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Originally published In Press as doi:10.1074/jbc.M306554200 on October 8, 2003

J. Biol. Chem., Vol. 278, Issue 51, 51291-51300, December 19, 2003
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Surface-exposed Glycopeptidolipids of Mycobacterium smegmatis Specifically Inhibit the Phagocytosis of Mycobacteria by Human Macrophages

IDENTIFICATION OF A NOVEL FAMILY OF GLYCOPEPTIDOLIPIDS*

Christelle Villeneuve, Gilles Etienne, Valérie Abadie, Henri Montrozier, Christine Bordier, Françoise Laval, Mamadou Daffe, Isabelle Maridonneau-Parini, and Catherine Astarie-Dequeker{ddagger}

From the Département "Mécanismes Moléculaires des Infections Mycobactériennes," Institut de Pharmacologie et de Biologie Structurale, Unité Mixte de Recherche 5089, Centre National de la Recherche Scientifique et Université Paul Sabatier, 31077 Toulouse, France

Received for publication, June 20, 2003 , and in revised form, September 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytosis by macrophages represents the early step of the mycobacterial infection. It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria. The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M. smegmatis and the opportunistic pathogen M. kansasii by human macrophages derived from monocytes. The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment. Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs). Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-rhamnosyl unit linked to the alaninol end of the molecules. These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M. smegmatis and M. avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization. At micromolar concentrations, the native GPLs also inhibit the uptake of both M. tuberculosis and M. kansasii. De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents. Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycobacterium spp. are responsible for several pathologies. For instance, Mycobacterium tuberculosis, the causative agent of tuberculosis and numerous other opportunistic mycobacterial pathogens, can cause respiratory infections (1, 2). Bacilli enter the organism by inhalation. Once in the lung, they infect alveolar macrophages, where they survive and multiply (3). After host cell lysis, bacilli released in the extracellular environment infect other macrophages. This cyclic infection is usually under the control of the immune system, which confines bacilli in multicellular structures, called granulomas, where mycobacteria can stay in latency for years. However, reactivation of the infectious cycle may occur under some immunocompromised situations, leading to an active pathology (4). Therefore, elucidation of the mechanisms involved in the interaction between macrophages and mycobacteria could help to develop new pharmacological strategies to prevent macrophage infection.

Pathogenic mycobacteria have the ability to persist in macrophages (5) that are usually meant to kill microorganisms. This successful parasitism involves mycobacterial strategies to protect themselves from potent host antimicrobial processes, such as restriction of lysosomal fusion with their phagosomes (6-8). The early process of recognition and internalization of mycobacteria in macrophages is essential in the outcome of infection. In human macrophages derived from monocytes (MDMs),1 we have demonstrated that the early step of infection by opportunistic mycobacteria did not evoke microbicidal responses, inducing neither a production of superoxide anions nor a fusion of phagosomes with a subpopulation of lysosomes (9). Interestingly, phagocytosis of nonpathogenic mycobacteria, such as M. smegmatis, did not elicit bactericidal responses (9). Finally, at the early step of infection, mycobacteria do not actively control their host cell because phagosomes containing live or heat-killed bacteria are indistinguishably refractory to fusion with lysosomes (10). In sharp contrast, when mycobacteria were serum opsonized, their phagocytosis was associated with an oxidative response (9) and a maturation of phagosomes toward a fusion with lysosomes (6, 11, 12). This led us to propose that, during the initial steps of the infection, mycobacteria have developed a common strategy, which consists of the use of receptors of non-opsonic phagocytosis uncoupled to bactericidal responses. In agreement with this proposal, we recently demonstrated that the mannose receptor, which efficiently participates in binding and internalization of pathogenic and nonpathogenic mycobacteria, was not coupled to bactericidal functions in human macrophages (9). Similarly, binding and internalization of the opportunistic pathogen M. kansasii through the complement receptor type 3 did not activate the NADPH-oxidase in macrophage cell lines (13).

The mycobacterial envelope is composed of a plasma membrane surrounded by a complex cell wall, which in turn is recovered by a superficial layer composed of proteins, carbohydrates and, to a lesser extent, lipids (14-16). This outermost structure, also called a capsule in the case of pathogenic species, represents a privileged interface between bacilli and their host cells. Some of its components have been implicated in the interaction with macrophages (17). The major carbohydrate constituent of the capsule, a glycogen-like glucan (14), has been shown to inhibit the binding of M. tuberculosis to complement receptor type 3-expressing Chinese hamster ovary cells (18). The purified mannose-capped lipoarabinomannan (Man-LAM), a cell envelope-associated glycoconjugate, interacts with the mannose receptor of macrophages (19). Other components of the mycobacterial cell envelope, e.g. sulfatides, phenolic glycolipids, glycopeptidolipids (GPLs), phosphatidylinositol mannosides (PIMs) induce various host cell responses (for a comprehensive review, see Ref. 20). Some of these biologically active molecules have been precisely localized in the outermost layer of the cell envelope (15, 21), but the precise location of others is still a matter of debate.

We extracted mycobacterial surface-exposed compounds by gentle surface abrasion with glass beads (15) and tested their role in the interactions between mycobacteria and MDMs to identify the most active bacterial partners of the phagocytic process. Because we have demonstrated that pathogenic and nonpathogenic strains use the same phagocytic receptors to infect cells (9, 10, 22, 23), this work was performed with the nonpathogenic species, M. smegmatis. Using this approach, we demonstrated that surface-exposed C-type GPLs inhibited the non-opsonic phagocytosis of GPL-containing mycobacteria and to a lower extent those of other mycobacteria.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Growth Conditions—M. smegmatis ATCC 607, M. tuberculosis H37Rv ATCC 27294, and M. kansasii ATCC 124478 were grown as surface pellicles on liquid Sauton's medium at 37 °C without agitation; M. avium (serovar 4) was cultured under shaking at 250 rpm in Middlebroock 7H9 medium supplemented with 10% albumin-dextrose-catalase enrichment (Difco). The growth curves were determined as described previously (24).

Single-cell suspensions were prepared with late log-phase cultures (6 days for M. smegmatis, 3 weeks for M. kansasii, M. tuberculosis, and M. avium), as described previously (23). When specified, M. smegmatis were isolated from early log phase culture (day 3). Briefly, pellicles (M. smegmatis and M. kansasii) were harvested by pouring off the medium, dispersed by gentle shaking for 30 s with 5 g of glass beads (4-mm diameter) and resuspended in PBS, pH 7.4. For M. tuberculosis, this procedure was repeated to further disaggregate the pellicles. In the case of M. avium, the culture was recovered by centrifugation at 10,000 x g for 10 min. To remove the remaining clumps, the bacterial suspensions were allowed to sediment for 10 min. The supernatants were collected and then centrifuged for 10 min at 200 x g. Up to 90% of mycobacteria were individualized, the remaining formed small aggregates containing two or three bacilli. The viability of mycobacterial cells was assessed by i) quantifying the level of isocitrate dehydrogenase, an indicator of autolysis, in the culture filtrates (24), ii) labeling cells with both propidium iodide and fluorescein diacetate (10), and iii) serial dilutions and plating on Middlebroock 7H10 medium. The percentage of viable mycobacteria averaged 85%. When specified, mycobacteria were labeled with FITC, as described previously (23).

Preparation of Surface-exposed Material—Surface pellicles of mycobacteria grown on Sauton's medium were treated with 10 g of glass beads (4-mm diameter) for 1 min and resuspended in distilled water (50 ml/flask). Bacilli were removed by filtration through a 0.2-µm-pore-size sterile filter (Nalgene). The crude filtrate, which contained the surface-exposed material, was then extensively dialyzed against distilled water with cellulose membrane (Mr 1000) (Spectra/Por 7, Spectrum). When specified, dialysis was performed using a Mr 100 or Mr 6000-8000 molecular weight cut-off membrane. The protein content of the sterile surface-exposed material was determined using the Bradford method (Bio-Rad). Surface extracts were treated at 37 °C with either 0.1 M NaOH for 4 h or 0.1 mg of Pronase or proteinase K per mg of protein overnight in the presence of chloramphenicol and gentamicin (100 µg/ml). Proteases were inactivated at 100 °C for 15 min. For both treatments, surface-exposed material was then dialyzed, and the pH was restored to 7.

Portions of the surface-exposed material were extracted with chloroform and methanol according to the Bligh and Dyer procedure (25). After drying, the organic phases were washed. The aqueous phases were re-extracted with chloroform, and the interphase was rinsed with a mixture of CHCl3:H2O (1:1 v/v). The three phases were then dried under vacuum, weighed, and resuspended in a volume of sterile water equal to the initial volume of the crude surface extract. The lipid components of the surface-exposed material were identified by TLC as described previously (26) and further fractionated as described below.

Extraction and Purification of Mycobacterial Lipids—Lipids were purified both from whole cells and surface-exposed material. In the former case, wet cells were extracted first with CHCl3:CH3OH (1:2 v/v) and then with CHCl3:CH3OH (1:1 v/v) at least three times. Surface-exposed material and pooled whole-cell lipid extracts were separately dried under vacuum and partitioned between water and chloroform (1:1 v/v). The organic phases were extensively washed with distilled water and evaporated to dryness. Portions of the chloroformic phases were used directly for biological assays. The remaining portions were dried, and lipids were resuspended in a minimal volume of chloroform and precipitated by trickling methanol. After standing for 2 h at 4 °C, methanol soluble lipids were recovered by centrifugation at 4 °C for 20 min (8000 x g). These lipids were then chromatographed on a Florisil (60-100 mesh) column (1.5 x 25-cm) irrigated with chloroform and then with a stepwise gradient of increasing concentrations of methanol and water in chloroform. GPLs and phospholipids, which were coeluted in polar fractions with a mixture of CHCl3:CH3OH:H2O (65:25:4 v/v/v), were further separated using an anion-exchange QMA-silica gel (Chromabond SB, Macherey-Nagel). GPLs were eluted with CHCl3:CH3OH:H2O (65:25:4 v/v/v), whereas phospholipids were eluted using 0.1 and 0.2 M ammonium acetate in CHCl3:CH3OH (1:2, v/v) and 0.2 M ammonium acetate in methanol. All of the purification steps were monitored by TLC on silica-Gel 60-precoated plates (0.25-mm thickness; Merck) developed with CHCl3:CH3OH (90:10 v/v). Sugar-containing compounds were visualized by spraying plates with 0.2% anthrone in concentrated sulfuric acid, followed by heating, whereas the Dittmer-Lester reagent was used to detect phosphorus-containing substances (27).

Analytical Procedures—Three different chemical degradation methods were applied to the purified GPLs (Fig. 1) (28), and the resulting products were analyzed by matrix-assisted laser-desorption/ionization-time of flight (MALDI-TOF) mass spectrometry: i) de-O-acylated and both de-O-acylated and {beta}-eliminated GPLs were obtained from treatment of native GPLs with 0.5 M sodium methanolate for 2 h at 37 °C; after neutralization with glacial acetic acid, the aqueous phase was extracted with chloroform, ii) perdeuteriomethylation of GPLs was carried out according to the method described by Blakeney and Stone (29) with trideuteriomethyl iodide (ICD3) as methylating agent, iii) the N-acyl-phenylalanyl moiety of GPLs was produced as methyl ester from native or perdeuteriomethylated GPLs by methanolysis with anhydrous 1.5 M CH3OH:HCl for 16 h at 80 °C; a portion of the native GPLs was further acetylated in 1:1 Ac2O/pyridine (100 °C, 1h) or its double bound was cleaved by a permanganate-periodate oxidation (30), iv) the partially methylated alditol acetates were obtained from perdeuteriomethylated GPLs after hydrolysis with 2 M trifluoroacetic acid (100 °C, 2 h), reduction with NaBH4, and acetylation with 1:1 Ac2O/pyridine (100 °C, 1 h).



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  		FIG. 1.
Schema of the chemical degradation procedures used to establish the structure of the GPL-like compounds. All the products shown in brackets have been analyzed by MALI-TOF mass spectrometry or GC/MS. R, CH3-(CH2)n-CH = CH-(CH2)m (m + n = 24 to 28); DHAB, dehydroaminobutyryl.

 
Characterization of the Succinyl Group—Five mg of GPLs III were treated by 0.5 ml of 0.5 M sodium methanolate for 2 h at 37 °C. After neutralization with glacial acetic acid, the mixture was dried under a stream of N2, dissolved in water, and desalted by filtration over a cation-exchange resin (Dowex 50, H+ form) column. After evaporation to dryness in the presence of CH3OH, the residue was methylated by diazomethane (CH2N2) for gas chromatography/mass spectrometry (GC/MS) analysis.

Spectrometric Methods—MALDI-TOF mass spectrometry analysis of lipids was performed as described previously (31). Sample solutions were prepared in chloroform at a concentration of 1 mM and were directly applied onto the sample plate as a 1-µl droplet, followed by the addition of 0.5 µl of the matrix solution (10 mg/ml 2,5-dihydroxybenzoic acid in CHCl3:CH3OH (1:1 v/v). Samples were then allowed to crystallize at room temperature. MALDI-TOF spectra were acquired on a Voyager-DE STR mass spectrometer (PerSeptive Biosystems) equipped with a pulsed nitrogen laser emitting at 337 nm and were analyzed in the Reflectron mode using an extraction delay time set at 100 ns and an accelerating voltage operating in positive ion mode of 20 kV. To improve the signal-to-noise ratio, 150 single shots were averaged for each mass spectrum, and typically, four individual spectra were accumulated to generate a summed spectrum. An external mass spectrum calibration was performed using the calibration mixture 1 of Sequazyme Peptide Mass Standards kit (PerSeptive Biosystems), including known peptide standards in a mass range from Mr 900 to 1600.

Electrospray ionization mass spectrometry and electrospray ionization mass spectrometry/tandem mass spectrometry (MS/MS) analyses were done by using a FinniganMat TSQ 700 triple quadrupole (ThermoFinnigan, San Diego, CA). Lipids were dissolved in 1% acetic acid in methanol. The mass spectra were obtained by direct infusion using a syringe pump (Harvard Apparatus, South Natick, MA) at a flow rate of 3 µl min-1. Full-scan spectra (positive mode, spray potential 4.6 kV) were acquired in the ion peak centroid or profile modes over the mass/charge range of 200-2000 at 3 s. MS/MS experiments were performed by conducting collision-induced dissociation in the radiofrequency-only collision cell of the triple quadrupole at a collision energy of 80 eV. Argon was used as a collision gas in the range of 1.5-2.5 mTorr. At least 15 scans were accumulated and averaged.

GC/MS analyses were performed on a Hewlett-Packard 5890 series II gas chromatograph, fitted with an OV1 fused-silica capillary column (12 m x 0.30 mm) and connected to a Hewlett-Packard 5989X mass spectrometer in electron-impact mode with an ionization potential of 70 eV. The temperature programs used were 100 °C (delay 3 min) to 290 °C at 8 °C/min or isotherm at 40 °C for alditol acetates or short acid methyl esters analysis, respectively.

One- and two-dimensional 1H-NMR spectra were recorded on a Bruker AMX-500 spectrometer using standard pulse sequences available in the Bruker software. The chemical shifts were expressed in parts per million relative to acetone as internal standard ({delta}H 2.22).

De-O-acylation of GPLs—GPLs were de-O-acylated with 0.1 N NaOH in CHCl3:CH3OH (50:50 v/v) for 30 min at 37 °C. After neutralization by glacial acetic acid, the mixture was dried under a stream of N2, then the de-O-acylated GPLs were extracted with chloroform and washed extensively with water. The effectiveness of the de-O-acylation was checked by MALDI-TOF mass spectrometry analysis.

Isolation and Culture of Human MDMs—Human peripheral blood monocytes were isolated as described previously (9) and cultured on sterile glass coverslips in 24-well tissue culture plates (5 x 105 cells/well) containing RPMI 1640 with 10% heat-inactivated fetal calf serum and antibiotics for 6-7 days at 37 °C in 5% CO2. The culture medium was renewed at the third day. Before use, MDMs were washed twice with fresh RPMI 1640 and equilibrated for 20 min at 37 °C in 5% CO2.

Opsonization of Bacteria and Zymosan—FITC-stained mycobacteria or zymosan were incubated with pooled human sera for 25 min at 37 °C, washed twice, and suspended in PBS, pH 7.4 (9, 23).

Infection of Adherent Macrophages and Phagocytosis Assay—When specified, MDMs were pretreated for 15 min at 37 °C with either the crude surface-exposed material, the different phases obtained after phase partition, or the purified lipid fractions resuspended in sterile apyrogenic water and sonicated for 10 min. All dilutions were performed in RPMI 1640. MDMs were then put in contact with bacilli for 45 min and washed twice with fresh medium to remove unbound particles. Phagocytosis of FITC-stained bacteria was determined as described previously (22). Briefly, MDMs were fixed with 3.7% paraformaldehyde in PBS containing 15 mM sucrose, pH 7.4 for 20 min at room temperature. After neutralization with 50 mM NH4Cl, extracellular mycobacteria were labeled with rabbit polyclonal antibodies directed against mycobacteria (Camelia antibody, 1:100) (22), revealed by a TRITC-conjugated secondary antibody. MDMs containing at least one FITC-stained mycobacterium were counted out of 100 cells in duplicate samples. MDMs having engulfed either bovine serum albumin-coated latex beads and zymosan for 1 h (50 particles/cell) or serum-opsonized zymosan for 30 min (50 particles/cell) were permeabilized in methanol for 6 min at -20 °C and washed in PBS containing 0.1% Tween 20. Phagosomes containing particles were stained with a lysosomal membrane marker, CD63, revealed with a fluorescent-conjugated secondary antibody as described previously (9).

Statistics—Data are presented as the means ± S.E. of the indicated number of experiments (n) performed in duplicate. The significance of the differences was determined by the paired or unpaired Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibitory Activity of Surface-exposed Material from M. smegmatis on Mycobacterial Phagocytosis—We first checked that the surface-exposed material of M. smegmatis readily affected the ability of macrophages to internalize bacilli. The crude surface-exposed material was arbitrary dialyzed using a Mr 100 molecular weight cut-off membrane to eliminate small molecules from the culture medium. The amount of surface-exposed material to be used, corresponding to bacilli in contact with MDMs (50 particles/cell), was rationalized by using a final concentration of 150 ng of protein/ml of surface-exposed material. Under these conditions, surface-exposed material isolated from early log-phase-grown M. smegmatis (day 3) decreased by 43 ± 2% (n = 2), the rate of phagocytosis of 3-day-old bacteria. Similarly, surface-exposed material isolated from late log-phase-grown M. smegmatis (day 6) decreased by 41 ± 10% (n = 4) internalization of 6-day-old bacteria. It was thus concluded that surface-exposed material exhibited an inhibitory effect on the phagocytosis of M. smegmatis that was independent from growth conditions.

Chemical Nature of the Inhibitory Compounds from the Surface-exposed Material—We estimated the approximate size of these molecules by dialysis. Although the dialysis of surface-exposed material using a cut-off of Mr 1000 did not affect its inhibitory effect (52 ± 4%, n = 9) (Fig. 2), no inhibition was observed with surface-exposed material dialyzed at a Mr 6000-8000 cut-off (4 ± 1% inhibition, n = 3). These data indicated that the inhibitory molecules possess a low molecular weight (between Mr 1000 and 8000).



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  		FIG. 2.
M. smegmatis surface-exposed material specifically inhibits the non-opsonic phagocytosis of mycobacteria. MDMs were incubated at 37 °C for 15 min with or without M. smegmatis dialyzed (Mr 1000 cut-off) surface-exposed material adjusted to 150 ng of protein/ml and then allowed to ingest particles. Macrophages were challenged for 45 min with FITC-stained mycobacteria (25 particles/cell): non-opsonized M. smegmatis (Msm) and M. kansasii (Mka) or human serum-opsonized M. smegmatis (OS). Cells were then washed and fixed in paraformaldehyde, and the remaining extracellular bacteria were revealed by rabbit anti-mycobacterial serum and TRITC-conjugated anti-rabbit IgG. Macrophages were also challenged with 50 particles/cell of bovine serum albumin-coated latex beads (LB) for 1 h, human serum opsonized zymosan (OZ) for 30 min, or non-opsonized zymosan (Z) for 1 h. Intracellular particles were visualized by staining phagosomal membranes with a lysosomal marker, CD63, revealed with a fluorescent-conjugated secondary antibody. Phagocytosis was determined by fluorescence microscopy as the percentage of macrophages having engulfed at least one particle. The inhibitions are expressed as the percentages of effect when compared with response under control conditions. Data are presented as the mean ± S.E. of 3-11 experiments performed in duplicate. Significance of the surface-exposed material effect was assessed by a paired Student's t test. *, p < 0.05; **, p < 0.01 when compared with untreated conditions.

 
Accordingly, the subsequent work was performed with surface-exposed material dialyzed with a Mr 1000 molecular weight cut-off membrane and isolated from 6-day-old cultures of M. smegmatis to get enough biological material for further fractionation experiments. Under these conditions, surface-exposed material from M. smegmatis inhibited the uptake of both heat-killed M. smegmatis (data not shown) and the opportunistic pathogen M. kansasii (Fig. 2). In sharp contrast, the internalization of mycobacteria opsonized with human serum was not affected (Fig. 2). Furthermore, surface-exposed material did not modify phagocytosis of various inert particles, such as latex beads and opsonized or non-opsonized zymosan (Fig. 2). It was thus concluded that surface-exposed material contained inhibitory molecules that specifically decrease the non-opsonic phagocytosis of mycobacteria but do not affect internalization of other particles.

An overnight incubation of surface-exposed material with nonspecific/broad-spectrum proteases, Pronase and proteinase K, did not influence its inhibitory effect on M. smegmatis internalization (Fig. 3a), suggesting that the observed inhibition was not attributable to proteins. In contrast, when surface-exposed material was treated with alkali, a treatment that hydrolyzes ester bounds, a pronounced decrease of its inhibitory activity was observed (Fig. 3a), implying that a significant portion of inhibitory molecules was alkali-labile. On the basis of the occurrence of ester linkages in most of the mycobacterial lipids, these molecules were isolated by partitioning surface-exposed material between chloroform, methanol, and water. TLC analysis of the organic phase showed the presence of GPLs, 6-monomycoloyltrehalose, PIMs, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol (data not shown) as reported previously (21). The interphase and the aqueous phase contained mostly carbohydrates and proteins. As depicted on Fig. 3b, the organic phase exhibited the highest inhibitory effect on phagocytosis of M. smegmatis. Although some inhibition was observed with the aqueous phase (Fig. 3b), an extensive extraction with organic solvents to eliminate residual lipids abolished this effect (data not shown). The inhibitory effect of the interphase was negligible (Fig. 3b). Altogether, these data indicated that most of the inhibitory activity of surface-exposed material was attributable to its lipid constituents.



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  		FIG. 3.
The inhibitory property of M. smegmatis surface-exposed material is attributable to its lipid content. MDMs were incubated at 37 °C for 15 min with: a, 150 ng of protein/ml of M. smegmatis surface-exposed material (SEM) treated overnight at 37 °C with either 0.1 mg of Pronase (P) or proteinase K (K) per mg of protein or with 100 mM NaOH for 4 h at 37 °C; or b, 150 ng/ml of protein equivalent of the phases obtained after surface-exposed material partition: organic phase (OP), interphase (IP), and aqueous phase (AP). Cells were then challenged for 45 min at 37 °C with M. smegmatis (25 particles/cell), and phagocytosis was determined by fluorescence microscopy. The inhibitions are expressed as the percentages of effect when compared with response under control conditions. Data are presented as the mean ± S.E. of 3 to 4 (a) and 4 to 8 (b) independent experiments performed in duplicate. Significance was assessed by an unpaired Student's t test. *, p < 0.05; **, p < 0.01 when compared with the effects of untreated surface-exposed material.

 
Purification and Identification of the Inhibitory Substances of M. smegmatis—Lipids from the surface-exposed material were fractionated by adsorption chromatography on a Florisil column. They were eluted with chloroform, increasing concentrations of methanol in chloroform, and finally with a mixture of chloroform, methanol, and water. Five lipid fractions were obtained and tested at 100 µg of lipid/ml for their ability to inhibit M. smegmatis phagocytosis. Only two of them exerted an inhibitory activity. The most polar fraction showed the highest inhibitory activity comparable with that of the organic phase (50 ± 6 and 47 ± 1%, n = 3, respectively). TLC analysis (data not shown) demonstrated that this fraction contained glycolipids that shared with GPLs the same "string-of-beads" aspect and green-blue coloration with anthrone, and phospholipids such as PIMs, phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine, suggesting that both surface-exposed GPLs and phospholipids possessed inhibitory activities. The second fraction that inhibited phagocytosis of M. smegmatis by 26 ± 4%, (n = 3) contained GPL-like molecules but not phospholipids (data not shown).

Because GPL-like substances were present in the two inhibitory fractions, we next focused on these compounds. Because GPLs are present in both the outermost layer of the mycobacteria cell envelope and the whole bacteria (26, 32), they were isolated from whole bacterial lipids to obtain more material. The extract was first enriched in GPL-like molecules by methanol precipitation, a procedure known to concentrate phospholipids and mycoloylated glycolipids in the methanol-insoluble fraction. As expected, the extract enriched in GPL-like molecules was able to inhibit the internalization of M. smegmatis (Fig. 4). At concentrations ranging from 1 pg to 100 µg of lipid/ml, GPLs induced a biphasic effect on phagocytosis of M. smegmatis. The inhibitory effect increased from 1 to 100 pg of lipid/ml, with a maximal effect at 100 pg of lipid/ml that remained stable up to 10 ng of lipid/ml and then progressively decreased (Fig. 4). This phenomenon possibly reflects the critical aggregatory concentration of lipid compounds. Therefore, a concentration of 10 ng of lipid/ml of GPLs was used for subsequent experiments. Lipids from the methanol-soluble extract were fractionated on a Florisil column into 21 fractions. As shown in Fig. 5a, inhibitory activities of fractions increased with the polarities of the solvent mixtures used. TLC analysis showed that fractions S8 to S20 contained GPL-like compounds, whereas S21 fraction was composed of both GPL-like compounds (Fig. 5b) and some phospholipids (data not shown).



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  		FIG. 4.
The enriched-GPL-like fraction induces a dose-dependent inhibition of M. smegmatis phagocytosis. MDMs were preincubated for 15 min with the methanol supernatant (CH3OH SN) of extractable lipids at concentrations ranging from 1 pg/ml to 100 µg/ml and put in contact with M. smegmatis (25 particles/cell) for 45 min at 37 °C. The inhibitions are expressed as the percentages of effect when compared with response under control conditions. One experiment representative of three independent experiments is shown.

 



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  		FIG. 5.
GPL-like compounds of M. smegmatis inhibit its phagocytosis. Methanol-soluble lipids extracted from M. smegmatis were separated according to their polarity by adsorption chromatography; elution was carried out with a stepwise gradient of increasing concentrations of methanol and water in chloroform (from 100:0:0 to 65:25:4). a, the 21 fractions (noted S1 through S21) obtained after elution of a Florisil column were preincubated at 10 ng/ml with MDMs for 15 min at 37 °C. Cells were then challenged for 45 min at 37 °C with M. smegmatis (25 particles/cell), and the percentage of phagocytosis was determined. The inhibitions are expressed as the percentages of effect when compared with response under control conditions. Data are presented as the mean ± S.E. of three independent experiments. Significance was assessed by a paired Student's t test. *, p < 0.05; **, p < 0.01 when compared with phagocytosis under untreated conditions. b, representative fractions were analyzed by TLC using CHCl3:CH3OH, 90:10 (v/v) as the solvent system and were revealed with anthrone.

 
To firmly establish the nature of the active compounds, S21 was chromatographed again on an anion-exchange column. The purified GPL-like compounds from S21 decreased by >60% the internalization of M. smegmatis (from 35 ± 7 to 13 ± 6%; n = 3). In contrast, the phospholipid fraction eluted from the ion-exchange column with ammonium acetate in CHCl3:CH3OH reduced the internalization of bacteria by only 30% (from 35 ± 7 to 23 ± 8%, n = 3). This led us to conclude that GPL-like molecules of S21 were the main compounds involved in inhibition of M. smegmatis phagocytosis. We finally checked by TLC that the various GPL-like molecules isolated from the whole lipid extract of M. smegmatis were also present in the surface-exposed material and showed by MALDI-TOF mass spectrometry that they were composed of the same species (Fig. 6).



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  		FIG. 6.
MALDI-TOF mass spectra of unfractionated GPL-like compounds. MALDI-TOF mass spectra of GPL-like-enriched methanol-soluble lipids were prepared from whole cells (a) or surface-exposed material (b) of M. smegmatis.

 
Because GPLs are species-specific (33, 34), the purified GPL-like compounds from S21 of M. smegmatis were tested in competition with M. avium, a species containing GPLs (33), and M. tuberculosis and M. kansasii, two species devoid of GPLs (16). As depicted in Fig. 7, pretreatment of MDMs with 10 ng of lipid/ml significantly decreased the rate of phagocytosis of M. avium (from 29 ± 4 to 20 ± 4%, n = 4; p < 0.01), whereas it did not significantly affect the internalization of M. tuberculosis and M. kansasii (from 55 ± 4 to 45 ± 9% and from 31 ± 9 to 31 ± 8%, respectively; n = 4). However, when lipid concentration was increased from 10 ng/ml to 10 µg/ml, phagocytosis of M. tuberculosis progressively decreased (data not shown). At 10 µg/ml the inhibitory effect was similar to that obtained with 10 ng of lipid/ml on M. smegmatis phagocytosis (Fig. 7). As a control, we checked that 10 µg of lipid/ml from M. smegmatis decreased to the same extent the phagocytosis of M. kansasii (from 27 ± 1 to 16 ± 2%; n = 2), whereas they did not interfere with the internalization of zymosan (Fig. 7). This led us to propose that the GPL-like molecules that composed S21 specifically inhibited the non-opsonic phagocytosis of mycobacteria. Because these compounds exhibited TLC mobilities similar to those of GPL-like substances found in fractions S13-18 (Fig. 5b), we postulated that subtle structural features might exist between the isolated compounds.



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  		FIG. 7.
Polar GPL-like compounds specifically inhibit the non-opsonic phagocytosis of mycobacteria. MDMs were incubated for 15 min at 37 °C with 10 nM () or 10 µM ({square}) purified GPLs from fraction S21. Cells were then challenged for 45 min at 37 °C with M. smegmatis (Msm), M. avium (Mav), M. tuberculosis (Mtu), M. kansasii (Mka), or zymosan (Z) (25 particles/cell), and the percentages of phagocytosis were determined. The inhibitions are expressed as the percentages of effect compared with control values. Data are presented as the mean ± S.E. of four independent experiments, except for effects of 10 µM GPLs on M. kansasii (n = 2). Significance was assessed by a paired Student's t test. **, p < 0.01 when compared with phagocytosis under untreated conditions.

 
Structural Features of GPL-like Substances—The structure of the various constituents of S8-21 was determined by NMR, MALDI-TOF mass spectrometry, and analysis of chemical degradation products according to the procedures shown in Fig. 1. Preliminary MALDI-TOF mass spectrometry analysis has led us to choose fractions S8, S13, and S20 as representative of three types of GPL-like compounds. Their one-dimensional (Fig. 8) and two-dimensional 1H-NMR spectra were very similar to those of the previously characterized C-type GPLs (33) and confirmed the presence of i) phenylalaninyl, threoninyl, alaninyl, and alaninol residues, ii) deoxysugar units, iii) {beta}-hydroxylated long-chain, iv) double bonds, and v) methoxyl groups in these compounds (35). Molecules in fractions S8, S13, and S20 were thereafter called GPLs I, II, and III, respectively (Fig. 9). In addition, signals at 2.62 and 2.70 ppm were seen in the 1H-NMR spectrum of S20 (GPLs III) (Fig. 8b) and absent from those of the other fractions (Fig. 8a). Strong acid methanolysis (CH3OH:HCl, 1.5 M, 16 h at 80 °C) of S8-S20 (Fig. 1), followed by the analysis of the resulting N-acyl-phenylalanyl methyl ester by MALDI-TOF mass spectrometry, demonstrated the occurrence of major pseudomolecular ion [M + Na]+ peaks corresponding to a mixture of hydroxylated C28, C30, and C32, and methoxylated C29, C31, and C33 saturated and unsaturated fatty acyl-phenylalanyl methyl esters. The presence of methoxylated homologs in the mixture was further ascertained by the analysis of the MALDI-TOF mass spectra of both the perdeuteriomethylated and peracetylated products (Fig. 1). Although perdeuteriomethylation of hydroxylated compounds induced a 34 atomic mass units shift of their [M + Na]+ values, those of methoxylated substances were shifted by only 17 atomic mass units (a CD3 on the nitrogen atom of the phenylalanyl residue). Similarly, peracetylation of the mixture of fatty acyl-phenylalaninyl methyl esters (Fig. 1) induced a 42-atomic mass unit shift of hydroxylated compounds, whereas no change was observed for methoxylated homologs. Oxidative cleavage of the double bond occurring in the fatty acyl-phenylalanyl moiety, followed by methanolysis and GC/MS analysis, led to the identification of C15-C17 fatty acid methyl esters. MALDI-TOF mass spectrometry analysis of the native and perdeuterioacetylated phenylalanyl diacid methyl esters resulting from the oxidative cleavage showed that the hydroxyl and methoxyl groups were located on the phenylalanyl diacid moiety of the unsaturated molecules. It followed then that fractions S8-20 contained all the structural features found in C-type GPLs (Fig. 9).



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  		FIG. 8.
Compared 1H-NMR spectra of GPLs I (a) and GPLs III (b). Spectra were obtained in CDCl3 with a GPL concentration of 10 mg/ml.

 



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  		FIG. 9.
Structures of the GPLs found in M. smegmatis. R1, acetyl, except for the GPLs IIa where R1 = H.

 
Structures of the Various GPLs of M. smegmatis—The MALDI-TOF mass spectrum of the native S8 (Fig. 10a) showed a series of major pseudomolecular ion [M + Na]+ peaks corresponding to the previously described apolar non-serovar-specific GPLs (GPLs I) of M. smegmatis (36). The [M + Na]+ species corresponded to substances differing from one another by the degrees of O-methylation of either the rhamnosyl unit or the hydroxyl group of the fatty acyl substituent (36-38). In addition, ion peaks differing by 14 atomic mass units, i.e. one methylene unit, were observed and reflected the variability in the chain length of the fatty acyl substituent that contains C28 to C32 (36) (Fig. 9). The complexity of the mass spectra was further enhanced by the variable occurrence of a double bound in the fatty acid chain (36), resulting in the presence of pseudomolecular [M + Na]+ ions differing from those of saturated chains by 2 atomic mass units. Perdeuteriomethylation of GPLs I followed by acid hydrolysis and analysis of partially methylated, partially acetylated alditol derivatives by GC/MS (Fig. 1) led to the identification of i) 1,5-di-O-acetyl-2,3,4-tri-O-CD3-6-deoxytalitol, ii) 1,5-di-O-acetyl-2,3,4-tri-O-CH3-rhamnitol, and iii) 1,5-di-O-acetyl-2-O-CD3-3,4-di-O-CH3-rhamnitol in a 1:0.7:0.3 ratio. The general structure of these substances is depicted in Fig. 9.



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  		FIG. 10.
MALDI-TOF mass spectra of purified GPLs. MALDI-TOF mass spectra of: a, apolar GPL I; b, polar GPL III (a and b subfamilies); and c, GPL III treated with 0.5 M sodium methanolate. 1, pseudomolecular ions (M + Na)+ of the de-O-acylated and {beta}-eliminated GPL IIIa subfamily; 2 and 3, pseudomolecular ions (M + Na)+ of the de-O-acylated GPL IIIa and GPL IIIb subfamilies, respectively.

 
The mass spectra of fractions S9 to S15 (GPLs II) contained two series of pseudomolecular ion peaks of similar intensity. The m/z values of the major [M + Na]+ peaks were 84 atomic mass units lower than those of GPLs I, i.e. at 1199.7 and 1213.7 m/z rather than 1283.8 and 1297.6 for GPLs I. This series corresponded to GPLs I devoid of the two acetyl groups (de-O-acetylated GPLs I or GPLs IIa subfamily). The other series of [M + Na]+ peaks, 174 atomic mass units higher than those observed for GPLs I, were also present in the mass spectra of fractions S9-15 (hyperglycosylated GPLs I or GPLs IIb subfamily; data not shown). Acid hydrolysis of these fractions, followed by GC and GC/MS analysis of the sugar derivatives (Fig. 1), identified them as alditol acetates 6-deoxytalosyl, 3,4-di-O-CH3-rhamnosyl, and 2,3,4-tri-O-CH3-rhamnosyl residues. On the basis of our previous observations (36), our data suggested that fractions S9 to S15 contained de-O-acylated GPLs I (GPLs IIa; Fig. 9), composed of a 6-deoxytalosyl residue linked to allo-threonyl and either a 3,4-di-O-CH3-rhamnosyl or 2,3,4-tri-O-CH3-rhamnosyl residue attached to the alaninol residue. The other components of fractions S9-15 were hyperglycosylated GPLs I (GPLs IIb; Fig. 9) in which a diglycosyl units composed of two 3,4-di-O-CH3-rhamnosyl residues modified the alaninol end of GPLs. While this work was in progress, this last class of compounds has been identified in M. smegmatis (39).

The most active fractions S17-21 contained GPL-like molecules (called GPLs III) exhibiting mobilities on TLC similar to those of GPLs II (Fig. 5b). This observation was unexpected because S17-21 were eluted from the gel only when water was added to the mixture of chloroform and methanol. When these fractions were analyzed by MALDI-TOF mass spectrometry, however, the observed [M + Na]+ peaks (Fig. 10b) were clearly different from those of both native and deacylated GPLs I. The major pseudomolecular [M + Na]+ ion peaks of GPLs III were seen at 1355.1, 1357.1, 1383.1, and 1385.1 m/z (Fig. 10b, GPLs IIIa subfamily). A minor series of peaks was observed at higher (174 atomic mass units) m/z values at 1515.1, 1517.1, 1543.1, 1545.1, 1557.1, and 1559.1 (Fig. 10b, GPLs IIIb subfamily). Treatment of GPLs III with 0.5 M sodium methanolate (Fig. 1) resulted in a 184-atomic mass unit downshift of the [M + Na]+ peaks of both series (Fig. 10c), indicating the occurrence of an alkali-labile substituent on native GPLs III in addition to the acetyl groups esterifying the 6-deoxytalosyl unit. The alkaline treatment (Fig. 1) also induced the {beta}-elimination of the sugar unit linked to the allo-threonyl residue, i.e. the 6-deoxytalosyl unit, with the additional loss of 174 atomic mass units. The [M + Na]+ peaks attributable to the remaining molecules were observed for the major series at 1008.9, 1036.9, and 1064.9 m/z (Fig. 10c). Perdeuteriomethylation of GPLs III followed by MALDI-TOF mass spectrometry analysis (Fig. 1) showed that hydroxylated compounds incorporated nine CD3 (including four on the peptide core), whereas only eight CD3 were incorporated in methoxylated molecules (data not shown). Acid hydrolysis of the perdeuteriomethylated products (Fig. 1) and analysis of the partially O-methylated, partially O-acetylated alditol derivatives by GC-MS led to the identification of i) 1,5-di-O-acetyl-2,3,4-tri--O-CD3-6-deoxytalitol, ii) 1,5-di-O-acetyl-3,4-di-O-CH3-2-O-CD3-rhamnitol, and iii) 1,2,5-tri-O-acetyl-3,4-di-O-CH3-rhamnitol in a 1:0.4:0.3 ratio. These data demonstrated that GPLs III were composed of a 6-deoxytalosyl unit linked to the allo-threoninyl residue and either a 3,4-di-O-CH3-rhamnosyl unit (for the GPLs IIIa subfamily; Fig. 9) or a disaccharide constituted two 3,4-di-O-CH3-rhamnosyl units (for GPLs IIIb subfamily; Fig. 9) attached at the alaninol end of the molecules. The proposed structure was consistent with the observed 14-atomic mass unit downshift of the mass values of the major series of de-O-acylated GPLs III when compared with those of the de-O-acylated GPLs I (data not shown), i.e. the replacement of the 2,3,4-tri-O-CH3-rhamnosyl residue of GPLs I by a 3,4-di-O-CH3-rhamnosyl unit in GPLs III. Likewise, the observed difference of 174 atomic mass units between the two subfamilies of GPLs III, in the MALDI-TOF mass spectra of both the native and alkali-treated GPLs III (Fig. 10, b and c), suggested the presence of a disaccharidyl residue composed of two 3,4-di-O-CH3-rhamnosyl units in the minor series of GPLs III (Fig. 9).

The chemical nature of the O-acyl residue that substituted the 3,4-di-O-CH3-rhamnosyl unit was guessed from the 1H-NMR spectrum of the GPLs III (Fig. 8). Two proton resonances at 2.62 and 2.70 ppm were present in the spectrum of GPLs III and absent from those of both GPLs I and their de-O-acylated forms (GPLs II). These deshielded resonances were attributed to methylene located near carboxylic functions. Furthermore, analysis of the two-dimensional homonuclear spectrum of GPLs III showed a correlation peak between the two resonances and no correlation with other resonances (data not shown). On the basis of the mass value deduced from the MALDI-TOF mass spectrum (101 atomic mass units), the O-acyl residue was postulated to be a succinyl group. Final identification of the O-acyl residue was performed by GC/MS analysis of the short-chain compounds released by alkaline methanolysis of GPLs III. These products were first desalted on a Dowex H+ column, and the putative carboxylic acids were methylated. GC analysis of the methyl esters using an isotherm program at 40 °C showed the presence of a peak whose mass spectrum gave an intense ion peak at 115 m/z that corresponded to the loss of a methoxyl group [M-31] and a fragment ion peak at 87 m/z attributed to the loss of a carboxyl methyl ester group [M-59]. On the basis of the structures proposed for GPLs I and III (Fig. 9), the only possible location for the additional O-acyl residue that accounts for 101 atomic mass units would be position 2 of the terminal di-O-CH3-rhamnosyl unit. This location was supported by the analysis of the MS/MS spectra on two [M + Na]+ of the native GPLs III at 1382.9 and 1557.8 m/z, respectively. The fragmentation pattern of the ion at 1382.9 m/z, representative of the major series of GPLs IIIa, showed a prominent peak at 1110.3 m/z that corresponded to the loss of succinylated anhydro-3,4-di-O-CH3-rhamnosyl residue. A similar fragmentation pattern was observed in the spectrum of the species at 1557.8 m/z, representative of the minor series of GPLs IIIb. The MS/MS spectrum showed two intense peaks at 1283.8 and 1109.7 m/z resulting from the loss of succinylated anhydro-3,4-di-O-CH3-rhamnosyl and anhydro-3,4-di-O-CH3-rhamnosyl-(1->2)-3,4-di-O-CH3-rhamnosyl residue, respectively.

Role of the Succinyl and Acetyl Substituents—GPLs III, the most inhibitory compounds of the family (Fig. 5), are characterized by the presence of a succinyl group on the terminal rhamnosyl unit. GPLs II, which were slightly less effective, differ from the nonactive GPLs I by the absence of the acetyl substituents (GPLs IIa) or the presence of a second rhamnosyl unit (GPLs IIb). GPLs I from S8 and GPLs III from S21 (Fig. 5) were de-O-acylated and analyzed for their inhibitory effect on M. smegmatis phagocytosis. The efficiency of the de-O-acylation procedure was checked by MALDI-TOF analysis. The alkaline treatment of GPLs I and GPLs III resulted in a 84-atomic mass unit downshift (loss of two acetyl groups) and a 184-atomic mass unit downshift (loss of two acetyl plus a succinyl group) of the [M + Na]+ peaks, respectively (data not shown). Compared with the native compounds, the inhibitory activity of the de-O-acetylated GPLs I was dramatically increased (8.67 ± 0.09 fold, n = 3). In contrast, the loss of the succinyl and acetyl substituents from GLPs III led to a decrease of the inhibitory effect (1.78 ± 0.05 fold; n = 3). Therefore, the succinyl substituent on the terminal rhamnosyl unit of GPLs III plays an essential role in their inhibitory activity as did the presence of free hydroxyl groups on positions 3 and 4 of the 6-deoxytalosyl moiety.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The outermost layer of mycobacteria represents the interface between bacilli and their host macrophages and probably specifies the intracellular fate of bacteria. Mycobacterial surface-exposed molecules are recognized by cognate cell surface receptors that enable bacteria to reach their habitable environment where they survive and multiply. A model of the outer leaflet proposed that glycolipids are embedded in a capsular layer of polysaccharides and proteins (40). A few purified molecules have been shown to play a role in the interaction between mycobacteria and host cells (40), but their surface location and their accessibility to their cell partners remain an unsolved question. To identify the nature of the putative mycobacterial partners of the phagocytic process, we used a step-by-step isolation procedure and were able to isolate surface-exposed molecules on the cell envelope of M. smegmatis that specifically prevent interactions between bacilli and macrophages. Among them, we identified phospholipids, including PIMs and C-type GPLs, which were the most active substances. The surface exposure of GPLs (32) and the recent demonstration of the impact of the absence of GPLs on the phagocytosis of M. smegmatis by human macrophages (26) are consistent with the implication of these molecules in the internalization of some non-tuberculous mycobacteria.

C-type GPLs are a family of species-specific lipids that typifies many non-tuberculous mycobacterial species (16, 33). In particular, they have been identified on the surface of M. smegmatis and M. avium but not on M. tuberculosis or M. kansasii (32). This observation certainly explains why nanomolar concentrations of GPLs from M. smegmatis decreased the internalization of M. avium, whereas much higher concentrations are necessary to affect phagocytosis of M. tuberculosis and M. kansasii. The four mycobacteria species tested herein have been shown previously to use common receptors, such as mannose receptors and complement receptor type 3, to infect macrophages (9, 10, 19, 41).2 Therefore, one can assume that mycobacteria express distinct ligands that can be recognized by a same receptor but with various ranges of affinity, a matter that remains to be addressed.

C-type GPLs share a common lipopeptidyl core composed of a long-chain fatty acyl linked to a tripeptide and terminated by alaninol. In all known C-type GPLs, the alaninol residue is substituted by a O-CH3-rhamnosyl or dirhamnosyl residue (33). In M. smegmatis, a di-O-acylated-6-deoxy-talosyl residue is linked to the allo-threoninyl residue. These simplest forms are found in all GPL-containing mycobacterial species and, accordingly, are called non-servar-specific GPLs or apolar GPLs. The allo-threoninyl-linked sugar unit of non-servar-specific GPLs may be further glycosylated in some GPL-containing mycobacteria, such as M. avium (33, 34). These discrete modifications confer to GPLs variable TLC patterns and antigenic properties (20, 33, 34). Detailed chemical analysis of the various purified GPLs that occur in M. smegmatis demonstrated the existence of a novel class of these molecules passed unnoticed in previous studies (Fig. 9). A combination of mass spectrometry, NMR, and chemical degradation techniques established the composition of the main constituents (GPLs IIIa) of this new family as a mixture of molecules possessing a core composed of the long-chain tripeptidyl amino alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-rhamnosyl unit linked to the alaninol end. Although various degrees of acetylation, methylation, or glycosylation exist in GPLs III, all of these molecules have in common the presence of a succinyl substituent.

Structure-function relationship analysis indicated that two structural features of GPLs play a critical role in their inhibitory activity. First, because the 3,4-de-O-acetylation of GPLs I led to an inhibitory effect, part of the GPLs II activity was attributable to the presence of free hydroxyl groups on the 6-deoxytalose moiety. Second, an alkali treatment of GPLs III, which removes the O-acetyl groups from the 6-deoxytalosyl residues and the succinyl group from the rhamnosyl unit, dramatically abolished the inhibition. This implies that the O-succinylation is the critical structural feature of the inhibition. This agrees with the absence of inhibition with GPLs I, which lack the succinyl substituent on the terminal rhamnosyl residue and are 3,4-O-acetylated on the 6-deoxytalosyl moiety. Such a dramatic phenotypical change after the succinylation of a glycoconjugate is not without precedent. For instance, strains of Rhizobium meliloti, which have lost their succinyl substituents on exopolysaccharides, became defective in alfalfa nodule invasion (42). Other succinylated glycoconjugates, such as a succinylated arabinomannan identified in the M. tuberculosis envelope (21), could share receptors with GPLs III. This would explain the inhibitory effect of M. smegmatis GPLs III on M. tuberculosis at high concentration. However, the new GPLs characterized in M. smegmatis exhibit other structural variations (Fig. 9) than succinylation, such as the presence of the second rhamnosyl unit. Therefore, a more detailed structure-function relationship study should be carried out to elucidate the precise role of these variations in biological activity of GPLs.

The mechanisms by which surface-exposed GPLs participate in the binding and phagocytosis of mycobacteria deserve consideration. GPLs from M. avium, essentially through its lipopeptide fragment, have been reported to disturb cell membrane ultrastructure and to change the expression of surface receptors of murine macrophages (43). In addition, mycobacterial GPLs are able to get inserted into phospholipid monolayers (44) and to disturb its properties (45). Such an insertion of molecules may alter interactions of mycobacteria with their host cell. However, although all GPLs share the same lipid core, only GPLs II and III were active. In addition, GPLs have no effect on the internalization of control particles such as zymosan, suggesting a specific recognition of structurally defined molecules. Taken together, these observations ruled out a nonspecific effect of GPLs under our experimental conditions. Further studies are clearly needed to elucidate the precise mechanisms by which GPLs affect the internalization of mycobacteria, either directly as ligands of receptor and/or indirectly as modulators of a specific receptor function.

In conclusion, the outermost layer of M. smegmatis contained new classes of C-type GPLs and phospholipids that efficiently inhibited the non-opsonic phagocytosis of mycobacteria. As such, these molecules may be of help in designing pharmacological drugs with a new therapeutic strategy, consisting of the inhibition of mycobacterial multiplication by preventing their cyclic internalization into macrophages. Mycobacteria are internalized by several receptors into human macrophages. Some of them are pattern recognition receptors, which can elicit different intracellular signals depending on the ligands used (13). Therefore, identification of surface-exposed mycobacteria ligands should also help to dissect the signaling pathways of receptors already known to internalize mycobacteria, as well as to discover new receptors involved in the infection of macrophages by mycobacteria.


   FOOTNOTES
 
* This work was supported in part by European Community Grant QLK 2CT 19990193 and Région Midi-Pyrénées. 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

{ddagger} To whom correspondence should be addressed: Institut de Pharmacologie et de Biologie Structurale, UMR 5089 (CNRS/UPS), 205 Route de Narbonne, 31077 Toulouse, France. Tel.: 33-561-17-54-55; Fax: 33-561-17-59-94; E-mail: Catherine.Astarie-Dequeker{at}ipbs.fr.

1 The abbreviations used are: MDM, macrophage derived from monocyte; GPL, glycopeptidolipid; PIM, phosphatidylinositol mannoside; FITC, fluorescein isothiocyanate; MALDI-TOF, matrix-assisted laser-desorption/ionization-time of flight; GC/MS, gas chromatography/mass spectrometry; MS/MS, electrospray ionization/tandem mass spectrometry. Back

2 C. Astarie-Dequeker, J. Castandet, C. Villeneuve, and I. Maridonneau-Parini, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. A. Lemassu (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France) for her expert advice in NMR and to C. Darribère for technical assistance.



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 ABSTRACT
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 DISCUSSION
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