Direct molecular mass determination of trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry

Yukiko Fujita, Takashi Naka, Takeshi Doi and Ikuya Yano

Japan BCG Central Laboratory, 3-1-5 Matsuyama, Kiyose-shi, Tokyo 204-0022, Japan

Correspondence
Yukiko Fujita
y-fujita{at}bcg.gr.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Direct estimation of the molecular mass of single molecular species of trehalose 6-monomycolate (TMM), a ubiquitous cell-wall component of mycobacteria, was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. When less than 1 µg TMM was analysed by MALDI-TOF mass spectrometry, quasimolecular ions [M+Na]+ of each molecular species were demonstrated and the numbers of carbons and double bonds (or cyclopropane rings) were determined. Since the introduction of oxygen atoms such as carbonyl, methoxy and ester groups yielded the appropriate shift of mass ions, the major subclasses of mycolic acid ({alpha}, methoxy, keto and wax ester) were identified without resorting to hydrolytic procedures. The results showed a marked difference in the molecular species composition of TMM among mycobacterial species. Unexpectedly, differing from other mycoloyl glycolipids, TMM from Mycobacterium tuberculosis showed a distinctive mass pattern, with abundant odd-carbon-numbered monocyclopropanoic (or monoenoic) {alpha}-mycolates besides dicyclopropanoic mycolate, ranging from C75 to C85, odd- and even-carbon-numbered methoxymycolates ranging from C83 to C94 and even- and odd-carbon-numbered ketomycolates ranging from C83 to C90. In contrast, TMM from Mycobacterium bovis (wild strain and BCG substrains) possessed even-carbon-numbered dicyclopropanoic {alpha}-mycolates. BCG Connaught strain lacked methoxymycolates almost completely. These results were confirmed by MALDI-TOF mass analysis of mycolic acid methyl esters liberated by alkaline hydrolysis and methylation of the original TMM. Wax ester-mycoloyl TMM molecular species were demonstrated for the first time as an intact form in the Mycobacterium aviumintracellulare group, M. phlei and M. flavescens. The M. aviumintracellulare group possessed predominantly C85 and C87 wax ester-mycoloyl TMM, while M. phlei and the rapid growers tested contained C80, C81, C82 and C83 wax ester-mycoloyl TMM. This technique has marked advantages in the rapid analysis of not only intact glycolipid TMM, but also the mycolic acid composition of each mycobacterial species, since it does not require any degradation process.


Abbreviations: MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; TDM, trehalose 6,6'-dimycolate; TMM, trehalose 6-monomycolate


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Cord factor (trehalose 6,6'-dimycolate; TDM) and trehalose 6-monomycolate (TMM) are among the most characteristic cell-surface components in mycobacteria and related micro-organisms (Bloch, 1950; Minnikin, 1982; Noll et al., 1956; Rastogi et al., 2001; Ryll et al., 2001). These molecules are believed to have a crucial role in the structure and function of the mycobacterial cell envelope, together with arabinogalactan mycolate or other mycoloyl glycolipids. Owing to their very long alkyl chain, they generate an extremely hydrophobic cell-surface environment and acid-fastness by their low fluidity and permeability. TMM is known to be a key precursor for the biosynthesis of TDM and, therefore, as an initial step, selected mycolic acids seem to be esterified to synthesize TMM (Barry et al., 1998; Walker et al., 1973). Recently, mycobacterial TMM has been reported to be a potent protein kinase C activator, resulting in tumour necrosis factor-{alpha} release in mouse lung tissues (Sueoka et al., 1995). Furthermore, in the host–parasite relationship, TDM, a trehalose diester of two mycolic acid molecules, has immunostimulatory (adjuvant) (Bekierkunst et al., 1971b; Silva et al., 1985), granulomagenic (Bekierkunst, 1968; Yarkoni & Rapp, 1977), antitumour (Azuma & Seya, 2001; Bekierkunst et al., 1971a; Orbach-Arbouys et al., 1983; Watanabe et al., 1999) and non-specific infection-prevention activities (Ribi et al., 1982; Yarkoni & Bekierkunst, 1976), via cytokine signalling processes, although in the mouse system they show lethal toxicity (Matsunaga et al., 1990, 1996; Ozeki et al., 1997; Yamagami et al., 2001). Recently, mycolic acids and mycoloyl glycolipids have been reported to be a unique lipid antigen restricted by the CD-1 antigen presentation molecule (Beckman et al., 1994; Moody et al., 1997, 1999; Niazi et al., 2001). Furthermore, it has been revealed that antisera against cord factor from either Mycobacterium tuberculosis or the M. aviumintracellulare group raised in rabbits preferentially recognized TDM from the species used as the antigen source (Fujiwara et al., 1999), suggesting that the underlying IgG antibodies were directed not against the carbohydrate backbone, but against the mycolic acid moiety; these results were subsequently confirmed with sera of human patients infected with M. tuberculosis or M. aviumintracellulare group (Enomoto et al., 1998; Pan et al., 1999). Therefore, determination of the precise molecular structure is essential. Since the mycolic acid composition of mycoloyl glycolipids differs greatly according to the species of mycobacteria and the physiological or growth conditions (Daffé et al., 1983; Minnikin, 1982; Minnikin et al., 1984a), structural determination of mycolic acids is important not only for chemical taxonomy, but also for virulence analysis of mycobacterial species. In fact, it has been reported that replacement of a cyclopropane ring by a double bond in the {alpha}-mycolate esterifying trehalose totally abolishes the formation of cords typical of virulent tubercle bacilli and thus affects the virulence of the mycobacterial strain (Dubnau et al., 2000; Glickman et al., 2000).

Since mycolic acids are very high molecular mass, branched-chain, 3-hydroxy fatty acids with 70 to 90 carbon atoms and possess a complicated alkyl chain with cyclopropane rings, methyl branches and a methoxy- or carbonyl group, it has been difficult to establish a strict relationship between molecular characteristics and immunopotentiating activities. Furthermore, these {alpha}-branched-chain 3-hydroxy fatty acids are unstable at high temperatures and are not amenable to gas chromatographic analysis. Wax ester-mycolates are easily broken down by alkaline hydrolysis to form dicarboxymycolic acid and secondary alcohol (Miquel et al., 1963; Toriyama et al., 1980, 1982). We intended to introduce a simpler, non-degradative technique for the molecular characterization of mycoloyl glycolipids, without hydrolysis or pyrolysis.

Previously, structural analysis of mycolic acids has mainly been performed by direct electron impact mass spectrometry (EI/MS) (Dubnau et al., 1997; Toubiana et al., 1979), fast atom bombardment mass spectrometry (FAB/MS) (Fujiwara et al., 1999; Watanabe et al., 2001) or gas chromatography mass spectrometry (GC/MS) (Tomiyasu & Yano, 1984; Toriyama et al., 1978; Yano et al., 1978) of mycolic acid ester derivatives or pyrolysis products (meromycolate). However, EI/MS and FAB/MS analysis were insufficient for molecular mass estimation of intact mycoloyl glycolipids such as TMM (molecular mass >1400 Da) and TDM (molecular mass >2600 Da), and GC/MS analysis is limited to shorter chain mycolic acids up to C50 to C60. Recently, Watanabe et al. (2001, 2002) described the separation and characterization of individual mycolic acids in representative mycobacteria and the determination of the location of functional groups in meromycolate chains in detail by FAB/MS and collision-induced dissociation mass spectrometry (CID/MS). Also, accurate molecular mass determination of mycolic acids was reported by highly sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Laval et al., 2001). However, molecular mass determination of intact mycoloyl glycolipids has not yet been performed successfully.

In the present study, we first applied MALDI-TOF mass spectrometry in a positive reflectron mode to molecular mass determination of intact mycoloyl glycolipid with molecular mass greater than 1400 Da. Prior to the complete analysis of TDM, we show the results of comparative MALDI-TOF mass analysis of intact TMM.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains.
Eleven strains from representative, human-virulent and non-virulent or rapidly and slowly growing mycobacteria were selected: M. tuberculosis H37Rv (=ATCC 27294), M. tuberculosis Aoyama B (=ATCC 31726), M. bovis BCG Tokyo 172 (=ATCC 35737), M. bovis BCG Connaught (=ATCC 35745), M. bovis No.10 (=ATCC 19210), M. intracellulare serotype 4 ATCC 35767, M. intracellulare serotype 16 ATCC 13950, M. kansasii ATCC 12478, M. smegmatis ATCC 19420, M. phlei ATCC 11758 and M. flavescens ATCC 14474.

Growth conditions.
M. tuberculosis, M. bovis (including BCG strains), M. smegmatis, M. phlei and M. flavescens were grown at 37 °C on Sauton medium as surface pellicles until early stationary phase. Members of the M. aviumintracellulare group and M. kansasii were grown on Middlebrook 7H9 medium with shaking at 37 °C until early stationary phase according to their growth rate.

Extraction, isolation and purification of TMM.
Bacterial culture was centrifuged after autoclaving at 121 °C for 15 min. Lipids were extracted from heat-killed, packed cells with 20 vols chloroform/methanol (2 : 1, v/v) three times with grinding. The two phases were separated in a funnel. After the lower phase containing major glycolipid was collected, the solvent was evaporated off with a rotary evaporator. The total lipids were first separated by solvent fractionation and then each acetone-soluble or chloroform-soluble or tetrahydrofuran-soluble or -insoluble fraction obtained was further separated by thin-layer chromatography (TLC) on silica plates (Uniplate; Analtech) with the solvent system of chloroform/methanol/water (90 : 10 : 1, by vol.) or chloroform/methanol/acetone/acetic acid (90 : 10 : 6 : 1, by vol.). Glycolipid spots were visualized with a 9 M H2SO4 spray followed by charring at 200 °C for analytical purposes or with iodine vapour for a few minutes for preparative purposes. TMM was recovered from the plate immediately after the iodine colour had disappeared by passing through a small glass column with the solvent chloroform/methanol (2 : 1, v/v). Finally, TMM was purified until a single spot was obtained by repeating TLC.

Analysis of mycolic acid methyl esters.
To confirm the possibility of direct analysis of intact molecular species composition of TMM by MALDI-TOF mass spectrometry, we also analysed mycolic acid methyl esters obtained from alkaline hydrolysis of TMM and other mycoloyl glycolipids. For this purpose, TMM, TDM and cell-wall-bound lipids were hydrolysed with 1·25 M NaOH in 90 % methanol at 70 °C for 1 h and the resultant mycolic acids were then extracted with n-hexane after acidification with HCl, followed by methylation with benzene/methanol/H2SO4 (10 : 20 : 1, by vol.) in a powerful fume cupboard under reduced pressure so as not to aspirate the vapour. Mycolic acid methyl esters from each lipid were fully separated into subclasses by TLC with the solvent system of benzene in the fume cupboard, considering the clear health hazards associated with benzene.

Sample preparation for mass spectrometry.
For MALDI-TOF mass analysis, TMM or mycolic acid methyl esters and 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix were dissolved in chloroform/methanol (2 : 1, v/v) at a concentration of 1 mg ml–1 (TMM or methyl mycolate) or 10 mg ml–1 (2,5-DHB). Aliquots of 5 µl of both samples and matrix were mixed and applied onto the sample plate as 1·5 µl droplets. The samples were then allowed to crystallize at room temperature.

Mass spectrometry analysis.
MALDI-TOF mass spectra (in the positive mode) were acquired on a Voyager-DE STR mass spectrometer (Applied Biosystems) with a pulse laser emitting at 337 nm. Samples were analysed in the reflectron mode with an accelerating voltage operating in positive ion mode of 20 kV. An external mass spectrum calibration was performed using calibration mixture 2 of the Sequazyme Peptide Mass Standards kit (Perseptive Biosystems), including known peptide standards in a mass range from 1290 to 5700 Da.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
TMM consists of one molecule of mycolic acid and one molecule of trehalose. The carbohydrate moiety is homogeneous; however, the mycolic acid composition has been considered to vary greatly by mycobacterial species. Based on the known mycolic acid subclasses ({alpha}-, {alpha}'-, methoxy-, keto-, wax ester- and epoxymycolic acid) and numbers of carbons and double bonds (or cyclopropane rings), we have deduced and identified each TMM molecular species.

MALDI-TOF mass spectrometry analysis of TMM from various mycobacterial species
Since natural mycolic acids occur generally as series of structurally related molecules differing from one another by two methylene units (28 a.m.u.), mycobacterial TMM generated complex mass spectra. Therefore, for the first approach in applying MALDI-TOF mass spectrometry to the analysis of TMM, we carefully took account of the calibration of pseudomolecular ions.

Fig. 1(a, b) shows positive MALDI-TOF mass spectra of TMM from two strains of M. tuberculosis. TMMs from M. tuberculosis H37Rv (Fig. 1a) and Aoyama B (Fig. 1b) showed similar pseudomolecular ion [M+Na]+ distributions ranging widely from m/z 1443 (nominal mass due to {alpha}-C75 TMM) to m/z 1725 (due to methoxy-C94 TMM). The characteristic feature is the biphasic distribution of pseudomolecular ions in this species, those due to {alpha}-mycoloyl TMM being in the lower mass range and those due to methoxy- or ketomycoloyl TMM in the higher mass range. A further characteristic is the occurrence of abundant odd-carbon-numbered (C77, C79, C81 and C83; in each case, the predominant member of each class is indicated in bold) {alpha}-mycoloyl TMM, which have not been reported so far in M. tuberculosis. Furthermore, these odd-carbon-numbered {alpha}-mycoloyl TMM showed monoenoic (or monocyclopropanoic) mycolate mass numbers, differing from even-carbon-numbered {alpha}-mycoloyl trehalose having dienoic (or dicyclopropanoic) mass numbers. This suggests that one of two cyclopropanation reactions occurs on the precursor destined to become dicyclopropyl {alpha}-mycolic acid at the level of TMM just before it is transferred to the cell wall, and not at the pre-{alpha}-meroacyl-S-acyl carrier protein level as previously considered (Takayama et al., 2005), although the precursor double-bond position should be clarified in future. TDM mycolate also contained a small but significant amount of odd-carbon-numbered monoenoic {alpha}-mycolates. Methoxy- and ketomycoloyl TMM were also identified based on the analytical results of [M+Na]+ ions of the intact TMM. In these TMM subclasses, C85, C87, C88 and C89 methoxy- and C87 and C88 ketomycoloyl TMM predominated.



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Fig. 1. MALDI-TOF mass spectra of TMM from M. tuberculosis complex strains M. tuberculosis H37Rv (a), M. tuberculosis Aoyama B (b), M. bovis BCG Tokyo (c) and M. bovis BCG Connaught (d). Samples were dissolved in chloroform/methanol (2 : 1, v/v) at a concentration of 1 mg ml–1 and applied on the sample plate as 0·75 µl droplets. 2,5-DHB was used as matrix. The accelerating voltage was 20 kV (TOF ion-trapping; positive reflectron mode).

 
TMMs from M. bovis No.10 and M. bovis BCG substrains differed from that of M. tuberculosis, although the subclass compositions of mycolic acids of M. bovis No.10 and M. bovis BCG Tokyo 172 were very similar, consisting of {alpha}-, methoxy- and ketomycolates. Fig. 1(c, d) shows distinctive distribution patterns of pseudomolecular mass ions of TMM from M. bovis BCG Tokyo 172 (Fig. 1c) and BCG Connaught (Fig. 1d). TMM of BCG Tokyo 172 consisted of even-carbon-numbered (C76 to C82) dienoic (or dicyclopropanoic) {alpha}-mycoloyl TMM centred at C78 and a smaller amount of odd-carbon-numbered (C77 to C81) monoenoic (or monocyclopropanoic) {alpha}-mycoloyl TMM, odd- (and even-) carbon-numbered (C81 to C91) methoxymycoloyl TMM centred at C85 and even- (and odd-) carbon-numbered (C79 to C89) ketomycoloyl TMM centred at C84. In contrast, BCG Connaught lacked methoxymycoloyl TMM entirely, as reported previously by Minnikin et al. (1984b). Instead, pseudomolecular mass ions due to {alpha}-mycoloyl TMM centred at C78 mycoloyl TMM and ions due to even- and odd-carbon-numbered ketomycoloyl TMM ranging from C82 to C91 were clearly demonstrated, thus forming the typical biphasic mass ion distribution pattern. Considering the relative intensity of mass ions due to {alpha}- and ketomycoloyl TMM, we can expect the corresponding accumulation of ketomycoloyl TMM, as reported by Dubnau et al. (1997).

Differing from M. tuberculosis and M. bovis, the M. aviumintracellulare group showed a unique pseudomolecular ion distribution due to the existence of wax ester-mycoloyl TMM, as shown in Fig. 2(a, b). In the lower mass range, substantial amounts of ions due to even-carbon-numbered dienoic {alpha}-mycoloyl TMM with C78 to C84 mycolate centred at C80 were found. However, in the higher mass range, a large amount of ions due to wax ester-mycoloyl TMM with odd- (and even-) carbon-numbered mycolate with C82 to C89 centred at C85 and ions due to ketomycoloyl TMM with the same carbon-numbered mycolate were demonstrated. A slight, but significant difference between M. aviumintracellulare serotypes 4 and 16 was observed in the relative amounts of keto- and wax ester-mycoloyl TMM.



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Fig. 2. MALDI-TOF mass spectra of TMM from the M. aviumintracellulare group and wax ester-mycolic acid-containing mycobacteria: M. aviumintracellulare serotype 4 (a), M. aviumintracellulare serotype 16 (b), M. phlei (c) and M. flavescens (d). Experimental conditions indicated in Fig. 1 were used.

 
M. phlei and M. flavescens, belonging to the rapidly growing mycobacteria and also to a group producing {alpha}-, keto- and wax ester-mycolates, showed a distinctive pattern in mass spectra. As shown in Fig. 2(c, d), the molecular species composition of {alpha}-mycoloyl TMM of M. phlei and M. flavescens was rather shorter, compared to those of the M. aviumintracellulare group; however, interestingly, keto- and wax ester-mycoloyl TMM possessed much shorter carbon chain mycolates than the latter species, based on pseudomolecular ions ranging from C74 to C86 centred around C81.

Tables 1 and 2 summarize the results of comparative studies of TMM molecular species from 11 strains of slowly and rapidly growing mycobacteria by MALDI-TOF mass analysis. Total numbers of carbons and double bonds (or cyclopropane rings) of major species of mycoloyl trehalose were determined by [M+Na]+ ions of {alpha}-, methoxy-, keto- and wax ester-mycoloyl TMM.


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Table 1. MALDI-TOF mass spectrometry data of individual mycolic acid types of TMM from representative species of the M. tuberculosis complex

Values represent the pseudomolecular mass [M+Na]+ of TMM. Types: I, {alpha} dienoic; II, {alpha} monoenoic; III, methoxy; IV, ketomycoloyl. Major homologues are shown in bold.

 

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Table 2. MALDI-TOF mass spectrometry data of the individual mycolic acid types of TMM from representative species of the M. aviumintracellulare group and rapidly growing mycobacteria

Type V, wax ester-mycoloyl TMM. Other details are given in the legend to Table 1.

 
{alpha}-Mycoloyl TMM from all mycobacterial species examined consisted of a series of even- and odd-carbon-numbered C75 to C85 mycolic acids. It was noted that, in the case of TMM, the ratios of even- and odd-carbon-numbered {alpha}-mycolate differed greatly according to the mycobacterial species. Furthermore, the homologues of even- or odd-carbon-numbered series may differ by 28 a.m.u.; however, the difference in mass number between even- and adjacent odd-carbon-numbered TMM must be 16 a.m.u. (between an even-numbered species and an odd-numbered species with one more carbon) or 12 a.m.u. (between an odd-numbered species and an even-numbered species with one more carbon). This indicates that even-carbon-numbered mycolates belong to dienoic or dicyclopropanoic series, while odd-carbon-numbered mycolates belong to monoenoic (plus monomethyl branch) or monocyclopropanoic series. For instance, the spectra of {alpha}-mycoloyl TMM from M. tuberculosis H37Rv and Aoyama B showed major mass ions at m/z 1472, 1500, 1528 and 1556 due to [M+Na]+ of C77, C79, C81 and C83, respectively, and peaks of lower abundance at m/z 1456, 1484, 1512, 1540 and 1568 due to C76, C78, C80, C82 and C84, respectively. The spectra of {alpha}-mycoloyl TMM from M. kansasii showed a similar mass pattern, having abundant odd-carbon-numbered monoenoic {alpha}-mycolates. In contrast, the mass spectra of {alpha}-mycolates of M. bovis TMM including BCG substrains showed [M+Na]+ ions due to major even-carbon-numbered dienoic or dicyclopropanoic (C76, C78, C80 and C82) series. A similar tendency in molecular species distribution in {alpha}-mycoloyl TMM was observed in the rapid grower group, such as M. phlei and M. smegmatis. To confirm these results, we carefully compared the {alpha}-mycolic acid molecular species composition of M. tuberculosis TMM by MALDI-TOF mass analysis of both intact TMM and mycolic acid methyl esters obtained from TMM, TDM and cell-wall-bound lipids. As shown in Table 3, {alpha}-mycolic acids from TMM contained abundant C77, C79, C81 and C83 monocyclopropanoic or monoenoic mycolic acids, and this was in accordance with the results of intact molecular analysis of TMM (Fig. 3). In contrast to TMM, TDM and cell-wall-bound lipids contained much smaller amounts of odd-carbon-numbered monoenoic {alpha}-mycolates, with more abundant even-carbon-numbered dienoic or dicyclopropanoic {alpha}-mycolate molecules. It was noted that the mycolic acid composition of each mycoloyl glycolipid in mycobacteria differs distinctively, and TMM contained a greater variety of mycolic acid molecular species, compared with TDM and cell-wall-bound lipids. To date, such a difference of mycolic acid molecular species composition among lipid classes has not been reported in mycobacteria. However, TMM is a key precursor for biosynthesis of mycoloyl glycolipids such as TDM or cell-wall-bound arabinose mycolate and, therefore, such a diversity of mycolic acid molecular species may exist. Since it appears that there are marked differences in the mycolic acid molecular species composition among mycoloyl glycolipid classes, it is especially interesting to investigate whether greater selection of a particular mycolic acid subclass or molecular species may occur at the biosynthetic stage for TDM or cell-wall-bound arabinogalactan mycolate.


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Table 3. MALDI-TOF mass spectrometry data of individual types of mycolic acid methyl esters from each mycoloyl glycolipid class in M. tuberculosis H37Rv

Details are given in the legend of Table 1.

 


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Fig. 3. Accurate mass measurement of {alpha}-mycolic acid methyl esters by MALDI-TOF mass analysis. {alpha}-Mycolic acid methyl esters of TMM from M. tuberculosis H37Rv (a), TDM from M. tuberculosis H37Rv (b) and bound lipid of cellular residues from M. tuberculosis H37Rv after organic solvent extraction (c). Experimental conditions indicated in Fig. 1 were used.

 
In the cases of methoxy- and ketomycoloyl TMM, the homologues of even- or odd-carbon-numbered mycolates differed by 28 a.m.u., and the difference of mass number between even- and adjacent odd-carbon-numbered mycoloyl TMM was exclusively 14 a.m.u. These results indicate that even- and odd-carbon-numbered mycolates belong to the same series, with the same number of double bonds or cyclopropanoic rings with or without a methyl branch.

Wax ester-mycolic acids are known to be a characteristic component in the M. aviumintracellulare group, M. phlei and other rapidly growing photochromogenic mycobacteria (Miquel et al., 1963). Owing to the alkali-instability of the intramolecular ester bond, intact molecule analysis of wax ester-mycolates has not been successful so far. Instead, analysis of dicarboxy mycolic acid and secondary alcohols derived from alkaline hydrolysis products of wax ester-mycolates has usually been performed (Toriyama et al., 1980, 1982). In the present investigation, we report the first results of molecular characterization of intact wax ester-mycoloyl TMM by MALDI-TOF mass spectrometry without any degradation or hydrolysis procedure. In the M. aviumintracellulare group, distinctive [M+Na]+ ions due to C83, C85 and C87 wax ester-mycoloyl TMM centred at C85 were clearly demonstrated at m/z 1586, 1614 and 1642 in the higher mass range, while, in M. phlei and M. flavescens, [M+Na]+ ions due to C78, C79, C80, C81, C82 and C83 wax ester-mycoloyl TMM were observed at m/z 1516, 1530, 1544, 1558, 1572 and 1586, respectively, in the lower mass range. Interestingly, as expected, numbers of carbons of keto- and wax ester-mycoloyl TMM were almost identical, reflecting the metabolic precursor–product relationship between the former and the latter, since the wax ester-mycolates are synthesized directly from ketomycolates by a biological Baeyer–Villiger type oxidation system (Toriyama et al., 1982).

The MALDI-TOF mass spectra of intact TMM extracted from each mycobacterium therefore give a characteristic fingerprint of the species (or subspecies) useful for chemical taxonomy or species identification and, furthermore, such molecular characterization may be especially important for the structure–immunomodulating activity relationship of cell-wall mycoloyl glycolipids for the host defence system.

Conclusions
Mycoloyl glycolipids such as TDM, TMM and arabinogalactan mycolate are extremely characteristic surface molecules in the mycobacterial cell wall. They play crucial roles not only in characterizing the mycobacterial surface structure, but also in affecting host immune responses as antigen, immunopotentiator or virulence factor at the site of infection. The present paper describes, for the first time, that direct MALDI-TOF mass spectrometry of TMM, the most simple mycoloyl glycolipid, can give precise information of the intact mycolic acid molecule without a degradation process. This technique is particularly promising for the analysis of more complicated mycoloyl glycolipids such as TDM or arabinogalactan polymycolate of the cell-wall skeleton, since it seems likely that the mycolic acid composition differs distinctively according to the mycobacterial species, growth conditions such as growth temperature and each glycolipid class, although analysis of TDM appears more laborious. Furthermore, the fast and easy separation of samples for MALDI-TOF mass spectrometry seems to be suitable for fingerprinting mycoloyl glycolipids from a given mycobacterial strain. It may also provide more information on the mechanism of biosynthesis of mycoloyl glycolipids in each mycobacterial species in the context of selective utilization of mycolic acid molecular species.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 26 November 2004; revised 28 January 2005; accepted 28 January 2005.



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