, characteristic of a di-acetylated, tetra-methylated inositol (46). EI/MS fragments at m/z 200, 191, and 75 allowed us to positively identify this compound as 2,6-diacetyl-1,3,4,5-tetramethylinositol moiety (20, 46). These results strongly suggest that TpaLAM possesses an MPI anchor characterized by a diglycosylated myo-inositol unit substituted at positions 2 and 6, as established for mycobacterial LAM (14, 47, 48). This was further supported by the identification of phosphatidyl-myo-inositol dimannosides (PIM2) species, considered to be LAM precursors, within T. paurometabola lipidic fractions (data not shown). Moreover, 1D 31P NMR analysis (15, 40) of TpaLAM (not shown) indicated the occurrence of MPI anchor acyl-forms bearing essentially diacylglycerol units.

TNF-{alpha} Production by Macrophages
The potency of TpaLAM, in comparison with a mycobacterial ManLAM (from M. bovis BCG), to stimulate the production of TNF-{alpha}, was investigated using murine J774 macrophage and human THP-1 monocyte cell lines. LPS, used as a positive control, induced the production of TNF-{alpha} by J774 macrophage cells (Fig. 6A). This production was completely inhibited by polymyxin B. ManLAM, known to be a poor inducer of proinflammatory cytokines, induced a very weak amount of TNF-{alpha}. In contrast, TpaLAM, when tested at concentrations of 10 and 20 µg/ml, consistently induced a dose-dependant production of TNF-{alpha} (Fig. 6A), which was around eight times higher than that induced by ManLAM, and furthermore, was not inhibited by polymyxin B, indicating that the observed cytokine induction was not due to LPS contamination. In a similar manner TpaLAM was also found to dose-dependently induce TNF-{alpha} by THP-1 cells, whereas ManLAM had no effect at all (Fig. 6B). It has been previously demonstrated that mycobacterial LM and PIM stimulate the production of TNF-{alpha} in a TLR-2-dependant fashion (49, 50). To investigate the TLR dependence of TNF-{alpha}-inducing TpaLAM activity, studies were conducted by measuring the inhibitory effect of cytokine production using specific anti-TLR-2 and anti-TLR-4 antibodies. As shown in Fig. 6C, whereas the anti-TLR-4 and an IgG2 isotype control antibodies had no affect on TNF-{alpha} production induced by TpaLAM, the anti-TLR-2 antibody completely inhibited this production. These data clearly underline the role of TLR-2 in mediating the stimulation of TNF-{alpha} production by THP-1 cells in response to TpaLAM.



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  		FIG. 6.
TNF-{alpha} production by murine J774 (A) and human THP-1 (B and C) macrophage cell lines in response to TpaLAM and various stimuli. A, ManLAM, TpaLAM, or mahTpaLAM were tested at 10 (black bars) and 20 (white bars) µg/ml, either in the presence (hatched bars) or not of polymyxin B, with J774 cells. LPS was tested at 0.2 µg/ml and induced more than 10 ng/ml TNF-{alpha}. B, ManLAM, TpaLAM, {alpha}TpaLAM, mahTpaLAM, or dTpaLAM were tested at 10 (black bars) and 20 (white bars) µg/ml with THP-1 cells. Polymyxin B, when previously added, had no effect on the amount of TNF-{alpha} released by these stimuli (not shown). LPS at a concentration of 0.2 µg/ml induced 1380 pg/ml TNF-{alpha}. C, TLR dependence of TpaLAM pro-inflammatory activity. TpaLAM and the various antibodies (anti-TLR-2, anti-TLR-4, and IgG2 isotype control) were added to THP-1 cells at a concentration of 10 µg/ml. LPS at a concentration of 0.2 µg/ml induced 680 pg/ml TNF-{alpha}. LPS was from E. coli 055:B5 and ManLAM from M. bovis BCG. {alpha}TpaLAM, {alpha}-mannosidase-treated TpaLAM; dTpaLAM, deacylated TpaLAM; mahTpaLAM, mild acidichydrolyzed TpaLAM.

 
To gain better insights into the structure/function relationships, TpaLAM was submitted to chemical or enzymatic degradations, and the resulting products were tested for their ability to induce TNF-{alpha} production. Deacylated TpaLAM (dTpaLAM) obtained after an alkaline treatment was unable to induce TNF-{alpha}, indicating that the fatty acids are required for activity (Fig. 6B). A TpaLAM variant devoid of mannose caps ({alpha}TpaLAM) was obtained by {alpha}-mannosidase treatment, with removal of the caps monitored by CE-LIF (not shown). {alpha}TpaLAM exhibited a TNF-{alpha}-inducing activity close to that possessed by the native molecule (Fig. 6B), indicating that the caps are not necessary for TpaLAM activity. Finally, and most interesting was the observation that the TpaLAM lipomannan core (mahTpaLAM), obtained after mild acidic treatment of TpaLAM, exhibited a TNF-{alpha}-inducing activity much stronger than that of native TpaLAM, clearly demonstrating that TpaLAM activity is predominantly mediated by its lipomannan moiety. One may explain the differences between mahTpaLAM and TpaLAM by reasoning that the mannan core in TpaLAM is likely to be partially veiled by the arabinan chain, leading to a reduced activity for native TpaLAM, when compared with the free lipomannan moiety.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To date a number of LAM variants have been isolated and characterized from a number of non-mycobacterial strains (24–30). These variants, although different in overall fine structure, exhibit a macro-structure in common with, but distinctly different from mycobacterial LAM. In the present report we have characterized the LAM from T. paurometabola. To date, TpaLAM exhibits the most similar architectural features when compared with mycobacterial LAM, a fact evidenced not only by their high molecular mass, with MALDI/MS providing an average molecular mass centered around 12.5 kDa but with their almost identical migration on SDS-PAGE when compared with mycobacterial LAM. The observation that TpaLAM is alike to mycobacterial LAM is interesting as Tsukamurella sp. are the most closely related species to mycobacteria (51), which corresponds with the notion that lipoglycan composition may be of some chemotaxonomic use (28).

TpaLAM has a similar carbohydrate composition to mycobacterial LAM, with an Ara:Man ratio being ~1:1.7. Per-O-methylation and detailed NMR studies revealed that TpaLAM was comprised from mainly linear ({alpha}1->6)-mannan and ({alpha}1->5)-Araf domains. The linear polymer of ({alpha}1->6)-Manp units lack any side chains, which is in complete contrast to mycobacterial LAM that exhibits a highly branched mannan domain (15). In addition, the arabinan domain is characterized by the presence of 2,5-{alpha}-Araf units, which were first identified in LAM from Amycolatopsis sulphurea (27). These branched Araf units differ from those found in mycobacterial LAM, where branching is fashioned from 3,5-{alpha}-Araf units. TpaLAM possessed a sophisticated arabinan domain, composed of a single ({alpha}1->5)-Araf chain, as opposed to the arabinan domain found in mycobacterial LAM, which is composed from a linear ({alpha}1->5)-Araf chain to which lateral side chains are attached. The lack of side chains in TpaLAM presumably arises from the fact that T. paurometabola lacks the relevant arabinosyltransferases found in mycobacterial strains. In addition, ~50% of these units were further substituted at the O-2 position by mannooligosaccharide units, characterized by the Manp-({alpha}->2)-Manp-({alpha}1-> dimannoside motif, depicted in Fig. 7.



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  		FIG. 7.
Structural model of TpaLAM. TpaLAM is comprised from a linear ({alpha}1->6)-mannan domain linked to a MPI anchor and a ({alpha}1->5)-Araf domain that is substituted at half of the O-2 positions by the Manp-({alpha}1->2)-Manp-({alpha}1-> dimannoside motif. From the molecular mass of 12.5 kDa and integration of anomeric protons signals, m, n, and p can be estimated at 11, 16, and 16, respectively. The proposed model does not account for the relative distribution of the substituted and non-substituted 5-Araf units.

 
The capping of the arabinan chain by mannooligosaccharide units directly onto the O-2 of the {alpha}-Araf chain is a further dissimilarity to that of mycobacterial LAM, where the mannooligosaccharide caps are located at the non-reducing end of lateral arabinan chains. 1H-13C HMBC NMR experiments revealed that the 2,5-{alpha}-Araf units were substituted at O-2 by the Manp-({alpha}1->2)-Manp-({alpha}1-> dimannoside motif, but the presence of longer {alpha}(1->2) oligosaccharide side chains could not be excluded due to the fact that the correlation observed between H-1 of 2-{alpha}-Manp with their own C-2 could be due to both intra- and intercyclic connectivities. Nevertheless, knowing that t-{alpha}-Manp units were present only in the caps, the similar intensity of the 1H anomeric signals of t-{alpha}-Manp, 2-{alpha}-Manp, and 2,5-{alpha}-Araf suggested the dimannoside as the most abundant motif. This was further confirmed by CE-LIF and CE/ESI-MS analyses on mild acid-hydrolyzed TpaLAM that showed Man-Man-Ara as the main reaction product (90%), with a very small amount of Man-Man-Man-Ara (10%) detectable.

1D 31P NMR analysis of TpaLAM showed that the MPI anchor contained essentially di-acylglycerol acyl forms. Furthermore, we identified 2,6-diacetyl-1,3,4,5-tetramethylinositol during per-O-methylation analysis, indicative of a MPI anchor characterized by a diglycosylated myo-inositol unit substituted at positions O-2 and O-6 (20, 46).

In summary, TpaLAM exhibits an original structure with the same core domains as described for mycobacterial LAM, but with some subtle differences, which explains the differences found in the relative molecular masses of TpaLAM and ManLAM. Indeed, lacking in TpaLAM, are first, the existence of several lateral arabinan chains found substituting the arabinan backbone, and second, the branching units of the mannan core. Nevertheless, at this time, we appear to have the most elaborated non-mycobacterial LAM molecule identified to date. The structural model proposed for TpaLAM is detailed in Fig. 7.

A current paradigm in mycobacteriology is that LAM molecules have immunomodulatory roles, conceivably during the pathogenesis of tuberculosis (14, 15, 47). More explicitly, Man-LAMs have the ability to inhibit the production of inflammatory cytokines such as TNF-{alpha} and interleukin-12 (21, 22), whereas PILAMs are able to stimulate such cytokines, via a TLR-2-dependent signaling cascade in macrophages (18, 23, 52). To date, their opposite activity has been attributed to the presence of either mannose or phosphoinositol caps. In the present study we found that native TpaLAM was able to induce the production of TNF-{alpha} in murine or human macrophage cell lines. This activity was unaffected after removal of the mannooligosaccharide units caps by an {alpha}-mannosidase treatment; however, it dramatically increased after a mild acidic treatment that yielded the lipomannan core. These results clearly demonstrate that the observed biological activity of TpaLAM is predominately initiated by its lipomannan core moiety. One might tentatively explain this observation by assuming that in the native molecule (TpaLAM) the mannan core is partly obstructed by the arabinan chain. In turn, this may decrease the frequency of receptor-ligand interactions, which may explain the above observations. Thus, the pro-inflammatory activity of TpaLAM can be paralleled to that of mycobacterial LM (50, 53) as well as that of ReqLAM (25), which resembled an LM-like molecule, except for half of the terminal mannose side chains being further substituted by Araf residues, with both being potent inducers of TNF-{alpha}. Altogether these data indicate that lipomannan-like and lipomannan-domain-containing molecules possess conserved structural motifs that are able to induce pro-inflammatory activity, however, for optimal induction, this structural moiety must be readily accessible to the host receptor(s). Indeed, ManLAM does not show any proinflammatory activity, despite the presence of a lipomannan-like motif in its structure. However, it is now clearly established that in mycobacterial LAM, the mannan core is hidden by the arabinan domain (54, 55). Moreover, it has been recently shown that chemical degradation of the ManLAM arabinan domain, yielding a lipomannan-like structure, restores the cytokine-inducing activity (52). Interestingly, in the present study we demonstrate that an ({alpha}1->6)-Manp chain is sufficient for providing pro-inflammatory activity and that the branched t-Manp units are not necessarily required.

Finally, in 1987 Ikeda-Fujita et al. (56) described the existence of a "novel amphipathic immunostimulator" found in the phenol-water extract of G. aurantiaca, i.e. T. paurometabola, that was composed of mannose, arabinose, and fatty acids consisting primarily of palmitic, stearic, and tuberculostearic acids, with a relative abundance in complete agreement with our data. This study was actually the first report of a LAM-like molecule possessing associated immunomodulatory activities. Indeed, this compound, which is clearly TpaLAM, was found to have the ability to stimulate the splenocytes of C3H/HeJ mice (LPS-Non responder). C3H/HeJ mice are now known to be Tlr4– (57). This is in agreement with our finding that TpaLAM induces TNF-{alpha} production in a TLR-2-dependent fashion, as previously reported for the mycobacterial manno-conjugates, PIMs (49) and LMs (50).


   FOOTNOTES
 
* This work was supported in part by grants from the CNRS (France) (to G. P.). 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. Back

A Lister Institute-Jenner Research Fellow and supported by grants from the Medical Research Council (UK) (G9901077 and G9901078) and the Wellcome Trust (058972). To whom correspondence should be addressed. Tel.: 0121-415-8125; Fax: 0121-414-5925; E-mail: g.besra{at}bham.ac.uk.

1 The abbreviations used are: PIM, phosphatidyl-myo-inositol mannoside; Araf, arabinofuranose; APTS, 1-aminopyrene-3,6,8-trisulfonate; AsuLAM, amycolatopsis sulphurea lipoarabinomannan; {alpha}TpaLAM, {alpha}-mannosidase-treated TpaLAM; CE-LIF, capillary electrophoresis-laser-induced fluorescence; CE/ESI, capillary electrophoresis/electrospray ionization; dTpaLAM, deacylated TpaLAM; GC, gas chromatography; HMBC, heteronuclear multiple bound correlation spectroscopy; HMQC, heteronuclear multiple quantum correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; IL, interleukin; LAM, lipoarabinomannan; LM, lipomannan; mahTpaLAM, mild acidic-hydrolyzed TpaLAM; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; Manp, mannopyranose; ManLAM, LAM with mannosyl caps; MPI, mannosylphosphatidyl-myo-inositol; MS, mass spectrometry; PILAM, LAM with phosphoinositide caps; ReqLAM, rhodococcus equi lipoarabinomannan; t, terminal; TLR, toll-like receptor; TNF-{alpha}, tumor necrosis factor {alpha}; TpaLAM, Tsukamurella paurometabola lipoarabinomannan. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Bénédicte Sichi and Jean-Dominique Bounéry (Institut de Pharmacologie et de Biologie Structurale, Toulouse) for expert technical assistance with TNF-{alpha} and GC/MS experiments, respectively. We thank Beckman (France) and ThermoFinnigan (France) for their supply of the CE/ESI-MS system.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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