Rapid structural characterization of the arabinogalactan and lipoarabinomannan in live mycobacterial cells using 2D and 3D HR-MAS NMR: structural changes in the arabinan due to ethambutol treatment and gene mutation are observed

Robin E.B. Lee2, Wei Li2, Delphi Chatterjee3 and Richard E. Lee1,2

2 Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 847 Monroe Ave. Rm. 327, Memphis, TN 38163; 3 Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523


1 To whom correspondence should be addressed; e-mail: relee{at}utmem.edu

Received on July 21, 2004; revised on September 13, 2004; accepted on September 14, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Differences between the species...
 Effects of EMB and...
 Discussion
 Materials and methods
 References
 
Mycobacteria possess a unique, highly evolved, carbohydrate- and lipid-rich cell wall that is believed to be important for their survival in hostile environments. Until now, our understanding of mycobacterial cell wall structure has been based upon destructive isolation and fragmentation of individual cell wall components. This study describes the observation of the major cell wall structures in live, intact mycobacteria using 2D and 3D high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR). As little as 20 mg (wet weight) of [13C]-enriched cells were required to produce a whole-cell spectra in which discrete cross-peaks corresponding to specific cell wall components could be identified. The most abundant signals of the arabinogalactan (AG) and lipoarabinomannan (LAM) were assigned in the HR-MAS NMR spectra by comparing the 2D and 3D NMR whole-cell spectra with the spectra of purified cellular components. This study confirmed that the structures of the AG and LAM moieties in the cell wall of live mycobacteria are consistent with structural reports in the literature, which were obtained via degradative analysis. Most important, by using intact cells it was possible to directly demonstrate the effects of ethambutol on the mycobacterial cell wall polysaccharides, characterize the effects of embB gene knockout in the M. smegmatis {Delta}embB mutant, and observe differences in the cell wall structures of two mycobacterial species (M. bovis BCG and M. smegmatis.) Herein, we show that HR-MAS NMR is a powerful, rapid, nondestructive technique to monitor changes in the complex, carbohydrate-rich cell wall of live mycobacterial cells.

Key words: arabinogalactan / HCCH-TOCSY / HR-MAS NMR / lipoarabinomannan / mycobacteria


    Introduction
 Top
 Abstract
 Introduction
 Results
 Differences between the species...
 Effects of EMB and...
 Discussion
 Materials and methods
 References
 
Mycobacterium is a medically important bacterial genus that possesses the causative agents of tuberculosis and leprosy along with many other human opportunistic infectious agents, including M. avium, M. marinum, M. kansasi, and M. ulcerans (Rastogi et al., 2001Go). Common to all mycobacteria is the unique and highly evolved carbohydrate- and lipid-rich cell wall that is believed to play a pivotal role in survival in hostile environments, including resistance to host defense mechanisms and to many antibiotic substances (Brennan and Nikaido, 1995Go; Lee et al., 1996Go). The carbonaceous materials within the cell wall have shown potent immunogenic and adjuvant properties that have led to their clinical utilization (Ribi et al., 1976Go). The complex structure of the mycobacterial cell wall has been the focus of much research as investigators have sought to identify and characterize its structural components through isolation and degradative analysis of the individual components (Brennan, 2003Go).

The major carbohydrate components associated with the mycobacterial cell wall are believed to be mycolyl arabinogalactan (mAG); lipoarabinomannan (LAM); lipomannan (LM); arabinomannan (AM); phosphatidylinositol mannosides (PIMs); acylated trehaloses, including cord factor and other glycolipids; phthiocerol dimycocerosates; and glucans. Although the relative amounts of these components are unknown, the most abundant material is believed to be the mAG complex, a unique, lipidated polysaccharide tethered to the peptidoglycan layer that forms the inner leaflet of a pseudo-outer membrane (Besra et al., 1995Go; Daffe et al., 1993Go; Liu et al., 1995Go). Closely associated with the cell wall is the immunogenic soluble polysaccharide LAM and its related component, LM (Chatterjee and Khoo, 1998Go; Nigou et al., 1999Go). PIMs, acylated trehaloses, phthiocerol dimycocerosates, and other glycolipids are associated with both the plasma membrane and the mycolic acid lipid layer (Lee et al., 1996Go). Mycobacterial glucans have also been described as abundant extracellular metabolites (Dinadayala et al., 2004Go).

The current challenges surrounding the mycobacterial cell wall are deciphering the cell wall biosynthetic pathways; establishing gene functions, virulence mechanisms, host interaction, and drug resistance mechanisms; and understanding how the key structural components interact as a system. Investigation and understanding these areas will facilitate the development of new tuberculosis drugs and treatment strategies. Annotation of several mycobacterial genomes suggests that a significant proportion of each genome is dedicated to the assembly of the cell wall. The postgenomic era has led to the development of many new genomic tools to study these gene functions, mycobacterial virulence, and resistance mechanisms, including rapid gene knockout technologies (Sassetti et al., 2003Go). However, the classical analytical techniques that complement these genomic tools can be limiting in several aspects. Primarily, antimicrobicide treatment or disruption of cell wall biosynthesis genes may cause the organism to grow very poorly or modify the cell wall significantly (Escuyer et al., 2001Go; Zhang et al., 2003Go). Thus preparing enough cell wall material for analysis can be difficult or even prohibitive. Subsequent investigation of the full complement of cell wall components by isolation and analysis of each macromolecule separately can be tedious and time-consuming, and artifacts may be introduced into cell wall structures during purification. Finally, by analyzing individual components, important interactions between the major macromolecules may be lost.

To address these issues, we have developed methods that use high-resolutiona magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) to analyze the cell wall components of intact, live mycobacterial cells. HR-MAS NMR permits the detection of cellular metabolites that are in a gel-like state and not normally visible under standard NMR conditions. Samples spin at 2500 Hz, at the magic angle, and a Carr-Purcell-Meiboom-Gill (CPMG) spin-echo-pulse sequence is employed to average out macroscopic inhomogeneites, which would otherwise cause significant line broadening. 2D HR-MAS NMR has been applied successfully to study lipopolysaccharides of intact Campylobacter and Yokenalla bacterial cells (Broberg and Kenne, 2000Go; Jachymek et al., 1999Go; Szymanski et al., 2003Go), but as yet this technique has not been applied to Mycobacteria. It is especially appropriate for mycobacterial studies due to the complex carbohydrate/lipid nature of the cell wall.

To evaluate HR-MAS as a tool for monitoring the cell wall structure of live mycobacterial cells, we chose to concentrate on assigning the carbohydrate signals corresponding to AG and LAM in the whole-cell spectra, followed by analysis of these structures under three conditions: treatment with the antituberculosis drug ethambutol (EMB), knockout of the embB gene, and comparison of two mycobacterial species. EMB inhibits arabinan biosynthesis in the cell wall of mycobacteria and EmbB is an arabinosyltransferase believed to be one of the primary targets for EMB. A member from the slow-growing M. tuberculosis complex, M. bovis bacillus-Calmette-Guérin (BCG), and a fast-growing mycobacteria, M. smegmatis, were chosen for demonstration of structural differences between species. In addition, this study describes the first use of 3D HR-MAS NMR for the structural analysis of live bacteria, which has proven to be a very powerful technique.


    Results
 Top
 Abstract
 Introduction
 Results
 Differences between the species...
 Effects of EMB and...
 Discussion
 Materials and methods
 References
 
Method development
Initial method development included 1D 1H HR-MAS and 2D 1H-13C heteronuclear single quantum coherence (HSQC) HR-MAS NMR experiments using unlabeled cells. The 1D proton spectrum was well defined with good line shape, but many peaks were overlapping. The 2D HSQC produced a spectrum with large numbers of discrete peaks in 24 h. The technique was modified to incorporate a [13C]-labeled carbon source, which greatly enhanced the sensitivity and significantly reduced the analysis time to under 1 h. Clearly visible in this spectrum were the signals associated with the carbohydrate signals ({delta}H 6.0–3.0 {delta}C 110–60 ppm), including the prominent signals characteristic of AG and LAM, as well as the lipid moieties ({delta}H 3.0–0.0 {delta}C 60–0 ppm) (Figure 1).



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Fig. 1. 1H-13C HR-MAS HSQC NMR spectra of whole-cell M. bovis BCG ({delta} 1H, 0.7–5.4, {delta} 13C 6–132) in D2O, 295 K, 500 MHz, spinning 2500 Hz at the magic angle. Clearly visible in this spectrum are the signals associated with the lipid moieties ({delta}H 3.0–0.0, {delta}C 60–0 ppm), and the carbohydrate signals ({delta}H 6.0–3.0, {delta}C 110–60 ppm). No other signals were observed outside this region.

 
Chemical shift assignment of AG and LAM signals in the whole-cell spectra
Assignment of all the AG and LAM signals in the whole-cell spectra was a prerequisite for defining changes observed in spectra of the EMB-treated and embB gene knockout cells. Many of the chemical shift assignments of AG and LAM from several mycobacterial species and other related species have been well established by several groups (Daffe et al., 1993Go; Garton et al., 2002Go; Gibson et al. 2003aGo,bGo; Gilleron et al., 1997Go, 1999Go, 2000Go, 2003Go; Guerardel et al., 2002Go, 2003Go; Khoo et al., 2001Go; Treumann et al., 2002Go; Venisse et al., 1993Go,1995Go). The current structural models for AG and LAM from M. tuberculosis are depicted in Figures 2 and 3 (Besra et al., 1995Go; Chatterjee et al., 1992Go; Chatterjee, 1997Go; Daffe et al., 1990Go; Nigou et al., 2003Go; Vercellone et al., 1998Go). However, the complex nature of the whole-cell NMR spectra made precise assignment based on assignments in the literature difficult due to subtle differences in chemical shift depending on the solvent, sample preparation, and the analytical parameters used. Therefore, unlabeled, solubilized AG and LAM were analyzed under the same conditions as the whole-cell experiments, as well as solution phase 1D and 2D HSQC, total correlation spectroscopy (TOCSY), heteronuclear multiple bond correlation (HMBC), and correlation spectroscopy (COSY) NMR. A direct comparison was made between these spectra and the whole-cell HR-MAS HSQC.



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Fig. 2. Current structure model of mycobacterial AG. AG is composed of an alternating ß(1-5), ß(1-6) galactofuranose chain with arabinan branching out from the galactan core. The galactan chain is tethered to the peptidoglycan via a rhamnose-GlcNAc linker. The arabinan consists of linear Ara{alpha}1->[5Ara{alpha}1 ->]n terminated at the nonreducing end with the branched Ara6 termini, Araß1->2Ara{alpha}1->5(Araß1->2Ara{alpha}1->3) Ara{alpha}1->5Ara{alpha}1->. Mycolic acids are esterified to the nonreducing termini of the arabinose motif.

 


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Fig. 3. Current structure model of mycobacterial LAM. The MPI anchor is linked to a mannan backbone with a {alpha}1->6Man core (20–26 residues) branched frequently at the 2-position with a single {alpha}Man. Attached to the mannan core is an arabinan domain (50–70 residues total) composed of linear Ara{alpha}1->[5Ara{alpha}1->]n terminated at the nonreducing end with Araß1->2Ara{alpha}1->to form a linear Ara4 motif. Branching off the linear 1->5-linked chains are Ara4 and Ara6 motifs. The point of attachment of arabinan to the mannan has not been elucidated. The nonreducing termini of the arabinan are capped with species specific sugars; 1–3 mannose units in M. tuberculosis and M. bovis, phosphatidylinositol in M. smegmatis.

 
Additionally, to reduce the complexity of whole-cell HR-MAS spectra, an insoluble cell wall fraction, rich in both mAG and LAM but mostly devoid of other cellular components, was used for complex, multidimensional NMR experiments. It produced a good, clean HR-MAS HSQC spectrum, particularly in the carbohydrate region (Figure 4). Seventeen major spin systems associated with AG and LAM were observed in the 2D and 3D spectra of both the insoluble standard and the whole-cell spectra. These were assigned to 10 types of Araf (one type of 3,5-{alpha}-Araf, four types of 2-{alpha}-Araf, three types of 5-{alpha}-Araf, two types of t-ß-Araf), three types of Galf (one type of 5-ß-Galf, one type of 6-ß-Galf, and one type of 5,6-ß-Galf), and four types of Manp (one type each of 2-{alpha}-Manp, 6-{alpha}-Manp, 2,6-{alpha}-Manp, and t-{alpha}-Manp).



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Fig. 4. 1H-13C HSQC spectra of the M. bovis BCG cell wall residues AG and LAM. Insoluble cell wall material was prepared by 2:1 chloroform:methanol extraction of 13C-labeled cells, followed by a partial sodium dodecyl sulfate extraction of the pellet. The remaining insoluble material was composed primarily of mAG and LAM. Glycosyl residues are denoted in Roman numerals and their carbons and protons in Arabic numerals. I, 3,5-{alpha}-Araf; II, 5-{alpha}-Araf; III, 2-{alpha}-Araf->3; III', 2-{alpha}-Araf->5; IV, t-{alpha}-Manp; V, t-{alpha}-Araf; VI, 6-{alpha}-Manp; VII, 2-{alpha}-Manp; VIII, 2,6-{alpha}-Manp; A, 5-ß-Galf; B, 6-ß-Galf; C, 5,6-ß-Galf; a, LAM; b, AG.

 
In the anomeric region of the 2D HR-MAS HSQC, most of the cross-peaks for the arabinan, galactan, and mannan can be differentiated due to their characteristic chemical shift. However, some signals do partially overlap, and some sugars have multiple anomeric signals (e.g., 5-{alpha}-Araf, 2-{alpha}-Araf, t-ß-Araf and t-{alpha}-Manp) due to differing chemical environments within the molecular structures of AG and LAM. Signals corresponding to the remaining ring carbons of the glycosyl residues, that is, the C2–C6 signals present in the carbohydrate region, generally overlapped significantly but all had at least one 1H-13C correlation in the spin system that was well separated and unambiguously resolved in both the whole-cell and the insoluble cell wall fraction. This allowed for definition of the presence and relative quantitation of a particular residue in the sample. Consequently, when the spectra of mutant or drug-treated cells were compared to wild-type or untreated cells, structural alterations could be identified.

The HR-MAS 3D HCCH-TOCSY was instrumental for the comprehensive assignment of all the AG and LAM signals, however acquisition took several hours. The 2D HR-MAS HSQC was found to be a valuable tool for rapid sample analysis as acquisition of the spectrum could be achieved in under 1 h. Therefore, it is relevant to report here the signals present in the 2D HSQC spectra, which are discrete enough to define the presence of individual linkages in a sample. Only these key signals, useful for identification in the 2D HSQC, are described next. Signals that overlap significantly in the 2D HSQC are not described but are listed in Table I and were assigned based on the 3D HCCH-TOCSY spectra. With the exception of 5,6-ß-Galf, all signals from the residues listed in Table I were observed in the 3D HCCH-TOCSY spectra of both whole cells and the insoluble cell wall fraction.


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Table I. 1H and 13C chemical shift assignment of AG and LAM from whole-cell M. bovis BCG, based on the interpretation of 2D HSQC, TOCSY, COSY, and 3D HCCH-TOCSY

 
Arabinan signals: 3,5-{alpha}Araf (Residue I). One anomeric signal for 3,5-{alpha}Araf (I) ({delta}H5.01/{delta}C107.38) was observed but it partially overlapped with the 5-{alpha}-Araf (II) anomeric signal. The signal for the C-2 of 3,5-{alpha}Araf ({delta}H 4.19/{delta}C 78.95) was well separated and can be unambiguously defined in the whole-cell spectra. This is a very informative signal, because its presence indicates branching of the 5-linked arabinan core.

5-{alpha}-Araf (Residue II). Two anomeric signals were observed for 5-{alpha}-Araf. The strongest signal, attributed to the anomeric signal of the arabinan units in the 5-linked arabinan core ({delta}H 4.99/{delta}C 107.48), overlapped partially with the weaker 3,5-{alpha}-Araf anomeric cross-peak. The anomeric signal ({delta}H 5.06/{delta}C 107.13), attributed to the 5-{alpha}-Araf residue branching off the arabinan core at the 3-position of 3,5-{alpha}-Araf, overlapped with the reported anomeric signal for 5,6-ß-Galf. This anomeric signal was useful, because it also indicated branching of the arabinan core. The signals for C-2 and C-4 of 5-{alpha}-Araf were observed in a region of heavily overlapping peaks. However, due to the high abundance of this residue in both AG and LAM, these were very strong signals, which were readily observable in all whole-cell and cell wall spectra we investigated.

2-{alpha}-Araf (Residue III). Four spin systems were observed for 2-{alpha}-Araf, which have been designated IIIa, IIIb, III'a, and III'b. Spin systems IIIa and IIIb arise from the 2-linked Araf residue substituting carbon three of 3,5-{alpha}-Araf in LAM and AG, respectively. Spin systems III'a and III'b arise from the 2-linked Araf residue substituting carbon five of the 3,5-{alpha}-Araf in LAM and AG, respectively. Only two anomeric signals were observed. The 2-{alpha}-Araf->3 (III: {delta}H 5.14/{delta}C 105.4) and the 2-{alpha}-Araf->5 (III': {delta}H 5.07/{delta}C 105.56) separate, but there is no distinction of AG and LAM signals. 2-{alpha}-Araf->3 and 2-{alpha}-Araf->5 signals were also differentiated by their C-4 signals (III: {delta}H 3.93/{delta}C 82.79, III': {delta}H 3.99/{delta}C 82.73), but again, there is no distinction of AG and LAM signals. The presence of 2-linked Araf residues in either LAM (IIIa, III'a) or AG (IIIb,III'b) was observed and differentiated by the C-2 and C-3 signals (C-2: AG {delta}H 4.10, LAM {delta}H 4.08; C-3: AG {delta}H 4.02, LAM {delta}H 3.99). Although the separation of these signals is not prominent, it was readily apparent in the HSQC spectrum. No distinction was observed between the C-2 signals for 2-{alpha}-Araf->3 (III) and 2-{alpha}-Araf->5(III'). Nor was there any distinction observed between the C-3 signals for 2-{alpha}-Araf->3 (III) and 2-{alpha}-Araf->5(III'). These shift changes indicate that the 2-{alpha}-Araf residues experience different environments in the cell wall, perhaps due to altered spatial conformations of AG and LAM or substitution of the nonreducing termini of arabinan with mycolic acids (AG).

t-ß-Araf and 5-ß-Araf (Residue V). Only one anomeric signal was observed for the ß-Araf residues. Although this residue can be either unsubstituted or substituted differently in AG (esterified with mycolic acids) and in LAM (glycosylated with mannose caps), no distinction between the AG and LAM anomeric signals was observed, thereby indicating that their environment in the whole cells was quite similar and that the substitution pattern did not affect the chemical shift of the C-1. However, the C-3, C-4, and C-5 of ß-Araf signals were informative for observing the differences between these two molecules. The signals for C-3 of ß-Araf separate partially (AG {delta}H 4.39/{delta}C 73.99, LAM {delta}H 4.02/{delta}C 73.93). The C-4 of ß-Araf signals separate dramatically in both the carbon and the proton chemical shifts (AG {delta}H 3.81/{delta}C 81.94, LAM {delta}H 3.92/{delta}C 79.59). The {delta}C of the C-5 of t-ß-Araf was shifted downfield by about 5.26 ppm when substituted with a mannose residue (AG {delta}H 3.61, 3.73/{delta}C 62.87, LAM {delta}H 3.59, 3.69/{delta}C 68.13) compared to the unsubstituted t-ß-Araf. This is a very useful signal when investigating the mannose capping of the arabinan. The C-5 signals of ß-Araf, when it is esterified with mycolic acids in AG, were not identified.

Mannose: t-{alpha}-Manp (Residue IV). A minor separation ({delta}H 0.01 ppm) of the t-{alpha}-Manp signal ({delta}H 4.93/{delta}C 102.19) was seen in the anomeric region. This represented the terminal mannose in the mannan core and the mannose caps on the termini of the arabinan of LAM. The C-2 signal of t-{alpha}-Manp also separated well from other cell wall signals ({delta}H 3.97/{delta}C 70.00), but as expected, no distinction between the caps and the core was detected.

6-{alpha}-Manp (Residue VI). One anomeric signal for 6-{alpha}-Manp was unambiguously defined ({delta}H 4.81/{delta}C 99.70), however, its chemical shift was very close to the HOD peak and was sometimes lost on saturation of the HOD. The C-6 (H6/H6') signals of 6-{alpha}-Manp partially separate from other signals ({delta}H 3.7, 3.87/{delta}C 65.26), but they were weak signals that were not always visible in the spectra.

2-{alpha}-Manp (Residue VII) and 2,6-{alpha}-Manp (Residue VIII). The anomeric signals for 2-{alpha}-Manp ({delta}H 5.02/{delta}C 98.08) and 2,6-{alpha}-Manp ({delta}H 5.05/{delta}C 98.13) overlapped partially, however, these two signals could be differentiated from each other. The C-2 cross-peaks for these two sugars overlapped completely with each other ({delta}H 3.92/{delta}C 78.62), but they were well defined from other signals in the spectrum. Similarly, the C-5 of 2,6-{alpha}-Manp and 6-{alpha}-Manp ({delta}H 3.53/{delta}C 73.00), and the C-5 of 2-{alpha}-Manp and t-{alpha}-Manp ({delta}H 3.68/{delta}C 73.20), overlapped with each other but were separate from other signals. The C-6 (H6/H6') signals for 2,6-{alpha}-Manp were also unambiguously defined ({delta}H 3.60, 3.89/{delta}C 65.56) but were sometimes weak signals. This could be due to relative abundance or reduced mobility resulting in shorter T2 times.

Galactan: 5-ß-Galf (Residue A). One strong anomeric signal for 5-ß-Galf was observed ({delta}H 4.92/{delta}C 107.77). The C-3 signal of 5-ß-Galf ({delta}H 4.00/{delta}C 76.61) overlapped partially with the C-2 of t-ß-Araf ({delta}H 4.05/{delta}C 76.18) and C-3 of 6-ß-Galf ({delta}H 3.96/{delta}C 76.30), but it could still be differentiated. The C-5 signal of 5-ß-Galf ({delta}H 3.86/{delta}C 75.5) was reasonably well isolated and also served as a good marker for this residue. The C-6 (H6/H6') cross-peak of 5-ß-Galf ({delta}H 3.69/{delta}C 60.94) was observed as a single peak that overlapped with many other signals in the heavily crowded CH2 region. However, it is worth noting that this signal becomes very strong and readily observable when cells were disrupted. This apparent increase in intensity could be due to enhanced mobility of the galactan or a decrease in the presence of overlapping signals in this region.

6-ß-Galf (Residue B). One strong anomeric signal of 6-ß-Galf was observed ({delta}H 5.12/{delta}C 106.98). The C-3 signal of 6-ß-Galf ({delta}H 3.96/{delta}C 76.30) slightly overlapped with the C-3s of 5-{alpha}-Araf ({delta}H 3.92/{delta}C 76.61) and 5-ß-Galf ({delta}H 4.00/{delta}C 76.61) but could still be differentiated in the 2D spectra. The C-5 ({delta}H 3.89/{delta}C 69.4) and the C-6 (H6/H6') signals ({delta}H 3.53, 3.79/{delta}C 69.2) of 6-ß-Galf partially overlapped with some of the 2-linked and 2,6-linked mannose residues but were readily observable in the spectra.

5,6-ß-Galf (Residue C). Unlike other peaks, the 5,6-ß-Galf signals could not be unambiguously defined in the 2D HSQC or the 3D spectra. This may be because of the low number of these molecules (three per AG moiety) or because these signals completely overlapped with other galactose and arabinose signals.

To address the overlapping peaks in the 2D HSQC, an HR-MAS 3D HCCH-TOCSY experiment was performed on both the BCG whole cells (Figure 5) as well as the insoluble cell wall material (data not shown). All the 13C(F2)–1H(F3) planes were combined into one 2D frame to construct a projection of the 3D spectra, allowing the visualization of all spin systems acquired in the whole cell at one time (Figure 5a). The proton–proton correlation is displayed along the F3 axis and the carbon–carbon correlation (constructed carbon–carbon "TOCSY") is displayed along the F2 axis. The analysis of only one spin system, t-ß-Araf, was selected for presentation here. The characteristic anomeric region is clearly visible in the projection ({delta}H 5.4–4.8/{delta}C 110–90 ppm) allowing straightforward identification of the anomeric t-ß-Araf signal ({delta}H 5.04/{delta}C 100.57). The value of the projection for the most part was limited to defining the precise chemical shifts of the anomeric carbons. However, the TOCSY {delta}H shift pattern for t-ß-Araf was readily observed for t-ß-Araf along the F3 axis, facilitating the identification of the spin system in the individual planes. The spin system of the t-ß-Araf was observed in five separate 13C(F2)–1H(F3) planes, which corresponded to the C1–C5 of t-ß-Araf (V). Partial sections of these planes are displayed in Figure 5b. The TOCSY {delta}H shift pattern for t-ß-Araf can be seen along the F3 axis ({delta}H C-1 5.04, C-2 4.05, C-3 4.39, C-4 3.81, C-5 3.59/3.69), and the carbon shift for each carbon in the spin system is displayed along the F2 axis ({delta}H C-1 100.57, C-2 76.18, C-3 73.99, C-4 81.94, C-5 62.87). Carbon and proton shifts were compared to 2D HR-MAS HSQC spectra of M. bovis BCG whole cells (Figure 5c), and the 2D HSQC spectrum of the base-solubilized AG standard (Figure 5d). From these data, the cross-peaks for the t-ß-Araf residue were clearly defined in the whole-cell HSQC spectra. The chemical shifts of these peaks were compared to previously assigned standards and to assignments reported in the literature. Subsequently, each of the residues present in LAM and AG (I–VIII, A and B) were defined in the same manner (Table I). The chemical shifts for 5,6-ß-Galf and the less abundant 5-{alpha}-Araf residues could not be unambiguously defined.



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Fig. 5. Analysis of the t-ß-Araf spin system using 3D HCCH-TOCSY and 2D HSQC HR-MAS NMR. (a) All the 13C(F2)–1H(F3) planes were combined into one 2D frame to construct a projection of the 3D spectra allowing the visualization of all spin systems acquired in the whole cell at one time. The proton–proton correlation is displayed along the F3 axis, and the carbon–carbon correlation is displayed along the F2 axis. The anomeric cross-peak for the t-ß-Araf was defined in the 3D projection (circled) and subsequently identified along with the TOCSY {delta}H shift pattern in the individual 3D planes. (b) Partial sections of the five separate 13C(F2)–1H(F3) planes, which corresponded to the C1–C5 of t-ß-Araf (V). The carbon and proton shifts were compared to (c) the 2D HSQC of M. bovis BCG whole cells and (d) the 2D HSQC spectrum of the base-solubilized AG standard. From these data, the cross-peaks for the t-ß-Araf residue were clearly defined in the whole-cell HSQC spectra (circled in c).

 

    Differences between the species M. bovis versus M. smegmatis
 Top
 Abstract
 Introduction
 Results
 Differences between the species...
 Effects of EMB and...
 Discussion
 Materials and methods
 References
 
The most striking difference between these species is in the intensity of LAM signals at the whole-cell level. The intensity of the LAM signals was much lower in M. smegmatis, whereas the relative intensities of AG appeared to be similar in both species (Figure 6). This finding substantiates the previous work by Daffe and colleagues (Lemassu et al., 1996Go) and others who have found experimentally that M. smegmatis has much less LAM in its cell wall than does M. bovis BCG. Additionally, the mannose capping of the terminal arabinose is readily detected in the M. bovis BCG whole-cell HSQC ({delta}H 3.59/3.69, {delta}C 68.13) but could not be detected at all in M. smegmatis. This observation was consistent with the analysis of isolated LAM from M. bovis BCG and M. smegmatis, because the arabinan of M. smegmatis is capped with phosphotidylinositol, not mannose (Nigou et al., 2003Go). Differences in the cell wall–associated molecules other than AG and LAM were also observed. The anomeric signals for the {alpha}-glucans (4-{alpha}-Glcp and t-{alpha}-Glcp {delta}H 5.11/{delta}C 99.5, 4,6-{alpha}-Glcp and 6-{alpha}-Glcp {delta}H 4.91/{delta}C 97.5) were detected as very strong signals in the BCG spectrum but were very weak or absent in the M. smegmatis spectrum.



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Fig. 6. 1H–13C HR-MAS HSQC spectra ({delta} 1H, 3.0–5.45, {delta} 13C 50–110) of (a) whole-cell M. bovis BCG and (b) M. smegmatis mc2 155. The relative intensities of the AG appear similar in both species, however the relative amount of LAM in M. smegmatis is significantly less when compared to the M. bovis BCG strain. Roman numerals (VIII: 2,6-{alpha}-Manp, IV: t-{alpha}-Manp) and asterisks (assigned in Figure 4) denote glycosyl signals present in LAM but not AG.

 

    Effects of EMB and embB mutation
 Top
 Abstract
 Introduction
 Results
 Differences between the species...
 Effects of EMB and...
 Discussion
 Materials and methods
 References
 
The antituberculosis drug EMB is known to act by inhibition of the mycobacterial arabinosyltransferases EmbA–C and in particular by affecting EmbB, which is required for formation of the 3-arm branch of the 3,5-{alpha}-Araf in the Ara6 motif found in AG (Escuyer et al., 2001Go). M. smegmatis, which has very little LAM, was used to examine the effects of EMB at the whole-cell level. Therefore, the effects on AG were observed, but not the effects on LAM. As expected from previous work in which the cell walls of EMB-treated mycobacteria were analyzed using degradative analysis, the HR-MAS HSQC spectra showed drastic reduction in all the arabinose signals of M. smegmatis whole cells treated with EMB for 6 h (Figure 7). Relative to the galactan signals, the signals for the 5-{alpha}-Araf (II) of the arabinan core and the weak signals for 2-{alpha}-Araf->5 (III') and t-ß-Araf (V) were still visible in the spectrum but at reduced intensity. However, most of the signals corresponding to branching of the arabinan, 2-{alpha}-Araf->3 (III) and 3,5-{alpha}-Araf (I), were almost completely lost. This indicates that after EMB treatment a 5-linked Araf core remains with some 2-{alpha}-Araf->5 and t-ß-Araf residues attached, but the branching into the Ara6 motifs had been inhibited.



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Fig. 7. 1H–13C HR-MAS HSQC spectra ({delta} 1H, 3.0–5.45, {delta} 13C 50–110) of whole-cell M. smegmatis. (a) Untreated, (b) treated with 50 µg/ml EMB for 6 h, and (c) M. smegmatis {Delta}embB mutant. The expanded region of the HSQC provides detail of the anomeric region ({delta} 1H, 4.8–5.4, {delta} 13C 95–110). The signals corresponding to the arabinan are significantly reduced in the spectrum of cells treated with EMB. Circled regions denote the cross-peak corresponding to the 2-linked {alpha}-Araf residue extending from the 3-arm of the 3,5-linked Araf in AG and LAM. This presence of this linkage is significantly reduced in the {Delta}embB knockout. Roman numerals correspond to the arabinan residues: I, 3,5-{alpha}-Araf; II, 5-{alpha}-Araf; III, 2-{alpha}-Araf->3; III', 2-{alpha}-Araf->5.

 
When HR-MAS NMR was performed on the M. smegmatis {Delta}embB mutant, the anomeric cross-peak corresponding to the 2-{alpha}-Araf->3 (III, {delta}H 5.14/{delta}C 105.4) signal extending from the 3-arm of the 3,5-{alpha}-Araf in the Ara-6 motif was almost completely missing, whereas the other arabinosyl signals were still readily observable in the spectrum (Figure 7). The 2-{alpha}-Araf->5 anomeric cross-peak (III', {delta}H 5.07/{delta}C 105.6) was still fairly strong, indicating only the 2-{alpha}-Araf->3 linkage had been inhibited. This is consistent with findings by Chatterjee and others obtained by characterization of base-solubilized AG from M. smegmatis {Delta}embB via standard degradative analytical techniques (Escuyer et al., 2001Go). The significance of this is that the effects of the gene knockout could be seen in the whole-cell spectra without any purification procedures. It is important to note here that only relative quantitation can be observed using HR-MAS due to the potential effects of T2 times and 13C uptake. Changes in the tertiary structure of the cell wall may lead to changes in mobility of the molecules, which may result in altered T2 times and subsequent changes of signal intensity. Therefore, although it was possible to observe a decrease in the amount of 2-{alpha}-Araf->3 with respect to 2-{alpha}-Araf->5, accurate quantitation of the total arabinan content was not possible in this study.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Differences between the species...
 Effects of EMB and...
 Discussion
 Materials and methods
 References
 
Herein we present the first described use of HR-MAS NMR to detect cell wall structures in live, intact mycobacterial cells. Previously, information at the whole-cell level has been restricted to examining the morphology under the microscope. Structural definition of the cell walls has been gained by prolonged extraction and purification process often requiring growth of large amounts (hundreeds of grams) of bacteria. The primary objectives of this study were to determine if specific metabolite populations of live mycobacterial cells are observable using this technique, assign and confirm the structure of the AG and LAM in whole cells, observe the effects of EMB and embB gene knockout on the cell wall of intact mycobacteria, and observe differences in cell wall structure between species. We chose to concentrate on deciphering the signals for AG and LAM, because these structures are abundant in the mycobacterial cell wall, detailed NMR data was already available in the literature for these components, direct structural effects of EMB treatment on AG and LAM have been previously characterized, and emb gene knockout strains were readily available. M. bovis BCG was chosen as a good model for investigating the cell wall because it is part of the M. tuberculosis complex and the cell walls of M. tuberculosis and M. bovis are highly similar. Indeed, this technique has proven to be effective as a means to view the metabolites of intact cells that would normally not be resolved by standard NMR techniques. A small amount of cells (20 mg) grown in [13C]-enriched media is all that is required to obtain complex 2D and 3D HR-MAS NMR spectra that may be used to simultaneously resolve signals from different mycobacterial macromolecular structures, including mAG, LAM, glucans, mycolates, glycolipids, and phospholipids.

Complete assignment of the AG and LAM signals in the whole-cell spectra required both 2D and 3D analysis. The 2D HR-MAS HSQC provided a useful method for rapid analysis of whole cells and crude cell wall preparations. In the anomeric region, the characteristic signals for AG and LAM were readily observed. These signals have been well defined in the literature, and the cross-peaks in this region were not very crowded, facilitating assignment. Conversely, the signals corresponding to the remaining ring carbons (C2–C6) were in a heavily crowded region. However, many of the signals were strong enough or separated well enough to be defined by comparing the whole-cell spectra to 2D HSQC of purified standards of AG and LAM acquired under the same conditions. To confirm the assignment of the whole-cell spectra and to define the chemical shifts of correlations not assigned in the 2D HSQC, the 3D HCCH-TOCSY was performed. This is the first known report of 3D HR-MAS NMR analysis of whole bacterial cells. It proved to be a powerful technique for analysis of the mycobacterial cell wall. Using this method, the 1H and 13C chemical shifts for the entire spin system of each glycosyl residue could be elucidated unambiguously. The exception for this was the 5,6-Gal, which could not be unambiguously defined from this experiment. This could be because of the low number of these molecules (three per AG moiety) or because these signals completely overlap with other galactan and arabinan signals.

Previously, the precise carbon and proton chemical shifts of the nonanomeric signals of AG have not been reported. These chemical shifts were determined in this study, which led to some interesting observations. It was found during the 3D analysis that spin system B, corresponding to the anomeric peak at {delta}H 5.12/{delta}C 106.98, was defined as 6-ß-Galf, not 5-ß-Galf as previously reported for M. smegmatis and M. tuberculosis (Daffe et al., 1993Go; Escuyer et al., 2001Go). Spin system A, corresponding to the anomeric peak at {delta}H 4.92/{delta}C 107.77, was defined as the 5-ß-Galf instead of the 6-ß-Galf. The reason for these differences has yet to be determined. More important, it was found that several of the signals corresponding to the structurally similar arabinan of AG and LAM could be differentiated in the 2D HSQC. This permitted the relative quantitation of AG and LAM in the cell wall, such as in the case of M. smegmatis or M. bovis BCG, and will allow differentiation between AG and LAM when investigating the biosynthesis pathways of these molecules or the effects of drug treatment. Characterization of mannose capping in LAM was also observed in the 2D HSQC, as the signals for the C3, C4, and C5 of the t-ß-Araf were shifted and distinct on substitution with mannose. This is of interest because the capping of arabinan in LAM varies significantly between species, and the mannose in the LAM of M. tuberculosis has been shown to be involved in host–cell interactions. Because significant differences were observed between the mycobacterial species M. smegmatis and M. bovis, studies are ongoing to determine if this method could be used to rapidly identify mycobacterial strains.

One of the most exciting results of this study was the ability to see the effects of EMB on the structure of the cell wall in live cells and then validate those results using the embB knockout mutant in M. smegmatis. Treatment of the mycobacterial cell with EMB is known to reduce the arabinan content of the cell wall by inhibiting the arabinosyltransferases, EmbA–C. The embCAB gene cluster was originally identified in M. smegmatis isolates that developed resistance in response to EMB treatment (Lety et al., 1997Go; Telenti et al., 1997Go). M. tuberculosis clinical isolates predominately have mutations in the embB gene (Ramaswamy et al., 2000Go; Telenti et al., 1997Go). Because overexpression of these genes leads to increased arabinosyltransferase activity and restoration of the branching of AG during EMB treatment (Belanger et al., 1996Go; Khoo et al., 1996Go), it was hypothesized that these were arabinosyltransferase genes. Therefore, Chatterjee and colleagues created knockout mutants in M. smegmatis for each gene in the embCAB operon (Escuyer et al., 2001Go; Zhang et al., 2003Go). These experiments have been carried out in M. smegmatis because gene disruption in M. tuberculosis has so far been unsuccessful, presumably because these genes are essential for tuberculosis. Analysis of the AG from these mutants by classical degradative analysis confirmed that arabinan in these mutants was truncated. The arabinan of AG in the {Delta}embB mutant lacked the 2-linked {alpha}-arabinose extending from the 3-arm branch of the 3,5-{alpha}-Araf in the Ara-6 motif (Escuyer et al., 2001Go). Indeed, when we performed the whole-cell 2D HR-MAS HSQC experiment with M. smegmatis {Delta}embB, the cross-peak corresponding to the 2-{alpha}-Araf linked to the C3 of the 3,5-Araf was barely visible in the spectra, whereas the cross-peak corresponding to the 2-{alpha}-Araf linked to the C5 of 3,5-Araf was readily observable. Although the {Delta}emb strain demonstrated the inhibition of a single linkage, treatment with EMB resulted in a significant decrease in intensity for most of the arabinose signals, indicating EMB inhibits multiple enzymes in the arabinan biosynthesis pathway. However, a considerable amount of 5-linked Araf was still present. This indicates that the arabinosyltransferase responsible for building the 5-linked core is not inhibited by EMB, which is consistent with previous studies (Lee et al., 1997Go). The ability to observe the effects of drug treatment and gene mutation on the cell wall was highly significant, as it demonstrated direct structural changes in the cell wall due to drug exposure or mutation can be rapidly observed in whole cells using HR-MAS NMR. This may help in defining mechanisms of action for other cell wall inhibitors, screening for new cell wall inhibitors, assigning gene function, and defining general stress response mechanisms.

Another challenge in these experiments is addressing mobility issues. Molecules that are very rigid may not be observed in the spectra because their T2 times are too short. We have observed this phenomenon with adjuvant preparations of insoluble mAG that have been rigorously purified and dried and were therefore almost a solid with limited mobility (data not shown). When the solid is made as a fine suspension in D2O and HR-MAS HSQC is performed, the arabinan signals were barely visible in the spectra, but the intensity of the galactan signals was good. After the lipids were removed by base solubilization of the mAG sample, the arabinan signals were readily observed along with the galactan residues. Presumably, in the mAG solid form, the long unbranched tail of the galactan chain (~30 residues) is exposed and mobile enough in the D2O, but the highly branched, mycolated AG has a very restricted mobility. Further development of experimental parameters may be needed to address this phenomenon.

The potential applications for mycobacterial cell wall analysis, beyond the examples presented here, are exciting. We may be able to detect modifications in cell wall structures as a result of host–cell interaction, growth phase, environmental conditions (particularly anaerobic conditions), and latency. By developing this technique further, elucidation of a more precise 3D structural model of the cell wall may be possible by analyzing the cell wall as a system and studying the interactions between the major structural macromolecules in the intact whole cells. Additionally, HR-MAS NMR was found to be suitable for the analysis of sample preparations of insoluble cell wall material, such as total cell wall preparations from broken cells, mAG, and mycobacterial cell wall samples used in adjuvant preparations. Current and future research on this study will explore these areas, provide a complete assignment of all signals observed in the whole-cell mycobacterial spectra, and involve the development of methods to detect signals not currently observed due to restricted mobility, T2 effects, or low abundance. We recognize that this technique cannot replace the traditional, elegant biochemical experiments that provide excellent quantitative data and linkage analysis. However, we show here that HR-MAS NMR is a powerful, rapid, nondestructive technique to observe the complex, carbohydrate-rich cell wall of live mycobacterial cells and study the effects of antimycobacterial agents and gene mutation on the cell wall structure. It adds yet another important new tool to compliment the investigator's repertoire of genomic, proteomic, and classical biochemical techniques in the pursuit of understanding the mycobacterial cell wall and developing new antimycobacterial treatments.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Differences between the species...
 Effects of EMB and...
 Discussion
 Materials and methods
 References
 
Mycobacterial strains and growth conditions
M. bovis BCG and M. smegmatis (mc2 155) were grown at 37°C in Middlebrook 7H9 with 0.05% Tween 80 (Difco, Detroit, MI), 0.2% glycerol, and 10% albumin-dextrose supplement. For 13C labeling, 0.2% glycerol and 0.2% glucose were replaced with 0.2% 13C-glycerol and 0.2% 13C-glucose (Cambridge Isotope Laboratories, Cambridge, MA). Cultures were grown to an OD600 of 0.6 and diluted in 13C-labeled media to an OD600 of 0.05. Cells were then harvested by centrifugation (3700 x g, 10 min, 4°C) when the OD600 reached ~ 0.8. The M. smegmatis {Delta}embB mutant was inoculated 1:100 from a glycerol stock into 5 ml Sauton media excluding glycerol, and grown at 37°C for 48 h. Cells were harvested by centrifugation for 15 min at 3000 rpm. The pellet was resuspended in 25 ml Sauton media prepared with 13C-glycerol, grown at 37°C for 48 h, and harvested.

Preparation of cells for NMR analysis
After harvesting, the cell pellets were washed three times in a volume of D2O (Cambridge Isotope Laboratories) representing 20% of the culture volume. The pellets were resuspended in a final volume of 2 µl of D2O per mg cells (wet weight). Fifty microliters of cell suspension was loaded into a glass MAS rotor and sealed.

Standard cell wall components
Purified lipoarabinomannan (M. tuberculosis, H37Rv) was obtained from Colorado State University under the NIH, NIAD contract NO1 AI-75320 titled "Tuberculosis Research Materials and Vaccine Testing." Purified AG (M. bovis BCG) was a generous gift from the McNeil laboratory at Colorado State University.

Preparation of insoluble cell wall material rich in AG and LAM
A 0.375-g (wet weight) 13C-labeled M. bovis BCG pellet was extracted twice in 2:1 chloroform:methanol at 37°C, shaking for 1 h. The sample was centrifuged at 3700 x g for 10 min. The supernatant was removed and the pellet was partially extracted in 2% sodium dodecyl sulfate at 85°C, resuspended in D2O, and heated at 85°C for 1 h followed by centrifugation at 20,000 x g for 5 min. The resulting pellet was resuspended in 55 µl of D2O, at which point it was very chunky. The entire pellet was loaded into the MAS rotor. All supernatants were stored at –20°C for further analysis.

EMB treatment
M. smegmatis mc2 155 was grown in 13C-labeling media as described. The culture was then diluted to an OD600 of 0.4 in 13C media, supplemented with 50 µg/ml EMB (Sigma-Aldrich, St. Louis, MO), and incubated at 37°C for 6 h. The cells were harvested by centrifugation at 3700 x g for 10 min at 4°C and prepared for NMR analysis.

NMR spectroscopy
All whole-cell HR-MAS NMR experiments and LAM analysis were performed on a Varian Inova 500 MHz spectrometer equipped with a 4 mm gHX Nanoprobe (Varian Inc., Palo Alto, CA). During 1D and 2D experiments, samples were spun at 2.5 kHz, and data were recorded at 295 K. Individual experimental parameters (90° pulse width, recycle delay, and solvent saturation parameters) were optimized for each experiment.

1D proton spectra were acquired by using a rotor-synchronized CPMG pulse sequence with water suppression. The total delay time counted as bt was 160 ms. 2D 1H-13C HSQC spectra were acquired using a standard HSQC sequence with rotor synchronization and an adiabatic decoupling pulse sequence. Typically 256 increments were acquired with two to four scans for each increment. Longer experiments at 512 increments and 24–48 scans were also conducted during method development. 3D HCCH-TOCSY spectra were acquired using an adiabatic mixing sequence. The spinning speed was set at 2000 Hz to synchronize all the gradient pulses (integer of 500 µs, the time for the rotor to rotate one cycle). 1001(T3) x 70 (T2) x 48 (T1) complex data points were acquired with 24 scans per free induction decay. The total mixing time for the TOCSY portion was 57 ms. Linear prediction was used in both the indirect dimensions with zero-filling before Fourier transformation. All data were processed with Varian's standard software with shifted sine bell weighting functions.


    Acknowledgements
 
The authors gratefully acknowledge helpful discussions with Drs. Jie Zheng, Richard Kriwacki, and Weixing Zhang of St. Jude Children's Research Hospital. This work was supported by NIH Grants AI-054798 (R.L. and W.L.) and AI-37139 (D.C.).


    Abbreviations
 
AG, arabinogalactan; BCG, bacillus-Calmette-Guérin; CPMG, Carr-Purcell-Meiboom-Gill; COSY, correlation spectroscopy; EMB, ethambutol; HR-MAS, high-resolution magic-angle spinning; HSQC, heteronuclear single quantum coherence; LAM, lipoarabinomannan; LM, lipomannan; mAG, mycolyl arabinogalactan; PIM phosphatidylinositol mannoside; TOCSY, total correlation spectroscopy


    References
 Top
 Abstract
 Introduction
 Results
 Differences between the species...
 Effects of EMB and...
 Discussion
 Materials and methods
 References
 
Belanger, A.E., Besra, G.S., Ford, M.E., Mikusova, K., Belisle, J.T., Brennan, P.J., and Inamine, J.M. (1996) The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc. Natl Acad. Sci. USA, 93, 11919–11924.[Abstract/Free Full Text]

Besra, G.S., Khoo, K.-H., McNeil, M., Dell, A., Morris, H.R., and Brennan, P.J. (1995) A new interpretation of the structure of the mycolyl-arabinogalactan complex of Mycobacterium tuberculosis as revealed through characterization of oligoglycosylalditol fragments by fast-atom bombardment mass spectrometry and 1H nuclear magnetic resonance spectroscopy. Biochemistry, 34, 4257–4266.[ISI][Medline]

Brennan, P.J. (2003) Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis. (Edinb.), 83, 91–97.[CrossRef][Medline]

Brennan, P.J. and Nikaido, H. (1995) The envelope of mycobacteria. Annu. Rev. Biochem., 64, 29–63.[CrossRef][ISI][Medline]

Broberg, A. and Kenne, L. (2000) Use of high-resolution magic angle spinning nuclear magnetic resonance spectroscopy for in situ studies of low-molecular-mass compounds in red algae. Anal. Biochem., 284, 367–374.[CrossRef][ISI][Medline]

Chatterjee, D. (1997) The mycobacterial cell wall: structure, biosynthesis and sites of drug action. Curr. Opin. Chem. Biol., 1, 579–588.[CrossRef][ISI][Medline]

Chatterjee, D. and Khoo, K.H. (1998) Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects. Glycobiology, 8, 113–120.[Abstract/Free Full Text]

Chatterjee, D., Hunter, S.W., McNeil, M., and Brennan, P.J. (1992) Lipoarabinomannan: multiglycosylated form of the mycobacterial mannosylphosphatidylinositols. J. Biol. Chem., 267, 6228–6233.[Abstract/Free Full Text]

Daffe, M., Brennan, P.J., and McNeil, M. (1990) Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C-NMR analyses. J. Biol. Chem., 265, 6734–6743.[Abstract/Free Full Text]

Daffe, M., McNeil, M., and Brennan, P.J. (1993) Major structural features of the cell wall arabinogalactans of Mycobacterium, Rhodococcus, and Nocardia spp. Carbohydr. Res., 249, 383–398.[CrossRef][ISI][Medline]

Dinadayala, P., Lemassu, A., Granovski, P., Cerantola, S., Winter, N., and Daffe, M. (2004) Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains. J. Biol. Chem., 279, 12369–12378.[Abstract/Free Full Text]

Escuyer, V.E., Lety, M.A., Torrelles, J.B., Khoo, K.H., Tang, J.B., Rithner, C.D., Frehel, C., McNeil, M.R., Brennan, P.J., and Chatterjee, D. (2001) The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan. J. Biol. Chem., 276, 48854–48862.[Abstract/Free Full Text]

Garton, N.J., Gilleron, M., Brando, T., Dan, H.H., Giguere, S., Puzo, G., Prescott, J.F., and Sutcliffe, I. C. (2002) A novel lipoarabinomannan from the equine pathogen Rhodococcus equi. Structure and effect on macrophage cytokine production. J. Biol. Chem., 277, 31722–31733.[Abstract/Free Full Text]

Gibson, K.J., Gilleron, M., Constant, P., Puzo, G., Nigou, J., and Besra, G.S. (2003a) Identification of a novel mannose-capped lipoarabinomannan from Amycolatopsis sulphurea. Biochem. J., 372, 821–829.[CrossRef][ISI][Medline]

Gibson, K.J., Gilleron, M., Constant, P., Puzo, G., Nigou, J., and Besra, G.S. (2003b) Structural and functional features of Rhodococcus ruber lipoarabinomannan. Microbiology, 149, 1437–1445.[CrossRef][ISI][Medline]

Gilleron, M., Himoudi, N., Adam, O., Constant, P., Venisse, A., Riviere, M., and Puzo, G. (1997) Mycobacterium smegmatis phosphoinositols-glyceroarabinomannans. Structure and localization of alkali-labile and alkali-stable phosphoinositides. J. Biol. Chem., 272, 117–124.[Abstract/Free Full Text]

Gilleron, M., Nigou, J., Cahuzac, B., and Puzo, G. (1999) Structural study of the lipomannans from Mycobacterium bovis BCG: characterisation of multiacylated forms of the phosphatidyl-myo-inositol anchor. J. Mol. Biol., 285, 2147–2160.[CrossRef][ISI][Medline]

Gilleron, M., Bala, L., Brando, T., Vercellone, A., and Puzo, G. (2000) Mycobacterium tuberculosis H37Rv parietal and cellular lipoarabinomannans. Characterization of the acyl- and glyco-forms. J. Biol. Chem., 275, 677–684.[Abstract/Free Full Text]

Gilleron, M., Quesniaux, V.F., and Puzo, G. (2003) Acylation state of the phosphatidylinositol hexamannosides from Mycobacterium bovis bacillus Calmette Guerin and mycobacterium tuberculosis H37Rv and its implication in Toll-like receptor response. J. Biol. Chem., 278, 29880–29889.[Abstract/Free Full Text]

Guerardel, Y., Maes, E., Elass, E., Leroy, Y., Timmerman, P., Besra, G.S., Locht, C., Strecker, G., and Kremer, L. (2002) Structural study of lipomannan and lipoarabinomannan from Mycobacterium chelonae. Presence of unusual components with alpha 1,3-mannopyranose side chains. J. Biol. Chem., 277, 30635–30648.[Abstract/Free Full Text]

Guerardel, Y., Maes, E., Briken, V., Chirat, F., Leroy, Y., Locht, C., Strecker, G., and Kremer, L. (2003) Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties. J. Biol. Chem., 278, 36637–36651.[Abstract/Free Full Text]

Jachymek, W., Niedziela, T., Petersson, C., Lugowski, C., Czaja, J., and Kenne, L. (1999) Structures of the O-specific polysaccharides from Yokenella regensburgei (Koserella trabulsii) strains PCM 2476, 2477, 2478, and 2494: high-resolution magic-angle spinning NMR investigation of the O-specific polysaccharides in native lipopolysaccharides and directly on the surface of living bacteria. Biochemistry, 38, 11788–11795.[CrossRef][ISI][Medline]

Khoo, K.H., Douglas, E., Azadi, P., Inamine, J.M., Besra, G.S., Mikusova, K., Brennan, P.J., and Chatterjee, D. (1996) Truncated structural variants of lipoarabinomannan in ethambutol drug-resistant strains of Mycobacterium smegmatis. Inhibition of arabinan biosynthesis by ethambutol. J. Biol. Chem., 271, 28682–28690.[Abstract/Free Full Text]

Khoo, K.H., Tang, J.B., and Chatterjee, D. (2001) Variation in mannose-capped terminal arabinan motifs of lipoarabinomannans from clinical isolates of Mycobacterium tuberculosis and Mycobacterium avium complex. J. Biol. Chem., 276, 3863–3871.[Abstract/Free Full Text]

Lee, R.E., Brennan, P.J., and Besra, G.S. (1996) Mycobacterium tuberculosis cell envelope. Curr. Top. Microbiol. Immunol., 215, 1–27.[ISI][Medline]

Lee, R.E., Brennan, P.J., and Besra, G.S. (1997) Mycobacterial arabinan biosynthesis: the use of synthetic arabinoside acceptors in the development of an arabinosyl transfer assay. Glycobiology, 7, 1121–1128.[Abstract]

Lemassu, A., OrtaloMagne, A., Bardou, F., Silve, G., Laneelle, M.A., and Daffe, M. (1996) Extracellular and surface-exposed polysaccharides of non-tuberculous mycobacteria. Microbiology, 142, 1513–1520.[ISI][Medline]

Lety, M.A., Nair, S., Berche, P., and Escuyer, V. (1997) A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis. Antimicrob. Agents Chemother., 41, 2629–2633.[Abstract]

Liu, J., Rosenberg, E.Y., and Nikaido, H. (1995) Fluidity of the lipid domain of cell wall from Mycobacterium chelonae. Proc. Natl Acad. Sci. USA, 92, 11254–11258.[Abstract/Free Full Text]

Nigou, J., Gilleron, M., Brando, T., Vercellone, A., and Puzo, G. (1999) Structural definition of arabinomannans from Mycobacterium bovis BCG. Glycoconj. J., 16, 257–264.[CrossRef][ISI][Medline]

Nigou, J., Gilleron, M., and Puzo, G. (2003) Lipoarabinomannans: from structure to biosynthesis. Biochimie, 85, 153–166.[CrossRef][ISI][Medline]

Ramaswamy, S.V., Amin, A.G., Goksel, S., Stager, C.E., Dou, S.J., El Sahly, H., Moghazeh, S.L., Kreiswirth, B.N., and Musser, J.M. (2000) Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 44, 326–336.[Abstract/Free Full Text]

Rastogi, N., Legrand, E., and Sola, C. (2001) The mycobacteria: an introduction to nomenclature and pathogenesis. Rev. Sci. Tech., 20, 21–54.[ISI][Medline]

Ribi, E., Milner, K.C., Granger, D.L., Kelly, M.T., Yamamoto, K., Brehmer, W., Parker, R., Smith, R.F., and Strain, S.M. (1976) Immunotherapy with nonviable microbial components. Ann. NY Acad. Sci., 277, 228–238.[ISI][Medline]

Sassetti, C.M., Boyd, D.H., and Rubin, E.J. (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol., 48, 77–84.[CrossRef][ISI][Medline]

Szymanski, C.M., Michael, F.S., Jarrell, H.C., Li, J., Gilbert, M., Larocque, S., Vinogradov, E., and Brisson, J.R. (2003) Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy. J. Biol. Chem., 278, 24509–24520.[Abstract/Free Full Text]

Telenti, A., Philipp, W.J., Sreevatsan, S., Bernasconi, C., Stockbauer, K.E., Wieles, B., Musser, J.M., and Jacobs, W.R. Jr. (1997) The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat. Med., 3, 567–570.[CrossRef][ISI][Medline]

Treumann, A., Xidong, F., McDonnell, L., Derrick, P.J., Ashcroft, A.E., Chatterjee, D., and Homans, S.W. (2002) 5-Methylthiopentose: a new substituent on lipoarabinomannan in Mycobacterium tuberculosis. J. Mol. Biol., 316, 89–100.[CrossRef][ISI][Medline]

Venisse, A., Berjeaud, J.M., Chaurand, P., Gilleron, M., and Puzo, G. (1993) Structural features of lipoarabinomannan from Mycobacterium bovis BCG. Determination of molecular mass by laser desorption mass spectrometry. J. Biol. Chem., 268, 12401–12411.[Abstract/Free Full Text]

Venisse, A., Riviere, M., Vercauteren, J., and Puzo, G. (1995) Structural analysis of the mannan region of lipoarabinomannan from Mycobacterium bovis BCG. Heterogeneity in phosphorylation state. J. Biol. Chem., 270, 15012–15021.[Abstract/Free Full Text]

Vercellone, A., Nigou, J., and Puzo, G. (1998) Relationships between the structure and the roles of lipoarabinomannans and related glycoconjugates in tuberculosis pathogenesis. Front. Biosci., 3, e149–e163.[Medline]

Zhang, N., Torrelles, J.B., McNeil, M.R., Escuyer, V.E., Khoo, K.H., Brennan, P.J., and Chatterjee, D. (2003) The Emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an N-terminal recognition region and a C-terminal synthetic region. Mol. Microbiol., 50, 69–76.[CrossRef][ISI][Medline]