Biosynthesis of Mycobacterial Lipoarabinomannan*

(Received for publication, February 28, 1997, and in revised form, May 12, 1997)

Gurdyal S. Besra Dagger , Caroline B. Morehouse Dagger , Christian M. Rittner Dagger , Charles J. Waechter § and Patrick J. Brennan Dagger

From the Dagger  Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523 and the § Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), are potent immunomodulators in tuberculosis and leprosy. Little is known of their biosynthesis, other than being based on phosphatidylinositol (PI), and they probably originate in the phosphatidylinositol mannosides (PIMs; PIMans). A novel form of cell-free incubation involving in vitro and in situ labeling with GDP-[14C]Man of the polyprenyl-P-mannoses (C35/C50-P-Man) and the simpler PIMs of mycobacterial membranes, reisolation of the [14C]Man-labeled membranes, and in situ chase demonstrated the synthesis of a novel alpha (1right-arrow6)-linked linear form of LM at the expense of the C35/C50-P-Man. There was little or no synthesis under these conditions of PIMan5 with its terminal alpha (1right-arrow2)Man unit or the mature LM or LAM with copious alpha (1right-arrow2)Man branching. Synthesis of the linear LM, but not of the simpler PIMan2, was susceptible to amphomycin, a lipopeptide antibiotic that specifically inhibits polyprenyl-P-requiring translocases. A mixture of P[3H]I and P[3H]IMan2 was incorporated into the linear LM, supporting other evidence that, like the PIMs, LM and LAM, it is a lipid-linked mannooligosaccharide and a new member of the mycobacterial glycosylphosphatidylinositol lipoglycan/glycolipid class. Hence, the simpler PIMs originate in PI and GDP-Man, but further growth of the linear backbone emanates from C35-/C50-P-Man and is amphomycin-sensitive. The origin of the alpha (1right-arrow2)Man branches of mature PIMan5, LM, and LAM is not known at this time but is probably GDP-Man.


INTRODUCTION

The cell wall of Mycobacterium spp. consists of a core composed of peptidoglycan linked to the heteropolysaccharide arabinogalactan which, in turn, is attached to the mycolic acids (1). Complementing the mycolyl residues is a variety of free lipids, and interspersed in this framework are lipoarabinomannan (LAM),1 lipomannan (LM), and various proteins (1). LAM has emerged as a major modulator of the host immune response in the course of tuberculosis and leprosy, through generalized suppression of immunity (2), induction of inflammatory cytokines (3), and the neutralization of potentially cytotoxic O2 free radicals (4). LAM may also act as a key ligand in the phagocytosis of Mycobacterium tuberculosis (5).

The biological importance of LAM dictates an understanding of its biosynthesis. Both LM and LAM are based on phosphatidylinositol (PI) (6, 7), specifically on monoacyl phosphatidylinositol dimannoside (Ac1PIMan2) (8, 9), a member of the PIM family with a characteristic myoinositol (Ino) 2,6-dimannosyl unit (10, 11). In the case of LM, a linear alpha 1right-arrow6-linked mannan with extensive alpha 1right-arrow2 Man branches emanates from position 6 of the Ino residue (8, 12). LAM contains an additional arabinan, similar but not identical to the arabinan of arabinogalactan (13, 14).

The structural progression from PI to PIMan2 to Ac1PIMan2 to LM and LAM has suggested a similar biosynthetic order (8); however, to date, this was mere speculation. In this present study, we have defined the origins of the Man units of the PIMs and LM and, in the course of the work, identified an alpha 1right-arrow6-linked linear form of LM, the apparent precursor of mature LM and LAM.


EXPERIMENTAL PROCEDURES

Preparation of Enzymatically Active Membranes and Cell Envelope

The transformable strain of Mycobacterium smegmatis, mc2155 (15), was grown in Bacto Nutrient broth (Difco Labs, Detroit, MI) to mid-log phase (about 24 h), harvested, washed with physiological saline, and stored at -20 °C. M. tuberculosis H37Ra (ATCC 25177) was grown in a liquid medium containing glycerol, alanine, and salts (16) for 14-16 days before harvesting. Mycobacteria (20 g, wet weight) were first washed and then resuspended at 4 °C in a buffer (20 ml) containing 50 mM MOPS (adjusted to pH 7.9 with KOH), 5 mM beta -mercaptoethanol, 10 mM MgCl2 (buffer A), 150 µg of DNase I (type IV, Sigma), and 250 µg of RNase (microsomal nuclease (Sigma)) and subjected to five passes through a French pressure cell (Aminco, Silver Spring, MD) at 10,000 p.s.i. The preparation was centrifuged initially at 600 × g to remove large particles and then at 27,000 × g for 20 min at 4 °C, and the membranes were obtained by centrifugation of the supernatant at 100,000 × g for 1 h at 4 °C. These (i.e. membranes arising from 20 g wet weight of cells) were resuspended in 1 ml of buffer A or buffer B (0.1 M Tris-HCl (pH 8.0), containing 0.25 M sucrose and 1 mM EDTA-Na2) (17)) to yield a total of ~20 mg of protein that was frozen in small aliquots; the enzyme activity in membranes was slightly diminished (~20%) by prolonged (2 months) storage at -20 °C. The pellet from the 27,000 × g centrifugation was resuspended in 7 ml of buffer A (20 mg protein/ml) or occasionally in the same volume of buffer B and used directly as an enzymatically active cell envelope fraction containing both cell walls and some membranes; the enzyme activity of this preparation was unaffected by prolonged storage (2 months) in small aliquots at -20 °C. Alternatively, the 27,000 × g pellet was resuspended in 40 ml of buffer A divided equally among four 40-ml centrifuge tubes to which were added 15 ml of Percoll (Pharmacia, Sweden) and centrifuged at 27,000 × g for 60 min at 4 °C (18). The particulate, upper diffuse band, containing both cell walls and membranes, was removed, collected by centrifugation, washed three times in buffer A, and finally resuspended in 4 ml of buffer A. The final concentration of this Percoll-60-purified cell envelope fraction (P-60) was 8-10 mg of protein/ml. Over 80% of the enzyme activity was lost on freezing and thawing, and hence, this fraction was freshly prepared.

Pulse Incorporation of Radiolabeled Man from GDP-[3H]Man or GDP-[14C]Man into Membrane Lipids

Early assays involved incubation of membranes (50 µl; 0.5 mg of protein) or cell envelope (50 µl; 1 mg of protein) in either buffer A or B in the presence of 10 µM MgCl2 and 10 µM (5 µCi) GDP-[2-3H]Man (35 Ci/mmol; NEN Life Science Products) in a total volume of 100 µl. Subsequent assays involved incubation of membranes (100 µl; 2 mg of protein) in buffer A with 9.7 µM (1 µCi) GDP-[U-14C]Man (321.4 mCi/mmol; NEN Life Science Products) and 62.5 µM ATP in a total volume of 320 µl.

Conditions for Further Metabolism of in Situ Labeled [14C]Man Membrane Glycolipids

Enzymatically active membranes containing in situ [14C]Man-labeled lipids, generated as a result of a 10-min pulse incubation with GDP-[14C]Man, were recovered by centrifugation of combined multiple reaction mixtures (either 10 or 20) at 100,000 × g, for 60 min. The supernatants from these centrifugations were removed, and the [14C]Man-labeled membranes were washed in place several times with cold buffer A to remove residual GDP-[14C]Man. The [14C]Man-labeled membranes were then carefully resuspended in buffer A (e.g. 1.0 ml for the proceeds from 10 replicate reactions), and 100-µl aliquots (2 mg of protein) were then redistributed into 10 fresh tubes, each containing 62.5 µM ATP and usually 2 mg (200 µl) of the Percoll-purified cell envelope (P-60) in a total volume of 320 µl and incubated further.

Pretreatment of Membranes with Amphomycin

Amphomycin (calcium salt) was a gift to C. J. Waechter from Bristol Laboratories and R. Bedensky, Case Western Reserve University, Cleveland, OH. The lipopeptide (up to 2 mg) was dissolved in 500 µl of 0.1 N acetic acid, and the solution was adjusted to 0.05 M sodium acetate (pH 7.0) with 0.1 N NaOH for a final concentration of 2 mg/ml (19). Membranes with or without the in situ labeled [14C]Man lipids were preincubated with amphomycin (10 µg/100 µl of reaction mixture) at 37 °C for 10 min prior to various manipulations such as the addition of the P-60-purified cell envelope fraction and further incubation.

Extraction of [3H]/[14C]Man-labeled Products from Reaction Mixtures

At the end of incubations, the reactions were terminated by the addition of CHCl3/CH3OH (2:1) (2.5 ml per 100 µl of reaction mixture) followed by centrifugation to separate the pellet (17, 20). The pellet was extracted once more with one-half the volume of CHCl3/CH3OH (2:1). The combined CHCl3/CH3OH (2:1) extracts were washed with 0.9% NaCl followed by CHCl3/CH3OH/H2O (3:48:47) to yield the washed CHCl3/CH3OH (2:1) lipids. The resulting insoluble pellet, in the case of reactions involving membranes with in situ labeled [14C]Man lipids, was directly extracted twice with CHCl3/CH3OH/H2O (10:10:3). The insoluble pellets derived from membranes that were directly labeled with GDP-[3H]Man or GDP-[14C]Man were first washed with 0.9% NaCl in CH3OH, H2O/CH3OH (1:1) and pure CH3OH (17) to remove residual GDP-[14C]Man before extracting with CHCl3/CH3OH/H2O (10:10:3). In an effort to determine whether the insoluble [14C]Man-containing products associated with the final insoluble residue were LM/LAM, it was successively extracted with refluxing 50% ethanol and 88% phenol, as described (7).

Preparation of Mycobacterial PIMan2s, P[3H]I, and P[3H]IMan2s

Characterization of the various PIMs followed earlier work (9, 11, 21) and also by comparison with well defined, two-dimensional TLC maps of the full spectrum of PIMs (22). Thus, characterizations were based on fast atom bombardment mass spectroscopy analysis (9) and chromatographic patterns of the intact PIMs (23, 24) and the deacylated forms (21), e.g. the deacylated PIMan2s produced a single product with an Rf compared with deacylated PI of 0.8 on paper chromatograms in 2-propyl alcohol/NH4OH (2:1), whereas the deacylated PIMan5s produced a single product with a comparative Rf of 0.30. To prepare P[3H]I and P[3H]IMan2s, M. smegmatis was grown in 100 ml of the glycerol/alanine/salts medium to mid-log phase (24 h) at which stage 250 µCi of [2-3H]myo-inositol (21 Ci/mmol; NEN Life Science Products) was added to the medium and growth continued for another 12 h to late-log phase. Cells were harvested aseptically, and the recovered medium was re-inoculated with fresh M. smegmatis and the cycle repeated. The combined harvested cells were washed several times with phosphate-buffered saline, lyophilized (184 mg), extracted several times with CHCl3/CH3OH (2:1), the extracts washed (25), dried (17 mg), and the phospholipids precipitated with acetone (8 mg; 2.1 × 107 cpm). Two-dimensional TLC, first in CHCl3/CH3OH/H2O (60:30:6) and then in CHCl3/CH3COOH/CH3OH/H2O (40:25:3:6), followed by treatment with EN3HANCE (NEN Life Science Products), and autoradiography demonstrated the presence of mostly P[3H]I and Ac2P[3H]IMan2 with small amounts of P[3H]IMan1 and P[3H]IMan2. To examine the incorporation of the P[3H]I and P[3H]Man2s into the linear LM, the dry preparation containing them was suspended by sonication in buffer A, and 500,000 cpm aliquots were added to each of five standard reaction mixtures (320 µl) containing membranes (8 mg), ATP, P-60 and buffer, which were incubated for 1 h, extracted with CHCl3/CH3OH (2:1) followed by CHCl3/CH3OH/H2O (10:10:3). Conditions for the preparation of the P[3H]IMan5s and PI[14C]Man5s from growing cells labeled with [3H]Ino and [14C]Glc, respectively, are described in the figure legends.

Selective Isolation of PIMan5s

This study resulted in an excellent protocol for the selective purification of the PIMan5s. We had observed that little of the PIMan5s (21) were present in the CHCl3/CH3OH (2:1) extracts of M. smegmatis. However, extraction of the resulting residue (from 40 g wet weight of cells) three times with CHCl3/CH3OH/H2O (10:10:3) resulted in 30.7 mg of material. Two-dimensional TLC, first in CHCl3/CH3OH/H2O (60:30:6) and then in CHCl3/CH3COOH/CH3OH/H2O (40:25:3:6), followed by staining with an alpha -naphthol-containing reagent (26), demonstrated the presence only of the two PIMan5s, PIMan5 and AcPIMan5 (27).

Analytical Procedures

TLC was conducted in one- and two-dimensions on aluminum-backed plates of silica gel 60 F254 (E. Merck, Darmstadt, Germany) in the solvents described in the text. An alpha -naphthol spray (26) and the molybdenum blue (28) dip reagent were used to detect carbohydrate and phosphorus in lipids, respectively. SDS-polyacrylamide gel electrophoresis and silver periodic acid-Schiff staining was performed as described (29). Autoradiograms were obtained by exposing chromatograms to Kodak X-Omat AR films at -70 °C, usually for 4-5 days. To locate 3H-labeled products, EN3HANCE (NEN Life Science Products) was used according to instructions. Plates were also scanned for radioactivity using the Bio-Scan System 200 Imaging Scanner with the Autochanger 3000 (plates were read stepwise, using the 10-mm collimator, in 3-mm stops, each stop reading for 15 min).

Mild acid hydrolysis of CHCl3/CH3OH (2:1)-soluble lipids was conducted in 0.5 N HCl tetrahydrofuran (1:4) for 2 h at 50 °C (30). Samples were neutralized with 200 µl of 0.1 N NaOH in 0.02 N sodium phosphate, dried, resuspended in CHCl3/CH3OH/H2O (4:2:1), centrifuged, and the aqueous and organic phases separated and analyzed. For mild acid hydrolysis of the CHCl3/CH3OH/H2O (10:10:3)-soluble lipids, samples (~100 µg) were suspended in 50 µl of 1-propyl alcohol, sonicated, treated with 100 µl of 0.02 N HCl at 60 °C for 30 min, neutralized with 15 µl of 0.2 N NaOH (20, 31), and subjected to gel filtration chromatography. For mild alkaline hydrolysis of both sets of lipids, samples were dissolved or suspended in 900 µl of ethanol by sonication, followed by the addition of 100 µl of 1 N NaOH and incubation at 40 °C for 1 h. The NaOH was neutralized with ethyl formate (~144 µl), held at 40 °C for 5 min, and dried. In the case of the CHCl3/CH3OH (2:1)-soluble lipids, the products were partitioned within CHCl3/CH3OH/H2O (4:2:1) and analyzed separately. The products from the CHCl3/CH3OH/H2O (10:10:3)-soluble lipids were not partitioned but were applied directly to columns. Gel filtration chromatography was conducted on columns (1 × 116-175 cm) of various Bio-Gel P resins (Bio-Rad) in 0.1 M sodium acetate (pH 6.5). Repeated efforts to fully methylate the linear LM by the NaOH method (32) were unsuccessful. Successful methylation was accomplished by the method of Hakomori (33) with modifications, particularly the additions of fresh CH3I and 4.8 M dimethyl sulfinyl carbanion. The final reaction mixture was applied to a C18 Sep-Pak cartridge (Waters, Milford, MA) and the permethylated linear LM recovered in the CH3CN eluant as determined by counting 1% of the eluants. The per-O-methylated linear LM was hydrolyzed in 2 M trifluoroacetic acid at 110 °C for 2 h, the acid evaporated, reduced with NaBH4, per-O-acetylated, and the alditol acetate characterized by gas chromatography/mass spectrometry as described (34).


RESULTS AND DISCUSSION

The Specific Question

The immediate question was the manner of biosynthesis of LM with a view to ultimate LAM synthesis. Work in the late 1960s provided possible clues. Brennan and Ballou (23, 24) and Ballou (35) demonstrated that membranes from M. smegmatis catalyzed the transfer of [14C]Man from GDP-[14C]Man to PI to produce PI[14C]Man2 with the further addition of palmitate residues from palmitolyl-CoA to yield a mixture of PIMan2, the monoacyl (Ac1PIMan2) and the diacyl (Ac2PIMan2) derivatives. It was later recognized that LAM, in its various forms, i.e. ManLAM and AraLAM (22), and LM contain a mannan core linked to PI similar to that in the family of PIMs (PIMan1-6) (6-8). Specifically, it was shown that these lipoglycans contain the D-myo-inositol 2,6-bis-alpha -Manp unit and thus to be based on PIMan2, and hence, it was assumed that PIMan2 (and probably PIMan3) was the immediate precursor of LM/LAM. Moreover, since Khoo et al. (9) demonstrated the presence of a palmitate substituent on C-6 of the Manp unit that is linked directly to C-2 of the Ino within LM/LAM, it was further assumed that Ac1PIMan2 was the precise precursor. Throughout, it was thought that GDP-Man was the immediate donor of all of the Manp units of LM/LAM simply because it was the demonstrated precursor of the Manp units of the PIMan2 family (23, 24). However, shortly after this initial work, Takayama et al. (36, 37) and Schultz and Elbein (38) described two alkali-stable mannophospholipids in M. tuberculosis and M. smegmatis, a mannosyl-1-phosphoryl-decaprenol (C50-P-Man) and a mannosyl-1-phosphoryl heptaprenol (C35-P-Man), which, in light of their group transfer potential and known role in mannolipid synthesis from other organisms (39), could be donors of polymerized Man in mycobacterial cell walls. Indeed, Schultz and co-workers (38, 40) demonstrated indirectly that C50/C35-P-Man was the Man donor of undefined polymers. In addition, more recently, Yokoyama and Ballou (41) demonstrated that the beta -mannosylphosphoryldecaprenol (C50-P-beta Man) was the direct Man donor of a series of alpha (1right-arrow6)-linked oligosaccharides, clearly not LM or LAM with their copious alpha (1right-arrow2) branches. Hence, the questions posed at the outset of this work concerned the metabolic relationships among GDP-Man, C35/C50-P-Man, the PIMs, LM/LAM, and the connection, if any, between the alpha (1right-arrow6)-linked mannooligosaccharides and LM/LAM.

Incorporation of GDP-[3H/14C]Man by M. smegmatis Membranes and Cell Wall

A variety of incubation conditions was applied as follows: those developed in the context of PIM biosynthesis (23, 24); those (17) designed to examine the specificity of dolichyl-P-Man-dependent mannosyltransferase activity in mammalian glycoprotein synthesis; those for the incorporation of Man from GDP-Man and C50-P-beta Man into the alpha (1right-arrow6)-linked oligosaccharides of M. smegmatis (41); and a more universal assay appropriate for the incorporation of [14C]GlcNAc, [14C]Rha, [14C]Gal, and [14C]Ara from their corresponding donors into the mycobacterial cell wall "core" (15, 18). Incorporation in all cases was quantitatively and qualitatively comparable. However, replacement of 10 mM MgCl2 (17) with 10 mM MnCl2 or 10 mM CaCl2 resulted in about 35% reduction of activity but no change in product profile; inclusion of 5 mM EDTA reduced incorporation by about 90%, pointing to the need for divalent cations for these mannosyltransferase activities.

Under all of these conditions, the majority (~89%; ~0.4 × 106 cpm/mg protein/reaction mixture) of the incorporated [3H]Man was present in the CHCl3/CH3OH (2:1)-soluble membrane lipids with only about 10% in other material (Table I). Substitution of membranes with active cell envelope preparations resulted in lower specific activity, but still ~80% of the incorporated [3H]Man appeared in the membrane lipids. Obviously, under these conditions, there was little or no synthesis of LM/LAM or large mannooligosaccharides which are not extracted by those solvents and would be in the insoluble residue. TLC autoradiography (see below) of the lipids synthesized by membranes and cell envelope preparations showed no significant differences in profile; about 75% of the total radioactivity was distributed between C50-P-Man and C35-P-Man, with the remainder in the PIMan2s. Short pulses (10 min) of membranes with GDP-[3H]/[14C]Man showed lesser synthesis of the PIMan2s; the C35-P-Man and C50-P-Man predominated. Longer incubations showed increased synthesis of the PIMan2s.

Table I. Incorporation of [3H]Man from GDP-[3H]Man into mycobacterial membrane or envelope mannolipids

Reaction mixtures contained 10 mM MgCl2, 1.0 × 106 cpm (10 µM) GDP-[2-3H]Man, membranes from M. smegmatis (100,000 × g pellet; 0.52 mg of protein) or cell envelope from M. smegmatis (27,000 × g pellet; 1.02 mg of protein) (50 µl) in buffer A in a total volume of 100 µl. Incubation was for 20 min at 37 °C. Reaction products after extraction with CHCl/CH3OH (2:1) were extensively washed with 0.9% NaCl in 50% CH3OH, 50% CH3OH, and CH3OH prior to extraction with CHCl/CH3OH/H2O (10:10:3).

Fraction Membranes Envelope Nature of products

cpm/assay cpm/assay
CHCl/CH3OH (2:1)-soluble lipids 0.41  × 106 (89%) 0.172  × 106 (83%) C35/C50-P-Man; PIMan2s
CHCl3/CH3OH/H2O (10:10:3)-soluble lipids 0.01  × 106  (2%) 0.017  × 106  (8%) Nondescript
Insoluble residue 0.04  × 106  (9%) 0.019  × 106  (9%)  ---

Effects of Amphomycin on Incorporation of [14C]Man from GDP-[14C]Man into Membrane Lipids

Amphomycin is one of several families of compounds that specifically disrupts the action of a variety of translocase enzymes, by chelating polyprenyl monophosphates in the presence of Ca2+ ion and thus inhibiting the transfer of a range of monomeric units to polyprenyl-P carriers (19, 42, 43). It was applied in the present context to determine (a) whether it would specifically inhibit the incorporation of [14C]Man from GDP-[14C]Man into C35/C50-P-Man, and (b) its effects on the synthesis of the PIMan2 family, i.e. to determine whether the Man units of PIMan2s arose in C35/C50-P-Man or GDP-Man. Preincubation of membranes with 10 µg of amphomycin prior to the addition of GDP-[14C]Man had a profound effect on the synthesis of the membrane mannophospholipids. First, there was a dramatic overall reduction in the synthesis of total membrane mannolipids (~60%). Second, inhibition was specific for the C35/C50-P-Man pair of lipids, and synthesis of the PIMan2 was unaffected (Fig. 1). Therefore, the immediate donor of the Man residues of PIMan2 is not C35/C50-P-Man, but GDP-Man, obviously reacting with PI as demonstrated previously (23, 24).


Fig. 1. Effects of amphomycin on incorporation of GDP-[14C]Man into membrane lipids. Membranes from M. smegmatis (2 mg of protein) were incubated in buffer A with GDP-[14C]Man (1 µCi) in a total volume of 320 µl for 10, 20, or 60 min in the absence and presence of amphomycin (10 µg/100 µl per reaction mixture in the presence of 0.1 M CaCl2; preincubated with membranes for 10 min), extracted with CHCl3/CH3OH (2:1), chromatographed in CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:3.6), and subjected to autoradiography.
[View Larger Version of this Image (23K GIF file)]

Chase of in Situ Labeled C35/C50-P-[14C]Man

To determine whether GDP-Man or C35/C50-P-Man was the donor for further mannosylation (e.g. in LM/LAM biosynthesis), a novel assay system was designed. Membranes were pulsed with GDP-[14C]Man during a short (10 min) incubation period, but instead of extracting with CHCl3/CH3OH, the [14C]Man-labeled membranes were re-harvested by centrifugation at 100,000 × g, washed by suspending in MOPS buffer, and again harvested. The [14C]Man-labeled membranes, shown to be devoid of GDP-[14C]Man, were then further incubated for various times with or without the cell envelope (P-60) prior to extraction with CHCl3/CH3OH/H2O (4:2:1) to form a biphase and provide the CHCl3/CH3OH (2:1)-soluble lipids for TLC (Fig. 2). The results were striking. At the zero chase time in the presence of membranes only, practically all of the radioactivity was associated with the CHCl3/CH3OH (2:1)-soluble lipids (310,800 cpm/mg protein), i.e. the C35/C55-P-Man combination and Ac1PIMan2 (Fig. 2, lane 1). Further incubation of membranes for 10 min resulted in little change in lipid radioactivity (Fig. 2, lane 2), whereas additional incubation for 60 min resulted in a significant loss of radioactivity (201,700 cpm/mg protein/reaction mixture) (Fig. 2, lane 3). However, the addition of the cell envelope (P-60) fraction to the reaction mixture in addition to the 60 min chase resulted in dramatic loss of lipid radioactivity (68,400 cpm/mg protein/reaction mixture), and TLC indicated that this loss was from the C35/C50-P-Man population, not the PIMan2 family (Fig. 2, lane 5). Thus, this form of in situ chase of the radioactivity demonstrated that C35/C50-P-Man was the source of the CHCl3/CH3OH (2:1)-insoluble Man-containing products.


Fig. 2. TLC autoradiography of the in situ labeled [14C]Man membrane glycolipids followed by chase with the cell envelope (P-60) fraction. Exact conditions for in situ labeling of membranous C35/C50-P-[14C]Man, subsequent chase induced by the cell envelope fraction (P-60), and extraction with CHCl3/CH3OH (2:1) are described under "Experimental Procedures." Lipids from 0 time, i.e. pulse-labeled [14C]Man lipids (lane 1); 10 min (lane 2) and 60 min of further incubation of membrane; 10 min (lane 4) and 60 min (lane 5) of further incubation of membranes with added cell envelope fraction (P-60).
[View Larger Version of this Image (27K GIF file)]

Properties of the End Products of the C35/C50-P-[14C]Man Chase

Efforts to identify the end products of the chase were based on this new assay in which these lipids were generated in situ through short (10 min) pulse labeling of membranes with GDP-[14C]Man to preferentially generate C35/C50-P-[14C]Man which were then further incubated for 1 h in the presence of the cell envelope (P-60) preparation. Twenty of these basic reactions were conducted, but this time, after extraction with CHCl3/CH3OH (2:1) and washing of the pellet to remove possible residual GDP-[14C]Man, the pellet was further extracted with CHCl3/CH3OH/H2O (10:10:3) in search of lipid-linked oligosaccharides, as possible intermediates on the pathway to LM/LAM. The results of this extraction are shown in Table II. A surprisingly large proportion of the incorporated radioactivity was solubilized by extraction with CHCl3/CH3OH/H2O (10:10:3).

Table II. Results of large scale chase of in situ labeled membranous C35/C50-P-[14C]Man into polymerized materials

A large batch of membranes were prepared from 20 g of M. smegmatis. These were divided among 20 tubes in the basic assay format, and each was incubated for 10 min with GDP-[14C]Man. The radiolabeled membranes were recovered by centrifugation at 100,000 × g, washed, and resuspended in 2 ml of buffer A, and 100 µl was added to each of 20 fresh tubes and further incubated for 1 h in the presence of 200 µl of cell envelope fraction (P-60). The reaction mixtures were then extracted with CHCl3/CH3OH (2:1) and CHCl3/CH3OH/H2O (10:10:3). The majority of the residual radioactivity was not extractable with either 50% ethanol or 88% phenol, indicating that it was not LM/LAM.

Fraction cpm/assay Percent of incorporated [14C]Man Nature of products

CHCl3/CH3OH (2:1)-soluble lipids 2.24  × 106 58.5 C35/C50-P-[14C]Man; PI [14C]Man2s
CHCl3/CH3OH/H2O (10:10:3)-soluble lipids 0.98  × 106 25.7 Linear-LM
Insoluble residue 0.60  × 106 15.7 Majority not soluble; apparently not LM/LAM

The results of a comparison of the TLC radioautography profiles of the CHCl3/CH3OH (2:1) and the CHCl3/CH3OH/H2O (10:10:3)-soluble lipids (Fig. 3) demonstrated the occurrence in the latter fraction of a family of gradated highly polar [14C]Man-containing glycolipids. This population was susceptible to alkali treatment but resistant to the mild acid conditions, indicating that they were possibly a family of PIMs intermediate in length between PIMan3-5 and LM. Since the chromatographable components of the CHCl3/CH3OH/H2O (10:10:3)-soluble lipids were minor, they were not further characterized. Clearly, the bulk of the CHCl3/CH3OH/H2O (10:10:3)-soluble material synthesized at the expense of C35/C50-P-[14C]Man remained at the origin in this solvent. Synthesis of this material also proved to be sensitive to amphomycin. When membranes were pretreated with amphomycin as described in Fig. 1 and then incubated with GDP-[14C]Man, there was a 90% reduction (compared with non-treated membranes) in incorporation of [14C]Man into the CHCl3/CH3OH/H2O (10:10:3)-soluble material remaining at the origin.


Fig. 3. TLC autoradiography of the [14C]Man glycolipids from a large scale GDP-[14C]Man pulse and C35/C50-P-[14C]Man chase experiment. The standard pulse-chase reaction was replicated 20 times, and 50,000 cpm were applied to each lane to demonstrate the minor reaction products. Lane 1, the CHCl3/CH3OH (2:1)-soluble lipids. Lane 2, the CHCl3/CH3OH/H2O (10:10:3)-soluble lipids.
[View Larger Version of this Image (60K GIF file)]

PIMan5 Is Not Appreciably Synthesized during the C35/C50-P-[14C]Man Chase

Although the PIMan5s and PIMan6 (21, 44) represent an appreciable proportion of the PIM population of mycobacteria, there was little evidence for their synthesis under the conditions of C35/C50-P-[14C]Man chase (Fig. 3). To establish categorically whether or not the higher PIMs were synthesized under these conditions, M. smegmatis was grown in the presence of [14C]Glc (45) or [3H]Ins, and the CHCl3/CH3OH/H2O (10:10:3)-soluble lipids from each harvest were compared with the lipids likewise extracted from the cell-free C35/C50-P-[14C]Man chase assays. Clearly, under standard growth conditions, the two PIMan5s (PIMan5 and Ac1PIMan5 (27)) were the only higher PIMs appreciably synthesized (Fig. 4, A and B) by M. smegmatis. However, there was no synthesis of these PIMan5s under the cell-free C35/C50-P-Man chase conditions (Fig. 4C). The implications were that C35/C50-P-Man is not a precursor of the alpha (1right-arrow2)Man unit that differentiates PIMan4-6 from the PIMan2s and PIMan3 (21). The results of the experiments described in Fig. 4 also demonstrated the presence of [3H]Ins in the material at the origin (Fig. 4B), indicating for the first time that the major CHCl3/CH3OH/H2O (10:10:3)-soluble product of the C35/C50-P-Man chase experiments may contain PI and may be a linear (alpha 1right-arrow6)-linked form of LM.


Fig. 4. Synthesis of PIMan5s during growth but lack of synthesis under conditions of cell-free C35/C50-P-[14C]Man chase. A, M. smegmatis (100 ml) at mid-log phase was grown in the presence of 1 µCi/ml [U-14C]Glc (250 mCi/mmol) for 2 h. The cells were harvested, washed with 0.9% NaCl, and extracted with CHCl3/CH3OH (2:1), 0.9% NaCl in CH3OH, and 50% CH3OH with probe sonication before extraction with CHCl3/CH3OH/H2O (10:10:3). The CHCl3/CH3OH/H2O (10:10:3) extract (10,000 cpm) was applied to TLC. Inset, two-dimensional TLC. B, M. smegmatis at early log phase was incubated with 1.8 µCi/ml [2-3H]Inositol (21.0 Ci/mmol) for 12 h before harvesting. The cells were extracted as described above, and 100,000 cpm of the CHCl3/CH3OH/H2O (10:10:3) extract were applied to TLC. C, membranes with in situ C35-P-[14C]Man were prepared by incubating 100 µl of membranes (2.5 mg/protein), 10 mM CaCl2, 1.22 mM CH3COONa buffer, and 0.0625 mM ATP for 10 min at 37 °C and then adding 1 µCi of [14C]GDP-Man (321.4 mCi/mmol) in a total volume of 320 µl/reaction mixture (total of 8 such reaction mixtures) and incubating for an additional 10 min. The reaction mixtures were combined and centrifuged at 100,000 × g for 1 h, washed, and resuspended to 800 µl. Eight new reaction mixtures were prepared containing 100 µl of the radiolabeled membranes, 200 µl of the cell envelope P-60 fraction (4 mg/protein), and 0.06 mM ATP in a total volume of 320 µl. The reaction mixtures were incubated at 37 °C for 1 h and then extracted with CHCl3/CH3OH (2:1) followed by CHCl3/CH3OH/H2O (10:10:3). The CHCl3/CH3OH/H2O (10:10:3) extract (25,000 cpm) was applied to TLC. One-dimensional TLC plates were run in the CHCl3/CH3OH, 1 M CH3COONH4/NH4OH/H2O (180:140:9:9:23) and analyzed for radioactivity using a Bio-Scan System 200 Imaging Scanner. Two-dimensional TLC was conducted, first in CHCl3/CH3OH/H2O (60:30:6) and then in CHCl3/CH3COOH/CH3OH/H2O (40:25:3:6).
[View Larger Version of this Image (18K GIF file)]

Characterization of CHCl3/CH3OH/H2O (10:10:3)-Soluble Polymer as an alpha (1right-arrow6) Linear Mannooligosaccharide Attached to PI

To characterize the CHCl3/CH3OH/H2O (10:10:3)-soluble [14C]Man lipids that remain at the origin in a variety of TLC solvents, the material was subjected to gel filtration before and after treatment with alkali and weak acid (Fig. 5). Gel filtration of the CHCl3/CH3OH/H2O (10:10:3), even on Bio-Gel P100 in 0.1 M CH3COONa, demonstrated that all of the radioactivity was excluded (Fig. 5A), possibly more a reflection of micelle formation than size. The deacylated fraction was included in Bio-Gel P100 (Fig. 5B) and Bio-Gel P10 (Fig. 5C) and, barely, in Bio-Gel P-2, generally demonstrating the retention volumes of eicosaccharides. Clearly, its size was smaller than that of deacylated LM and deacylated LAM (Fig. 5, B and C).


Fig. 5. Gel filtration chromatography of the CHCl3-CH3OH-H2O (10:10:3)-soluble lipids from the membranous C35/C50-P-[14C]Man chase experiment. Experimental conditions are described in Fig. 3. A, the intact CHCl3/CH3OH/H2O (10:10:3) extracted [14C]Man lipids were suspended in 0.1 M CH3COONa with sonication and applied to a column (1 × 120 cm) of Bio-Gel P-100 in 0.1 M CH3COONa. For each of the subsequent columns, 20,000 cpm of the [14C]Man lipid fractions were treated with 1 N NaOH at 40 °C for 1 h, neutralized, counted (typical recovery >80%), and applied to columns (1 × 130 cm) of Bio-Gel P-100 (B) and Bio-Gel P-10 (C).
[View Larger Version of this Image (20K GIF file)]

A large scale, 14-fold reaction mixture was prepared in which the membranes from 20 g of cells were preincubated with 14 µCi of GDP-[14C]Man, harvested, washed, and then further incubated with the cell envelope fraction from the same batch of cells for 1 h. The CHCl3/CH3OH/H2O (10:10:3)-soluble lipids were obtained after pre-extraction with CHCl3/CH3OH (2:1). About 1 × 106 cpm were recovered. SDS-polyacrylamide gel electrophoresis and subsequent autoradiography of the dried gels showed that this material had mobility properties intermediate between that of the family of PIMs and LM, pointing again to a product intermediate between the higher PIMs and LM.

To directly demonstrate the presence of PI in the CHCl3/CH3OH/H2O-soluble polymer, the membranes containing the in situ labeled C35/C50-P-[14C]Man (from five reaction mixtures) were further incubated with P[3H]IMan2 (5 × 500,000 cpm) for 1 h and extracted with CHCl3/CH3OH (2:1) followed by CHCl3/CH3OH/H2O (10:10:3). The latter extract was treated with alkali and applied to Bio-Gel P-2 (Fig. 6). The [3H]Ino and the [14C]Man counts were coincident, suggesting the presence of P[3H]IMan2 in the de novo synthesized product.


Fig. 6. Simultaneous incorporation of in situ C35/C50-P-[14C]Man and exogenous P[3H]I/P[3H]/Man2s into linear LM. The membranes containing in situ C35/C50-P-[14C]Man were obtained from five of the standard incubations. These were distributed into five new reaction mixtures to each of which was added 500,000 cpm of the P[3H]IMan2 preparation. Incubation was for 1 h. The reaction mixture was extracted with CHCl3/CH3OH/H2O (10:10:3), treated with alkali, and applied to a column (1 × 123 cm) of Bio-Gel P-2 and fractions counted for [14C]Man and [3H]Ino.
[View Larger Version of this Image (14K GIF file)]

All of the evidence pointed to the synthesis of a new form of LM (linear LM) under the in vitro conditions. To examine the linkage pattern of Man in the newly synthesized product, the CHCl3/CH3OH/H2O (10:10:3)-soluble material was subjected to a number of methylation attempts. Only the dimethylsulfinium carbanion-catalyzed method (33) was successful, with about 50% conversion of the original 1 × 106 cpm into a full permethylated product. This was purified on a small column of SepPak, hydrolyzed with 2 M CF3COOH, reduced with NaB[2H]4, acetylated, and the alditol acetates analyzed by gas chromatography/mass spectrometry with simultaneous counting of radioactivity (15). Only two products were obtained, the terminal Manp derivative (1,5-di-O-Ac-2,3,4,6-tetra-O-Me-mannitol) and the 6-linked Manp derivative (1,5,6-tri-O-Ac-2,3,4-tri-O-Me-mannitol); there was no evidence of any 2-linked Man derivative. Thus, the combined evidence points to the synthesis of an alpha (1right-arrow6) linked mannolipooligosaccharides linked to PI.

Synthesis of the Linear LM in M. tuberculosis

In situ labeling of the C35/C50-P-[14C]Man population of M. tuberculosis membranes was accomplished in like fashion. However, the C50-P-[14C]Man product represented the majority, ~90%, of the material synthesized. Likewise, further incubation of these [14C]Man-labeled membranes with a corresponding P-60 fraction from M. tuberculosis demonstrated incorporation into CHCl3/CH3OH/H2O-soluble material with all of the characteristics of linear LM, i.e. 6-linked Manp only, the presence of [3H]Ins, and in vitro incorporation of P[3H]IMan2.

Conclusions

This present body of research solves several questions left in abeyance for many years but now of importance in light of the role of LAM in phagocytosis of M. tuberculosis and generalized immunosuppression. The structural relationship between LAM/LM and the PIMs was first recognized with the discovery of the presence of the basic PI (7) and PIMan2 (8) units in the molecules. However, the biosynthetic origins of the PIMs had always been in doubt and that of LM/LAM had never been explored. Ballou and colleagues (23, 24, 35, 46) had clearly demonstrated that GDP-Man was the source of the Man units of PIMan2 and PI was a suitable acceptor of [14C]Man from GDP-[14C]Man. However, this evidence emerged prior to the recognition of the existence of a heptaprenyl (3,7,11,15,19,23,27-heptamethyl-2,6,10-octacosatriene-1-ol)-P-Man and a decaprenyl-P-Man (the structure of the decaprenol is probably similar to that in the decaprenyl-P-Araf (47)) in mycobacterial membranes (37) and their possible roles in polymer synthesis (38, 40). The use, in this present work, of amphomycin, a member of the mureidomycin family known to inhibit a variety of translocases by chelating to a variety of polyprenol monophosphates (19), resulted in complete inhibition of the synthesis of the polyprenyl-P-Man, but allowed continuing synthesis of the PIMan2s, demonstrating that the pathway proposed by Brennan and Ballou (23) prevails and that suggested by others (48) is incorrect.

The first definitive evidence of the precursor role of a polyprenol-P-Man, the decaprenyl-P-Man, in mycobacterial mannan synthesis came from the work of Yokoyama and Ballou (41), definitive in that it involved the isolation of pure C50-P-[14C]Man, its incubation with membrane preparations, and characterization of end products. Clearly, the newly synthesized products were alpha (1-6)-linked mannooligosaccharides, and there was no synthesis of (1right-arrow2)Man branches. However, the relationship of these mannooligosaccharides to the present linear LM, or LM, or LAM proper is not clear. Whether these mannooligosaccharides were lipid-linked or even reducing is also not clear. However, it would seem from earlier work (49) that at least one of these is an (alpha 1-6)Man-linked eicosaccharide, non-reducing, and hence with no apparent direct relationship to LM or LAM. These mannooligosaccharides may be natural autolysis products of linear LM; mycobacteria contain a non-lipidated, reducing mannan (50), apparently identical to the mannan within LM. Regardless of any relationship, the present body of work and that of Yokayama and Ballou (41) demonstrate that the alpha (1right-arrow6)-linked Man backbone of the mannooligosaccharides, the linear LM, and presumably mature LM and LAM arise in C50-P-Man, and the (alpha 1-2)-linked branches of PIMan5, LM, LAM, and other mannooligosaccharides arise elsewhere. Present tentative evidence indicates that GDP-Man is the source of these external Man residues as in the case of the PIMan2s. Further incubation of GDP-[14C]Man-pulsed, washed and chased membranes with fresh GDP-[14C]Man resulted in modest incorporation of [14C]Man into products indistinguishable from LM and LAM (results not shown). Thus, the combined evidence from the past and the present points to the pathway outlined in Fig. 7 for the biosynthesis of the native branched LM. Separately, we have shown that the arabinan of LAM arises by the single transfer of Araf units from C50-P-Araf (47) to an endogenous acceptor (18). Whether or not this acceptor is endogenous LM or linear LM has not been established.


Fig. 7. Postulated pathways for the biosynthesis of the PIMs, linear LM, native LM, and LAM of mycobacteria.
[View Larger Version of this Image (13K GIF file)]


FOOTNOTES

*   This work was supported by Grant AI-18357 from the NIAID, National Institutes of Health, and a Cooperative Agreement AI-38087 from the National Cooperative Drug Discovery Groups for the Treatment of Opportunistic Infections Program, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Tel.: 970-491-6700; Fax: 970-491-1815; E-mail: pbrennan@vines{at}colostate.edu.
1   The abbreviations used are: LAM, lipoarabinomannan; LM, lipomannan; PI, phosphatidylinositol; PIMs/PIMans, phosphatidylinositol mannosides; PIMan2, phosphatidylinositol dimannoside; Ac1PIMan2, monoacylphosphatidylinositol dimannoside; Ac2PIMan2, diacylphosphatidylinositol dimannoside; PIMan5, phosphatidylinositol pentamannoside; Ino, inositol; Dol-P-Man, dolichyl-P-Man; MOPS, 4-morpholinepropanesulfonic acid.

ACKNOWLEDGEMENTS

We thank Katarína Mikusová for technical help in aspects of this work and Marilyn Hein for preparing the manuscript.


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