Variation in Mannose-capped Terminal Arabinan Motifs of Lipoarabinomannans from Clinical Isolates of Mycobacterium tuberculosis and Mycobacterium avium Complex*

Kay-Hooi KhooDagger , Jyh-Bing TangDagger , and Delphi Chatterjee§

From the § Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523 and the Dagger  Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

Received for publication, May 10, 2000, and in revised form, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The unique terminal arabinan motifs of mycobacterial lipoarabinomannan (LAM), which are mannose-capped to different extents, probably constitute the single most important structural entity engaged in receptor binding and subsequent immunopathogenesis. We have developed a concerted approach of endoarabinanase digestion coupled with chromatography and mass spectrometry analysis to rapidly identify and quantitatively map the complement of such terminal units among the clinical isolates of different virulence and drug resistance profiles. In comparison with LAM from laboratory strains of Mycobacterium tuberculosis, an ethambutol (Emb) resistant clinical isolate was shown to have a significantly higher proportion of nonmannose capped arabinan termini. More drastically, the mannose capping was completely inhibited when an Emb-susceptible strain was grown in the presence of subminimal inhibitory concentration of Emb. Both cases resulted in an increase of arabinose to mannose ratio in the overall glycosyl composition of LAM. Emb, therefore, not only could affect the complete elaboration of the arabinan as found previously for LAM from Mycobacterium smegmatis resistant mutant but also could inhibit the extent of mannose capping and hence its associated biological functions in M. tuberculosis. Unexpectedly, an intrinsically Emb-resistant Mycobacterium avium isolate of smooth transparent colony morphology was found to have most of its arabinan termini capped with a single mannose residue instead of the more common dimannoside as established for LAM from M. tuberculosis. This is the first report on the LAM structure from M. avium complex, an increasingly important opportunistic infectious agent afflicting AIDS patients.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

One of the most prominent macromolecular entities of all mycobacterial cell walls is the arabinan, a common constituent of both the arabinogalactan (AG)1 and lipoarabinomannan (LAM) (1). In the chemical setting of mycolylarabinogalactan-peptidoglycan complex, AG forms an integral part of the cell wall proper, whereas the wall associated LAM has been implicated as a key molecule involved in the virulence and immunopathogenesis of tuberculosis (2). The arabinan of LAM is attached to a mannan core that extends from a phosphatidylinositol mannoside anchor at the reducing end. Of major biological significance is the degree and chemical nature of capping functions and other substituents on the arabinan. It was demonstrated that the arabinan chains of LAMs from Mycobacterium tuberculosis, as well as the attenuated Mycobacterium bovis BCG vaccine strain and Mycobacterium leprae, the etiological agent of leprosy, are mannose-capped to varying degrees (40-70%). In contrast, the majority of the arabinan termini from the rapidly growing, noninfective Mycobacterium smegmatis are uncapped, whereas a minor portion terminates with inositol phosphate (3-6). The mannose-capped LAM isolated from M. tuberculosis is generally less effective at stimulating macrophages than the non-mannose-capped LAM from fast growing strains (2), which may owe its potency in inducing cytokine production entirely to its inositol phosphate capping (5, 6). The presence of mannose caps on LAM has also been implicated to be essential in mediating the binding to human macrophage mannose receptors (7-9) and lung surfactant proteins (10, 11), thereby affecting the adherence of whole bacterium to macrophage host and its subsequent route of entry.

The arabinan represents a valid target for antimycobacterial drug because disruption of its biosynthesis would lead to dual dismantling of both the mycolyl-AG-peptidoglycan cell wall complex and LAM. Ethambutol (Emb), an effective antimycobacterial drug known to inhibit the biogenesis of the cell wall components, is now understood to act by inhibiting the arabinan biosynthesis (12-14), but its precise site or mechanism of action is still unknown. Interestingly, an Emb-resistant mutant derived from M. smegmatis apparently made normal cell wall AG but truncated LAM of smaller size in an Emb dose-dependent manner when grown in the presence of this drug (15). It is not known whether this phenomenon of truncation extends to mannose-capped LAMs from M. tuberculosis, which has a very similar underlying arabinan structure. Specifically, it is of interest to know whether Emb inhibition of arabinan synthesis would result in concomitant reduction in the extent of mannose capping on prematurely terminated arabinan chains, thereby compromising their biological activities.

LAM is a notoriously difficult biomolecule for precise structure and function definition because of its size, extreme heterogeneity, and the chemical nature of the arabinan. Further, it is not known whether our current understanding based on LAM samples from well established laboratory strains, such as M. tuberculosis H37Ra and H37Rv or M. bovis BCG and M. smegmatis, truly reflects in vivo scenario of infection. At present, virtually nothing is known about the branching pattern and capping function of LAMs from drug-resistant clinical isolates that are difficult to obtain in large quantities. Given the particular biological relevance of the terminal arabinan structures, a rapid and high sensitivity method of defining their relative profiles is imperative to further understanding of the consequence of drug action and resistance. Toward this end, our own crude endoarabinanase preparation (16) provides the only presently available tool to release intact the variably capped terminal oligoarabinosyl motifs from LAM (5, 17) without resorting to chemical cleavage methods that would otherwise degrade the labile arabinofuranosyl bonds.

We demonstrated in this paper that endoarabinanase digestion of intact LAM followed by direct high pH anion exchange chromatography (HPAEC) and mass spectrometry (MS) analysis can be a quick and effective way to assess the branching nature and capping functions of the arabinan motifs. For more detailed characterization, the enzymatically released oligomers can be fluorescently labeled at the reducing end and fractionated on normal phase HPLC. The liquid chromatography based analysis afforded a useful quantitative map of various digested products, whereas MS analysis allowed firm identification based on molecular mass, along with any novel substituents. These two approaches are therefore highly complementary and were used here to gain an insight into the arabinan framework of each LAM under investigation. We provided the first pictures of the native construct of the arabinan terminal motifs in LAMs from clinical isolates of M. tuberculosis and M. avium complex and highlighted their substantial differences in the nature of mannose capping. We also demonstrated, for the first time, that mannose capping on LAM from the laboratory M. tuberculosis strain can be completely abolished when grown in the presence of Emb at a concentration that would be ineffective against the drug resistant clinical isolates.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemical Reagents-- All chemical reagents were of highest grade from Aldrich/Fluka unless otherwise specified. Milli-Q® water was used for all chemical reactions and also for isolation of lipoglycans and column chromatography.

Growth Conditions of Mycobacteria Isolates-- The Emb-resistant M. tuberculosis isolate CSU19 (from Korea) was propagated on 7H11 plates. Isolated colonies were then grown in 5 ml of glycerol-alanine salts broth for 2-3 weeks at 37 °C on a shaker. Large scale cultures were initiated by using 100 µl of broth culture to inoculate 5 ml of fresh glycerol-alanine salts followed by 1 ml of culture into 400 ml of broth. M. tuberculosis, H37Ra, was grown in glycerol-alanine salts broth in the presence of 0.5 µg of Emb. M. avium, strain 2151, was grown in Middlebrook 7H11 agar plates with oleic acid/albumin/dextrose/catalase enrichment supplement to maintain stable colony morphotypes (18). The smooth transparent (SmT) type was used in this study.

Extraction of LAM-- Wet cells (~10 g) were disrupted mechanically using either a French Pressure Cell or a Bead Beater as required for over 90% breakage of cells. The initial breaking buffer for disruption contained Triton X-114 (Pierce) in phosphate-buffered saline in the presence of a protease inhibitor mixture (pepstatin, phenylmethylsulfonyl fluoride, and leupeptin) and DNase, RNase. The suspension after cell breakage was centrifuged at 27,000 × g for 1 h at 4 °C. The cell pellet was resuspended in breaking buffer and rocked overnight at 4 °C. The cell pellet was resuspended in breaking buffer and centrifuged as before. The combined supernatant was placed at 37 °C to initiate biphase (19) and centrifuged at 12,000 × g for 15 min at room temperature. The aqueous layer and the detergent layer were back extracted a second time, the detergent layer was separated, and the combined detergent layer was precipitated with 9 volumes of acetone. The acetone precipitate was collected, dried, and partitioned between phenol and water. The aqueous layer that contained majority of cellular LAM and lipomannan was dried and used for further purification (3).

Size Fractionation and SDS-PAGE-- Sephacryl S-200 (Amersham Pharmacia Biotech) column was prepared by using methods as described before (15). The gel was suspended in a buffer containing 0.2 M NaCl, 0.25% deoxycholate, 1 mM EDTA, 0.02% sodium azide, and 10 mM Tris, pH 8.0 (3). SDS-PAGE was used to monitor the elution profile of fractions containing LAM and lipomannan, which were then pooled accordingly and dialyzed at 37 °C without detergent followed by water for several days. LAM/lipomannan thus recovered was reanalyzed by SDS-PAGE to check for purity prior to detailed analysis. SDS-PAGE and silver-periodic acid Schiff staining were performed essentially as described (4). Sample concentrations were maintained at 1-2 µg in 10 µl of sample buffer.

Digestion with Endoarabinanase and Subsequent Analyses-- Selective growth of a soil microorganism, Cellulomonas gelida, from which the endoarabinanase was purified, has been described (16). The crude enzyme preparation (containing both endo and exo glycosidases) was fractionated on a Q-Sepharose column, and the eluents were examined for their ability to digest AG. The active fractions were then applied to a Cibacron Blue 3GA Affinity column (Sigma) and eluted with a step gradient of 0.2-1 M NaCl. The fractions were then desalted using Centricon, and enzyme activity was remonitored. The suitable fractions were pooled and dialyzed overnight against 100 mM Tris/HCl. After a final concentration of the enzyme dialysate in 20% polyethylene glycol, the concentrated solution is stored and maintained at 4 °C in the Tris buffer. The activity of the enzyme reduced significantly when the initial dialysis was carried out in water as opposed to the Tris/HCl.

To ensure a directly comparable and consistent result, all LAM samples (10 µg for initial mapping; 50 µg for enzyme digestion and subsequent analysis by 2-AB tagging and HPLC/MS analysis) were treated with same amount of the enzyme preparation for 4 h at 37 °C. An aliquot from the digestion product mixtures that contained both mannan core and the released oligoarabinosides were analyzed directly by HPAEC. Analytical HPAEC was performed on a Dionex LC system fitted with a Dionex Carbopac PA-1 column (15), and the oligoarabinosides were detected with a pulse amperometric detector (PAD-II). The remaining sample was frozen and dried down in SpeedVac (Savant) for subsequent MS analysis. For larger preparation, the reaction mixtures were boiled briefly to terminate the digestions and desalted directly to remove the Tris salts and the mannan core, as described in the "2-Ab Labeling and Normal Phase HPLC".

2-AB Labeling and Normal Phase HPLC-- Endoarabinanase released oligomers were first separated from resistant LAM core and desalted simultaneously with a HyperSep Hypercarb poros graphitized carbon (PGC) column (ThermoQuest). Samples were dissolved in water and loaded onto the cartridge. After washing with 3 ml of water, the oligosaccharides were eluted with 3 ml of 25% acetonitrile, whereas the mannan core was recovered in the subsequent 3-ml fraction of 50% acetonitrile containing 0.1% trifluoroacetic acid. The purified oligosaccharides were then incubated with 5 µl of the labeling reagents (0.35 M 2-aminobenzamide and 1 M sodium cyanoborohydride in 70:30 dimethyl sulfoxide:glacial acetic acid) at 65 °C for 2 h, after which the reaction mixtures were directly spotted onto a GlycoClean S cartridge (Glyko). After 15 min, the disc was washed sequentially with 1 ml of acetonitrile, 5× 1 ml of 96% acetonitrile, and finally the 2-AB-labeled glycans were recovered by eluting with 3× 0.5 ml of water. Normal phase HPLC was performed on a Hewlett Packard 1100 series LC system fitted with a thermostat column compartment and a PalPak type N column (Takara, 4.6 × 250 mm). Solvents A and B are acetonitrile and 250 mM aqueous formic acid, respectively. 2-AB-labeled glycans were size fractionated with a gradient of t = 0, 20% B, t = 40 min, 40% B, t = 65 min, 85% B, at a flow rate of 0.5 ml/min, and detected by fluorescence with excitation and emission wavelengths set at 310 and 410 nm, respectively.

Chemical Derivatization and MS Analysis-- Samples were permethylated using the NaOH/dimethyl sulfoxide slurry method as described by Dell et al. (20). FAB-MS and ESI-MS analyses were performed on an Autospec orthogonal acceleration-time of flight mass spectrometer (Micromass, Manchester, UK), and fitted with a magnet bypass flight tube and interchangeable FAB and ESI source assemblies. For FAB-MS experiments, the fitted cesium ion gun was operated at 26 kV, and the source accelerating voltage was operated at 8 kV. The permethyl derivatives were redissolved in CH3OH for loading onto the probe tip coated with m-nitrobenzylalcohol as matrix. Collision-induced dissociation FAB-MS/MS was performed by introducing argon gas to the collision cell to a reading of ~1.2 × 10-6 millibars on the time of flight ion gauge, at a lab frame collision energy of 800 eV and a push-out frequency of 56 kHz for orthogonal sampling. A 1-s integration time per spectrum was chosen for the time of flight analyzer with a 0.1-s interscan delay. Individual spectra were summed for data processing. For ESI-MS, the accelerating voltage was maintained at 4 kV. The permethyl derivatives were dissolved in CH3OH, and 10-µl aliquots were injected through a Rheodyne loop into the mobile phase (methanol/water/acetic acid, 50:50:1, v/v/v), delivered at a flow rate of 5 µl/min into the ESI source by a syringe pump. Underivatized native samples analyzed by ESI-MS were first subjected to clean up using the PGC cartridge described to remove salts and mannan core.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our previous work has shown that the alpha 1-5 arabinan chains of LAM can be digested by endoarabinanase from C. gelida into di-arabinosides, Arafalpha 1right-arrow5Araf (Ara2). However, the characteristic chain termination with a Arafbeta 1right-arrow2Araf renders the adjacent Arafalpha 1right-arrow5Araf unit resistant to endoarabinanase digestion, leading instead to the recovery of variably capped nonreducing terminal tetra- (Ara4) and hexa-arabinosyl (Ara6) units (Fig. 1). Thus, for an extensively mannose-capped LAM such as that from M. tuberculosis Erdman, the major products have been isolated by size fractionation on a Bio-Gel P-6 column and structurally characterized as Ara2, Man2Ara4, and Man4Ara6 because the most abundant cap on each arabinan terminus is Manalpha 1right-arrow2Man (17). On the other hand, endoarabinanase digestion of non mannose-capped LAM gave Ara2, Ara4, and Ara6 as major products (5).



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Fig. 1.   The nonreducing terminal oligoarabinosyl structural motifs of LAM. The formation of the characteristic mannose-capped linear Ara4 and branched Ara6 motifs (boxed) by endoarabinanase cleavages are indicated by vertical down arrows at site I. beta 2-Ara chain termination with and without additional alpha 3-branching will render site II resistant to cleavage. The remaining of the arabinan chains are mostly digested into Ara2. Heterogeneity in digestion products mainly reflects the heterogeneity in the size of the mannose cap on each terminus.

HPAEC Mapping of Endoarabinanase Digestion Products-- LAM from M. tuberculosis H37Rv (RvLAM) has previously been shown to be very similar in structure to that from Erdman, although the relevant ManxAran terminal units have not been isolated intact. To rapidly map these nonreducing terminal motifs, the endoarabinanase digested RvLAM was analyzed directly by HPAEC, and the profile obtained (Fig. 2A) was compared against that from the same sample further treated with alpha -mannosidase to remove the caps (Fig. 2B). The latter was found to give a simpler profile with three major peaks, which eluted at retention times corresponding to Ara2, Ara4, and Ara6 standards (peaks 1-3 in Fig. 2B). Thus, the major endoarabinanase digestion products of RvLAM as observed in Fig. 2A could be assigned as mannose-capped Ara4 and Ara6. This was further corroborated when Man2Ara4 and Man4Ara6 isolated from a Bio-Gel P-6 column and identified by MS analysis were found to elute at positions corresponding to peaks 4 and 5, respectively (data not shown). The major mannose-capped Ara4/Ara6 motifs, namely Man2Ara4 and Man4Ara6, could therefore be adequately resolved from the uncapped Ara4 and Ara6, respectively. Interestingly, mannose substitution at 5-OH of the beta -Ara results in earlier retention time of the oligoarabinosyl motifs, indicating that separation by HPAEC is both structure- and size-dependent. Direct analysis by HPAEC of the endoarabinanase digested LAM will thus give a rapid indication of whether the LAM under investigation does terminate with the Ara4/Ara6 motifs and whether it is characteristically capped with a dimannoside.



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Fig. 2.   HPAEC profiles of the endoarabinanase digestion products of RvLAM (A) and after alpha -mannosidase digestion (B). Peak 1, Ara2; peak 2, Ara4; peak 3, Ara6; peak 4, Man2Ara4; peak 5, Man4Ara6. The huge peak at around 6 min in B is mannose residue released by alpha -mannosidase.

MS Profiling of Endoarabinanase Digestion Products-- To provide a more definitive identification of the oligoarabinosyl motifs, the digestion product mixture of RvLAM was dried down directly, permethylated, chloroform extracted to remove most of the salt buffers used, and analyzed by MS. Rather unexpectedly, the m/z values of the major molecular ions did not correspond to the calculated molecular masses of the expected molecular species. Instead, they could be assigned as incorporating a Tris moiety at the reducing end. Thus, the expected [M+Na]+ molecular ions for Man2Ara4 and Man4Ara6 are only observed as minor signals at m/z 1117 and 1845, respectively, whereas the corresponding ones assigned as incorporating a Tris adduct afforded strong M+ signals at m/z 1254 and 1982 (Fig. 3A). Other Tris-incorporating molecular ion signals observed include Ara2, Ara4, Ara6, Man3Ara4, Man2Ara6, and Man5Ara6 at m/z 526, 846, 1166, 1458, 1574, and 2186, respectively. The assigned molecular composition clearly demonstrated that most of the heterogeneity associated with the digestion products could be attributed to the mannose capping, which ranges from Man1 to Man3 or no capping on each terminus. MS/MS analysis of the Man2Ara4 peak at m/z 1254 yielded a series of fragment ions at m/z 380, 540, 700, 860, and 1064, corresponding to reducing terminal ring cleavage ions Ara1 + Tris, Ara2 + Tris, Ara3 + Tris, Ara4 + Tris, and Man1Ara4 + Tris, respectively (Fig. 3B). Together with additional fragment ions, the MS/MS data supported a linear Man2Ara4 structure as drawn (Fig. 3B) and also indicated a covalent incorporation of Tris at the reducing terminus.



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Fig. 3.   MS analysis of the endoarabinanase-digested RvLAM. A, the reaction mixtures were dried down and permethylated directly for FAB-MS analysis. B, FAB-MS/MS analysis of the parent ion at m/z 1254 from A with a schematic drawing showing the probable cleavage pathways. The insets show the ESI-mass spectra for the native samples, focusing on the two major components i.e. Man2Ara4 and Man4Ara6, to show the effects of sample processing on Tris incorporation. Samples which were dried down first (inset C) have a high propensity to incorporate a Tris molecule coming from the concentrated enzyme buffer. Signals at m/z 996, 1584, and 1746 correspond to [M+Na]+ of Tris-incorporating Man2Ara4, Man4Ara6, and Man5Ara6, respectively. The adducts could be avoided if the reaction mixtures were first passed through a PGC column to remove the salts prior to drying down and analyzed by MS (inset D). The [[M+Na]+ signals were shifted to 103 units lower as compared with (inset C). The schematic drawing showing Tris incorporation at the reducing end only serves to illustrate how it affects the m/z values observed. The exact chemical structures have not been proven, and Tris may also be transglycosylated through one of the OH groups instead of the amine.

Further investigation demonstrated that Tris incorporation could be largely avoided if the enzyme digested mixture was first passed through a PGC column for salt removal instead of drying down directly, which probably concentrated the Tris buffer while the enzyme was still active (Fig. 3, insets C and D). Thus, for a specific purpose when it is imperative to maintain the free reducing end or for a large scale preparation, the digestion products could be desalted directly using a PGC or gel filtration column, which also render the sample clean enough for direct ESI-MS analysis (Fig. 3D). However, when sample amount is limited, permethylation of the dried down digestion products is preferable because it avoids the additional step of work up and improves sensitivity of detection by MS, probably because of creation of a fixed charge on the quaternary ammonium during permethylation. Thus, although it is yet unclear how the released oligoarabinosyl motifs could be transglycosylated with Tris present in the enzyme buffer, its stable incorporation clearly did not interfere with MS analysis. In fact, the dried down sample was equally amenable to further clean up with PGC column for ESI-MS analysis (Fig. 3C). The m/z values of the observed [M+Na]+ molecular ion signals for the Tris incorporating native samples further validated our assignment of the molecular compositions of the various oligoarabinosyl motifs.

Emb and Mannose Capping in M. tuberculosis LAM-- As shown previously, LAM from the avirulent H37Ra (RaLAM) was very similar to that from the virulent Erdman or H37Rv strain in that it was also mannose-capped. The HPAEC and MS profiles of endoarabinanase digested RaLAM (Fig. 4A) were indeed similar to those of RvLAM, with Man2Ara4 (m/z 1254) and Man4Ara6 (m/z 1982) being the dominant products. Other molecular ions afforded by ESI-MS analysis of the permethyl derivatives attested to the variation in mannose capping, corresponding to Ara4 (m/z 846) with shorter (Man1Ara4, m/z 1050) and longer (Man3Ara4, m/z 1458) mannose caps and Ara6 with a total of 0, 2, 3, 5, and 6 Man residues at m/z 1166, 1574, 1778, 2186, and 2390 respectively. In addition, Ara5 (m/z 1006) and Man2Ara5 (m/z 1414) were also detected.



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Fig. 4.   HPAEC and MS analysis of the endoarabinanase-digested RaLAM. A, ESI-mass spectrum of the permethyl derivatives of digested RaLAM from H37Ra grown under normal conditions. The inset shows the direct HPAEC profile of the digestion products. B, ESI-mass spectrum of the permethyl derivatives of digested RaLAMemb from H37Ra grown in the presence of 0.5 µg of Emb. The inset shows the direct HPAEC profile of the digestion products (upper panel) as compared with that of normal RaLAM further treated with alpha -mannosidase (lower panel). The HPAEC peaks 1-5 are as assigned for RvLAM (Fig. 2). The identity of the late eluting peak in the HPAEC profile of RaLAMemb is unknown at present but was unaffected by further alpha -mannosidase treatment.

Interestingly, the RaLAM derived from H37Ra propagated and maintained in an Emb concentration of 0.5 µg/ml (minimal inhibitory concentration = 1 µg/ml) (RaLAMemb) afforded a HPAEC profile that resembled that of alpha -mannosidase-treated samples, namely, instead of the ManxAran peaks, RaLAMemb yielded mainly the noncapped Ara4 and Ara6 (Fig. 4B, inset). In comparison with alpha -mannosidase-treated normal RaLAM, the amount of Ara4 and Ara6 terminal motifs were clearly reduced relative to Ara2, indicating that a larger proportion of the arabinan of RaLAMemb terminated prematurely, concomitant with loss of mannose capping. In support of the HPAEC analysis, glycosyl composition analysis showed that the Ara:Man ratio for RaLAMemb was 2.03, whereas that of a "normal" RaLAM was 0.95. ESI-MS analysis of the permethyl derivatives of the digestion products further confirmed that the major oligoarabinosyl motifs detected were devoid of Man capping (Fig. 4B). In addition to Ara2, Ara4, and Ara6, significant amounts of Ara7 (m/z 1326), Ara8 (m/z 1486), and larger oligoarabinosides (Ara9 up to at least Ara14) were also detected. Together, the data clearly showed that application of 0.5 µg/ml of Emb to the M. tuberculosis, H37Ra culture has drastically led to complete inhibition of mannose capping, concomitant with significant alteration in the underlying arabinan framework.

Mannose Capping in Drug-resistant Clinical Isolate of M. tuberculosis-- The LAM isolated from Emb-resistant CSU19 clinical isolate (TbLAM) grown without any Emb showed endoarabinanase digestion/HPAEC and MS profiles (Fig. 5) distinct from that of RvLAM. TbLAM was deduced to be more heterogeneous in the spread of size, ranging from full size to severely truncated even in the absence of Emb (results not shown). Its average Ara:Man molar ratio of 1.44 is somewhat intermediate between a normal mannose-capped LAM (~1.0) and the noncapped RaLAMemb (2.03). The HPAEC profile of its endoarabinanase digestion products indicated that peaks corresponding to Man2Ara4 (peak 4) and Ara4 (peak 2) were almost of equal abundance, whereas the Man4Ara6 peak (peak 5) was reduced in relative abundance (Fig. 5, inset). Interestingly, after alpha -mannosidase digestion, the relative ratio of the resultant Ara2, Ara4, and Ara6 peaks was comparable with that of RvLAM and RaLAM, indicating that the difference was mainly due to mannose capping. FAB-MS analysis of the TbLAM digestion products corroborated this observation (Fig. 5). Qualitatively, all peaks observed for RvLAM were also present in TbLAM but with significant changes in relative intensities. Most obvious was the enhanced abundance of molecular ions corresponding to Ara4 (m/z 846) relative to Man2Ara4 (m/z 1254), whereas Man4Ara6 (m/z 1982) was significantly reduced, and Man5Ara6 was hardly detected.



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Fig. 5.   HPAEC and MS analysis of the endoarabinanase digested TbLAM. The reaction mixtures were dried down and permethylated directly for FAB-MS analysis. The inset shows the direct HPAEC profile of the digestion products (upper panel) as compared against that of the same sample further treated with alpha -mannosidase (lower panel). The HPAEC peaks 1-5 are as assigned for RvLAM (Fig. 2).

To further confirm the observed changes in the relative amounts of the ManxAran oligomers, the endoarabinanase digestion products were subjected to fluorescent tagging with 2-AB after removal of the resistant lipomannan core and other salt contaminant including Tris, which would affect the reducing end tagging. The 2-AB-tagged products were then size fractionated by normal phase HPLC on an amine column. Each major peak detected was collected and analyzed by MS to confirm its identity by molecular mass (Table I). In comparison with similarly analyzed RaLAM (Fig. 6A), the HPLC profile of TbLAM (Fig. 6C) clearly showed that the peak corresponding to Ara4 (peak 2) was much enhanced in abundance relative to Man2Ara4 (peak 6) and that both Man4Ara6 (peak 8) and Man5Ara6 (peak 9) were reduced in abundance. MS analysis also showed that peak 7 for RaLAM was Man3Ara4, whereas the corresponding peak 7' for TbLAM was found to contain mostly Man2Ara6. For RaLAMemb (Fig. 6B), in agreement with the HPAEC and MS data (Fig. 4), all mannose-capped oligoarabinoside peaks (peaks 6-9) were absent. Instead, Ara7 (peak 4) and Ara8 (peak 5) were identified as additional peaks not found in RaLAM or TbLAM.


                              
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Table I
Identification and quantitation of 2-AB-tagged ManxAran oligomers produced by endoarabinanase digested LAM from M. tuberculosis and fractionated by normal phase HPLC (Fig. 6)



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Fig. 6.   Normal phase HPLC profiles of the 2-AB derivatives of the endoarabinanase digested products of RaLAM (A), RaLAMemb (B), and TbLAM (C). Peaks were detected by fluorescence, and those numbered 1-9 were identified by MS analysis (Table I). Because of a prior clean up of the endoarabinanase digestion products by passing through a PGC cartridge, a significant amount of Ara2 was lost from the retained fractions used for 2-AB tagging and HPLC. Peak 1 was additionally contributed by other fluorescent contaminants and therefore could not be used as a quantitative representation of Ara2.

Because the detector response of fluorescently labeled oligomers was based on the fluorophore incorporated at the reducing end of each molecule, it is relatively independent of the overall size or structure of the oligosaccharides. The HPLC profile therefore provided a more accurate representation of the relative abundance of each oligoarabinosyl motifs in comparison with the HPAEC profile, where the response factor is more structure-dependent. Integration of the areas for each major peak detected therefore provided a way to estimate their relative abundance. It was found that the molar ratio of Man2Ara4 to Ara4 for TbLAM was about 1:1 (50% capping for the linear Ara4 termini), whereas in RaLAM, the total of Man2Ara4 and Man3Ara4 relative to noncapped Ara4 was 17.6:1, or approximating 95% capping. For the branched Ara6 termini, the total of Man4Ara6 and Man5Ara6 relative to uncapped Ara6 (peak 3) in RaLAM was 2.35:1 or about 70% capping. In contrast, the total of mannose-capped Ara6 (Man2Ara6, Man4Ara6, and Man5Ara6) relative to noncapped Ara6 in TbLAM was 1.2:1 or about 55% capping. Based on these estimations, the total Ara4:Ara6 ratio (capped and uncapped) in RaLAM was ~0.74, whereas in TbLAM the ratio was 1.4. The comparative figure for RaLAMemb was 0.45. These calculated figures did not take into consideration other minor peaks or cases where mannose-capped Ara4 and Ara6 coeluted together within a single fluorescently detected peak, for example peak 7' (Fig. 6C). Nevertheless, the major conclusion derived from three independent approaches was fairly consistent. It can be seen from the HPAEC profile of alpha -mannosidase-treated samples that for RaLAM, the Ara6 peak was marginally more abundant than the Ara4 peak, whereas the reverse is true for TbLAM. For "normal" RaLAM, the Ara4 termini was highly capped (95%), whereas the degree of mannose capping in Ara6 termini was only 70%, therefore yielding a significant amount of noncapped Ara6 in the digestion products. For the Emb-resistant clinical isolate, both termini were only 50-55% mannose-capped. A slightly lower degree of branching was also observed, which gave rise to more linear Ara4 relative to Ara6.

Single Mannose Capping in Clinical Isolate of M. avium-- The HPAEC profile of digested LAM from a clinical isolate of M. avium (AvLAM) (Fig. 7B) indicated that it was rather different from the M. tuberculosis LAM described above. However, the same characteristic Ara2, Ara4, and Ara6 peaks were produced after alpha -mannosidase digestion (Fig. 7C), suggesting that the underlying arabinan motifs remain similar and that structural difference may again reside on the variation in mannose capping. Further information was derived from MS analysis (Fig. 7A) in conjunction with HPLC fractionation of the 2-AB-tagged digestion products (Fig. 7D). The single most important difference as revealed by ESI-MS analysis of the total digestion products was the dominance of Man1Ara4 (m/z 1050) with very little Man2Ara4 (m/z 1254) detected. Of Ara6-containing species, the most prominent were Man1Ara6 (m/z 1370) and Man2Ara6 (m/z 1574), although Man3Ara6 (m/z 1778), Man4Ara6 (m/z 1982), and Man5Ara6 (m/z 2186) could also be detected. This was well reflected by the normal phase HPLC profile of the 2-AB-tagged sample (Fig. 7D). The most abundant peak 3' was clearly shown by FAB-MS and MS/MS analysis to be 2-AB-tagged Man1Ara4 with a fragmentation pattern consistent with a linear Ara4 capped at the nonreducing end by a single Man (Fig. 8C). Importantly, the fragment ions at m/z 219 (Hex+) and 187 (Delta Hex+) indicated that the nonreducing terminal was a Man and not Ara, which would otherwise give prominent fragment ions at m/z 175 (Pent+) and 143 (Delta Pent+) as afforded by Ara4 (Fig. 8A) and Ara6 (Fig. 8B). These nonreducing terminal oxonium ions were characteristically very abundant, even in the daughter ion spectrum of other weaker parent ions, which did not afford any other significant fragment ions apart from m/z 355 (HO)Ara1-AB. Thus, additional nonreducing terminal ion at m/z 391 corresponding to Delta Hex-Hex+ was afforded by Man2Ara4 and indicated that it was a linear hexamer with a dimannoside at the nonreducing end (Fig. 8D). Interestingly, only fragment ions at m/z 187 and 219 were detected for Man2Ara6 (Fig. 8E) and not ions that would correspond to terminal Ara+ (m/z 175/143) or Man2+ (m/z 391). The latter ion was, however, present in the MS/MS spectrum of Man4Ara6 (Fig. 8F). It may thus be concluded that the arabinan in AvLAM terminate in the same way as either the linear Ara4 or branched Ara6 motifs. The amount of Ara2 relative to the terminal Ara4 and Ara6 was, however, not as high as in LAM from M. tuberculosis, as shown by the HPAEC profile of alpha -mannosidase-treated sample (Fig. 6C). Very little of these termini were exposed. Instead, our data suggested that most were capped with a single mannose residue, yielding Man1Ara4 and Man2Ara6 as the most abundant endoarabinanase digestion products, although Man2 cap was also found.



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Fig. 7.   Analysis of the endoarabinanase digested AvLAM. A, the reaction mixtures were dried down and permethylated directly for ESI-MS analysis. B, the direct HPAEC profile of the digestion products as compared against that of the same sample further treated with alpha -mannosidase (C). The HPAEC peaks 1-5 are as assigned for RvLAM (Fig. 2). D, the normal phase HPLC profile of the 2-AB derivatives of the digested products after a further PGC column clean up. The numbering of the peaks is as in Fig. 6. A prime is used to distinguish peaks of same retention time containing different or additional components: Peak 3', mainly Man1Ara4 with small amount of Ara6; peak 6', Man1Ara6 and Man2Ara4; peak 7', contains mainly Man2Ara6.



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Fig. 8.   FAB-MS/MS analysis of the permethyl derivatives of 2-AB-tagged Ara4 (A), Ara6 (B), Man1Ara4 (C), Man2Ara4 (D), Man2Ara6 (E), and Man4Ara6 (F) from AvLAM. For the latter two, only the low mass regions containing the diagnostic fragment ions were shown. Because of very weak parent ions, other fragment ions were either too weak or absent. Sections of the mass spectra are magnified to show the detected peaks more clearly. Signals at m/z 143, 175, 187, 219, and 391 correspond to nonreducing terminal oxonium ions as described in the text. Other ions are reducing terminal beta -cleavage or ring cleavage ions. Man0,1,2Ara4 afforded a (OH)1Ara-Ara-AB ion at m/z 515, which could not be produced from ManxAra6 because the second Ara from the 2-AB-tagged end is the branch point in the latter.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In summary, we demonstrated that direct endoarabinanase digestion followed by HPAEC analysis could rapidly give a first indication as to the presence or absence of the expected ManxAran motifs. Using the well characterized RvLAM as the standard reference, a "normal" mannose-capped LAM is expected to yield predominantly Ara2, Man2Ara4, and Man4Ara6 as the endoarabinanase digestion products. Subsequent treatment with alpha -mannosidase would confirm that both the linear Ara4 and branched Ara6 are indeed synthesized and that their relative ratios could be compared with that of RvLAM. A departure from this norm could be rapidly fingerprinted, as demonstrated with analysis of other LAMs reported here. However, because of complexity and heterogeneity of the products that would result from endoarabinanase digestion, a further confirmatory analysis such as that using MS is required, particularly to define the unknown peaks produced when analyzing a whole spectrum of LAMs from different sources.

For MS analysis, we found that permethylation followed by a simple organic phase partition was sufficient to yield good quality spectrum. Of the ionization methods tried, FAB-MS and ESI-MS both yielded comparable molecular ion profiles with the latter affording better signal to noise ratio especially for larger components. However, the relative abundance would depend on the cone voltage employed, which could be tuned to favor the high mass or low mass components. ESI-MS could also be used to analyze native samples, but in general, all samples for ESI-MS should be desalted prior to injection. The presence of Tris in the incubation buffer could result in its covalent incorporation into the digestion product if the mixtures were concentrated and dried down directly prior to further processing. It is as yet unclear what is the mechanism or the degree of incorporation that appeared to vary from batch to batch. However, as demonstrated here, this did not interfere but actually facilitated the MS analysis. By creating a fixed charge through permethylation, the various Tris-containing species ionized as M+ instead of the usual M+Na+ molecular ions normally afforded by ESI-MS and FAB-MS when the matrix was not doped with acid. Collision-induced dissociation MS/MS analysis of the permethyl derivatives of Tris-containing species did not result in dissociation of the Tris moiety but yielded a series of reducing terminal fragment ions retaining the Tris and hence the charge. Additional ESI-MS analyses of native samples further showed that Tris incorporation was not induced by permethylation. It should be noted that probably only a fraction of each of the digestion products actually had Tris covalently incorporated at the reducing end but nevertheless ionized preferably over the non-Tris containing ones and hence appeared to be the dominant molecular ions in both FAB- and ESI-mass spectra. We further found that the Tris-containing species could be reduced in the same way the non-Tris containing species got reductively aminated with 2-aminobenzamide. After reducing end tagging, both 2-AB-tagged and Tris-tagged species could be detected directly or after permethylation by MS analysis. To avoid formation of Tris-tagged species, it is necessary to first desalt the digestion mixtures prior to drying down, which would minimize the Tris incorporation and leave the reducing end free for maximum and quantitative tagging with 2-AB.

Although the quantitative yield of 2-AB tagging using the well established protocols for glycans from glycoproteins (21) has not been rigorously proven for the oligoarabinosides, the HPLC profile of the resultant products based on fluorescence detection agreed well with that by HPAEC PAD response. The 2-AB-tagged oligomers can be fractionated by normal phase HPLC and conveniently collected for further analysis without any desalting step because only volatile buffer system was used. In comparison with HPAEC using the Dionex LC system, we achieved a better separation of the noncapped Aran peaks from those that were mannose-capped. In HPAEC, Ara4 and Man2Ara4 eluted close to one another, whereas Man1Ara4 eluted in between. In the HPLC of 2-AB-tagged sample, Man1Ara4 was not well resolved from Ara6. LC mapping by either mode is therefore insufficient but, when complemented by MS screening, proves to be highly effective for quantitative comparison of the various structural motifs. Although the 2-AB derivatives could be analyzed native by MS, we prefer the permethyl derivatives for better sensitivity and to facilitate interpretation of MS-MS data.

Using these approaches, we demonstrated that LAMs from Emb-resistant M. tuberculosis and M. avium clinical isolates showed a significant reduction in Man2-capped Ara4 and Man4-capped Ara6 motifs. In the case of TbLAM, the preferred cap is still Man2, but more arabinan termini, especially Ara4, were exposed. In M. avium LAM, the preferred cap appeared to be Man1. Consequently, Man1Ara4 and Man2Ara6 appeared as the major digestion products. However, up to Man3Ara4 and Man4Ara6 could be detected. In contrast, no mannose capping could be detected in LAM obtained from the H37Ra strain grown in the presence of Emb. All LAMs do maintain the basic framework of the arabinan structural motifs, as revealed by alpha -mannosidase removal of the mannose caps.

The inhibitory effect of Emb on the biosynthesis of arabinan have been demonstrated at several levels (12, 14, 22), whereas the phenomenon of truncated LAM was reported for the fast growing, Emb-resistant M. smegmatis grown in the presence of Emb (15). Our studies on LAMs from M. tuberculosis clinical isolates as reported here represent the first of a series of work initiated to ascertain the relationship, if any, between their arabinan structure and Emb resistance. Our results clearly showed that variation in mannose capping exists among different mycobacteria isolates that can be further affected by the application of Emb during growth. Because mannose capping would only be effected on a properly beta -Ara terminated chain, it can be envisaged that inhibited elongation and proper termination of arabinan chains would also inhibit subsequent mannose capping. It remains to be established whether such reduction in mannose capping is wide spread among M. tuberculosis isolates and how it may affect the dissemination and survival of this intracellular pathogen of macrophage, not least in the context of mediating binding to macrophage mannose receptor or lung surfactant proteins (9, 10, 23). Even more intriguing is how it may translate into Emb resistance. The reduction in mannose capping in the presence of Emb argues that Emb does perturb the machinery for a complete assembly of the arabinan in LAM. It follows that an Emb-resistant strain must therefore be able to somehow encounter this effect by altering one or more parts of this machinery that could conceivably lead indirectly to alteration in mannose capping.

The mechanism of drug resistance in M. avium complex is further complicated by the additional presence of the strain-specific glycopeptidolipids (GPL) and different colony morphotypes when grown on solid media, namely, smooth opaque (SmO), SmT, and rough. The search for the basis of morphological variations in M. avium has been met with little enlightenment. It has been shown that the rough strains are devoid of GPLs, whereas SmT and SmO forms express these GPLs in similar quantities. In addition, SmT morphotype is usually more virulent and shows more innate drug resistance than the SmO type. Although the GPLs of M. avium have been studied extensively and have been implicated in drug resistance and impermeability of the organism, structural or functional studies on LAM have never been undertaken prior to this study. It is not known whether the predominance of single Man as the preferred cap in LAM from the strain 2151 SmT investigated here is common among different M. avium strains of distinct colony morphotypes and drug resistance profiles. From the limited data obtained thus far, it would seem that different mycobacteria species evolve different resistance mechanism in neutralizing the inhibitory effect of Emb on the assembly of their quintessential arabinan. Variation in mannose capping may be a manifestation or consequence of such adaptation but with important implications on their presumed biological roles.


    ACKNOWLEDGEMENTS

We thank Israel Muro and Jerry Tews for growth of the M. tuberculosis, H37Ra, and CSU 19 strains and isolation and purification of LAM described in this manuscript. We also thank Dr. Yi Xin for providing the endoarabinanase arabinan-degrading enzyme.


    FOOTNOTES

* This work was supported by Grants AI-37139 and TW 00943 from the NIAID, National Institutes of Health (to D. C.) and in part by Academia Sinica and National Science Council (Taipei, Taiwan) Grant NSC 89-2311-B-001-090 (to K. H. K.).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.: 970-491-7495; Fax: 970-491-1815; E-mail: delphi@lamar.colostate.edu.

Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M004010200


    ABBREVIATIONS

The abbreviations used are: AG, arabinogalactan; 2-AB, 2-aminobenzamide; Emb, ethambutol; ESI, electrospray ionization; FAB, fast atom bombardment; GPL, glycopeptidolipid; HPAEC, high pH anion exchange chromatography; LAM, lipoarabinomannan; MS, mass spectrometry; PGC, poros graphitized carbon; SmO, smooth opaque; SmT, smooth transparent; AvLAM, LAM from M. avium 2151; RaLAM, LAM from M. tuberculosis H37Ra; RvLAM, LAM from M. tuberculosis H37Rv; RaLAMemb, LAM from M. tuberculosis H37Ra grown in the presence of Emb; TbLAM, LAM from M. tuberculosis CSU19; HPLC, high pressure liquid chromatography.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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