1 Japan BCG Central Laboratory, 3-1-5 Matsuyama, Kiyose-shi, Tokyo 204-0022, Japan
2 Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA
Correspondence
Yukiko Fujita
y-fujita{at}bcg.gr.jp
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ABSTRACT |
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INTRODUCTION |
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In our previous paper, we reported for the first time the direct MALDI-TOF MS analysis of trehalose monomycolate, a precurser of TDM biosynthesis, from 11 species of mycobacteria (Fujita et al., 2005). By the introduction of MALDI-TOF mass analysis of the intact glycolipid, the molecular characterization of ester-bound mycoloyl glycolipid of the mycobacterial cell envelope became possible. Although MALDI-TOF MS is a powerful tool, the structure of the intact TDM, with two molecules of mycolic acids, is very complex and its analysis would be very laborious. Therefore, in the present study, we first isolated the mycolic acid subclasses of TDM from nine species of representative mycobacteria and identified them by MS, and then based on these results we directly analysed the intact TDM from the same species of mycobacteria by MALDI-TOF MS for the first time.
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METHODS |
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Growth conditions.
M. tuberculosis, M. bovis (including BCG strains), M. phlei and M. flavescens were grown at 37 °C on Sauton medium as surface pellicles until early stationary phase. M. aviumintracellulare group and M. kansasii were grown on Middlebrook 7H9 medium with shaking at 37 °C until early stationary phase.
Extraction, isolation and purification of TDM.
The procedures as described for trehalose monomycolate in our previous paper (Fujita et al., 2005) were used.
Analysis of mycolic acid methyl esters from TDM.
Mycobacterial TDM without wax ester mycolic acid was hydrolysed with 1·25 M NaOH in 90 % methanol at 70 °C for 1 h, and the resultant mycolic acids were extracted with n-hexane after acidification with HCl, followed by methylation with benzene/methanol/H2SO4 (10 : 20 : 1, by vol.). For the separation of keto- and wax ester mycolate subclasses, TDM containing wax ester mycolic acid was added to 5 % tetrabutylammonium hydroxide (TBAH, 1 ml) and hydrolysed at 100 °C for over 8 h, then mixed with water/CH2Cl2/CH3I (10 : 20 : 1, by vol.) (Minnikin, 1988). After centrifugation, the lower organic phase was kept and washed with 1 M HCl and water. Finally, the resultant mycolic acids were extracted with diethyl ether after acidification with glacial acetic acid, followed by reduction of ketomycolate to hydroxymycolate with tetrahydrofuran (2 ml) and NaBH4 (10 mg) at 37 °C overnight. Mycolic acid methyl esters derived from each TDM were fully separated into subclasses by threefold TLC with benzene as solvent. For reasons of health and safety, experiments using benzene were done with care, in a high-power negative-pressure chamber.
Sample preparation for MS.
For MALDI-TOF mass analysis, mycolic acid methyl esters, TDM 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 ml1 (mycolic acid methyl esters and TDM) or 10 mg ml1 (2,5-DHB). Samples and matrix (5 µl each) were mixed and applied onto the sample plate as 1·5 µl droplets. The samples were then allowed to crystallize at room temperature.
Mass spectrometric analysis.
MALDI-TOF mass spectra (in the positive mode) were acquired as described by Fujita et al. (2005).
We determined the molecular mass of mycobacterial TDM based on the quasimolecular mass ions [M+Na]+ by the reflectron mode with higher resolution power. Since TDM is a high-molecular-mass (2500 Da) glycolipid, C172H330O17 [in the case of diketo 80-(monocyclopropanoic or monoenoic)-mycolyl TDM], there are marked differences between the nominal and accurate mass numbers in one molecule of TDM. If we calculate the mass of the above TDM, the nominal mass number becomes [(Cx172)+(Hx330)+(Ox172)]=2666, while the accurate number is [(12·0107x172)+(1·00794x330)+(15·9994x172)]=2670·4504, the difference being 4·4504. Therefore, we had to correct each molecular (nominal) mass number, and then determine the numbers of C, H and O experimentally.
Structural analysis of TDM.
Based on the analytical results of mycolic acids obtained after alkali or very mild alkali hydrolysis of parent TDM, the possible and most abundant combination(s) of mycolic acids were deduced.
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RESULTS AND DISCUSSION |
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As shown in Fig. 3, positive MALDI-TOF mass spectra of
-mycolic acid methyl esters of TDM from M. tuberculosis H37Rv showed the most abundant mass ion at m/z 1201 due to C80
-mycolate. Mass ions due to C78, C82, C84 and C86
-mycolates were also detected clearly. On the other hand, the mass spectra of methoxy- and ketomycolates were shifted higher, by about 100 a.m.u., corresponding to an increase of 57 carbon atoms and introduction of one oxygen atom. Thus, the most abundant mass ions are at m/z 1289 and 1317 due to C85 and C87 methoxymycolic acid and at m/z 1315 due to C87 ketomycolic acid. The carbon numbers of methoxy- and ketomycolate ranged between C80 and C92 in the former and between C79 and C91 in the latter in TDM from M. tuberculosis H37Rv. Essentially the same composition of subclasses and molecular species of mycolic acids were demonstrated in the case of TDM from M. tuberculosis Aoyama B.
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Differing from M. tuberculosis, M. bovis and M. kansasii, the TDM of the M. aviumintracellulare group possessed characteristic -, keto- and wax ester mycolic acid subclasses (Fig. 4
). Since the RF values of keto- and wax ester mycolic acid methyl esters were almost identical on TLC, and further, wax ester mycolates were broken down to dicarboxylic acid and secondary alcohol by the conventional alkali hydrolytic procedure, we methylated the free mycolic acids obtained after very mild alkali hydrolysis with TBAH, followed by NaBH4 reduction without cleavage of intramolecular ester bond of wax ester mycolate (Minnikin, 1988
). The resultant (k+H2)-mycolic acid methyl ester derived from ketomycolate was separated clearly from wax ester mycolic acid methyl ester on TLC. Major molecular species of
-mycolate of TDM from M. aviumintracellulare serotype 4 were identified as C80 (m/z 1201), C82 (m/z 1229) and C83 (m/z 1243)
-mycolate, indicating an
-mycolate composition of TDM resembling those of other slow-growing mycobacterial species. The major molecular species of (k+H2)-mycolate of TDM were C85 (m/z 1289) and C87 (m/z 1317), and those of wax ester mycolate of TDM were also C85 (m/z 1303) and C87 (m/z 1331), suggesting that these two subclasses are in precursorproduct relationships biosynthetically. Mass spectra of mycolate of TDM from M. aviumintracellulare serotype 16 also showed essentially the same results.
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Table 1 and Table 2
summarize the results of comparative MALDI-TOF mass analysis of mycolic acid of TDM from the nine species of mycobacteria. Total carbon and cyclopropane ring (or double bond) numbers of mycolic acids were determined by the mass ions of
-, methoxy-, keto- and wax ester mycolic acid, respectively.
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In this study, we first introduced very mild alkali hydrolysis with TBAH followed by NaBH4 reduction to isolate wax ester mycolate in an intact form and to detect ketomycolate as a reduced form (k+H2). Since the effect of such procedures on the intact molecules of mycolic acids has not been fully investigated, we compared the alkali hydrolysis products, especially ketomycolate obtained by classical and novel techniques. The results showed both ketomycolates to be essentially the same.
MALDI-TOF MS analysis of TDM
Generally, mycobacterial TDM consisted of a diverse mycolic acid molecular species with 6090 carbon atoms; thus the mass spectra of TDM showed wide ranges of distribution of mass ions. Based on the precise information on the mycolic acid composition of each TDM from nine representative species of mycobacteria, we have deduced possible combinations of two molecules of mycolic acids and TDM molecular structure.
M. tuberculosis complex and M. kansasii, belonging to the slow-growing mycobacteria, produce -, methoxy- and ketomycolic acid subclasses, among which there is a difference of five to eight carbons between
- and polar mycolates. Therefore, in one cluster of mass ions, ions due to di-
-mycolyl TDM were detected in the lowest mass range, ions due to
- and methoxy- or
- and ketomycolyl TDM in the middle, and ions due to dimethoxy-, methoxy- and keto- or diketomycolyl TDM in the highest mass range.
Fig. 5(a) shows positive MALDI-TOF mass spectra of TDM from M. tuberculosis H37Rv. Diverse pseudomolecular ions (mass ion, [M+Na]+) of TDM from both M. tuberculosis H37Rv and Aoyama B strains were detected, ranging from m/z 2601 (due to di-
78-mycolyl TDM) to m/z 3013 [due to methoxy (m)92 and keto (k)91(1)-mycolyl TDM] [(1) means monocyclopropanoic or monoenoic]. The characteristic feature is the normal and almost symmetric distribution of mass ions in these species centring at m/z 2773. Although this ion at m/z 2773 is the most abundant among the quasimolecular ions of TDM from M. tuberculosis, there were possibly multiple combinations of two molecules of mycolates. Based on the analytical results of pre-separated mycolic acid, ions at m/z 2773 was probably due to
78- and m89-,
80- and m87- or
82- and m85-mycolyl TDM, as shown in Table 3
. In the lower mass ranges, ions due to di-
-mycolyl TDM predominate, while in the middle mass ranges, ions due to
- and methoxy- or
- and ketomycolyl TDM were abundant. On the other hand, in the higher mass ranges, ions at m/z 2887 seemed to be due to m85- and k89(1)-, m87- and k87(1)- or m88- and k86(1)-mycolyl TDM, although there were numerous minor components with combinations of molecular species other than the major ones. Thus, in the TDM from M. tuberculosis, three groups of mass ion distributions, due to di-
-,
-, and polar and dipolar mycoloyl TDM, were demonstrated.
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Differing from the M. tuberculosis complex and M. kansasii, the M. aviumintracellulare group showed a unique mass ion distribution due to the occurrence of TDM containing wax ester mycolate besides - and ketomycolates, as shown in Fig. 7
. The lowest mass range of TDM from M. aviumintracellulare serotype 4 (Fig. 7a
) consisted mainly of small amounts of ions due to di-
-mycolyl TDM with C80, C82 and/or C83 (m/z 2657 due to di-
80-, m/z 2685 due to
80- and
82-, m/z 2713 due to di-
82-, m/z 2727 due to
82- and
83-mycolyl TDM) (see Table 4
). In the higher mass ranges, a large amount of ions due to TDM containing keto- and wax ester or di-wax ester mycolate were demonstrated clearly [m/z 2845 due to k85(1)- and wax ester(w)85-, m/z 2861 due to di-w85-, m/z 2873 due to k87(1)- and w85-, m/z 2889 due to w85- and w87-mycolyl TDM]. In the middle mass range, a large amount of ions due to TDM containing
- and keto- or
- and wax ester mycolate were detected distinctively [m/z 2759 due to
80- and w85-, m/z 2785 due to
83- and k85(1)-, m/z 2787 due to
80- and w87- or
82- and w85-mycolyl TDM]. Thus, in the case of TDM from M. aviumintracellulare serotype 4, the distribution of mass ions of TDM showed a triphasic pattern (m/z 26292727, 27312829 and 28452943). A similar pattern was also observed in the case of TDM from M. aviumintracellulare serotype 16 (Fig. 7b
). The mycolic acid subclass compositions of the TDM from M. aviumintracellulare serotypes 4 and 16 were almost the same, but the mass ion distribution of these types of TDM differed substantially. The mass ion distribution of TDM from M. aviumintracellulare serotype 16 was shifted lower than that of serotype 4, and TDM with di-
-mycolate molecules (m/z 2657 due to di-
80-mycolyl TDM) predominated.
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Conclusion
TDM is the most prominent and best-studied mycolic-acid-containing glycolipid of mycobacteria. The structure varies greatly among mycobacterial species, and the mycolyl moiety has been linked to toxicity, antigenicity or intracellular survival, thereby constituting potential virulence or immunostimulating mechanisms. In order to establish the structureactivity relationship, simple and precise analysis of the high-molecular-mass glycolipids is essential.
To establish the intact molecular structure analysis of TDM by MALDI-TOF MS, we first analysed mycolic acid subclasses after alkali hydrolysis of TDM. The results showed marked differences in subclasses and molecular species composition of mycolic acids according to the mycobacterial species. -Mycolic acid was a ubiquitous component among the mycobacterial species, with carbon numbers ranging from C74 to C88 and with two cyclopropane rings or equivalent double bonds. Ketomycolates were also widely distributed, with carbon numbers ranging from C76 to C91, but the ranges of the carbon number of ketomycolates differed greatly among the species. Methoxymycolates were a more characteristic component in particular species of mycobacteria such as M. tuberculosis, M. bovis BCG Tokyo 172 and M. kansasii. Wax ester mycolates were also specific for the M. aviumintracellulare group and chromogenic rapid growers such as M. phlei and M. flavescens, differing in the ranges of distribution of carbon numbers.
In the case of slow growers such as M. tuberculosis, M. bovis BCG, M. kansasii and the M. aviumintracellulare group, chain length of keto-, methoxy- or wax ester mycolate was much longer than those of -mycolate, while in the case of rapid growers such as M. phlei and M. flavescens, the chain lengths of both
- and polar mycolates were similar.
The carbon numbers of keto- and wax ester mycolic acid were almost identical, this reflecting the metabolic precursorproduct relationship between the former and the latter, since the wax ester mycolates are synthesized directly from ketomycolates by a direct biological BaeyerVilliger type oxidation system (Toriyama et al., 1982).
Based on such precise information on mycolic acids and their possible combination of two molecules of mycolic acids, we deduced the molecular structure of each TDM from the direct MALDI-TOF mass spectra. MALDI-TOF mass analysis enabled us to reveal whole molecular structures of TDM without a degradative procedure, especially the carbon, cyclopropane ring or double bond numbers, and oxygenated functions such as carbonyl, methoxy or intramolecular ester group (although the positions or configurations of cyclopropane rings or double bonds should be determined further). The MALDI-TOF mass fingerprint of the whole TDM molecule from a given mycobacterial strain appears to be a very promising tool for rapid analysis, facilitating the identification of mycobacterial species and the elucidation of the relationship between structure and immunopotentiating activity.
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REFERENCES |
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Beckman, E. M., Porcelli, S. A., Morita, C. T., Behar, S. M., Furlong, S. T. & Brenner, M. B. (1994). Recognition of a lipid antigen by CD1-restricted + T cells. Nature 372, 691694.[CrossRef][Medline]
Bloch, H. (1950). Studies on the virulence of tubercle bacilli; the relationship of the physiological state of the organisms to their pathogenicity. J Exp Med 92, 507526.
Crowe, L. M., Spargo, B. J., Ioneda, T., Beaman, B. L. & Crowe, J. H. (1994). Interaction of cord factor (,
'-trehalose-6,6'-dimycolate) with phospholipids. Biochim Biophys Acta 1194, 5360.[Medline]
Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle, M. A., Yu, K., Quemard, A., Smith, I. & Daffé, M. (2000). Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol Microbiol 36, 630637.[CrossRef][Medline]
Enomoto, K., Oka, S., Fujiwara, N., Okamoto, T., Okuda, Y., Maekura, R., Kuroki, T. & Yano, I. (1998). Rapid serodiagnosis of Mycobacterium avium-intracellulare complex infection by ELISA with cord factor (trehalose 6,6'-dimycolate), and serotyping using the glycopeptidolipid antigen. Microbiol Immunol 42, 689696.[Medline]
Fujita, Y., Naka, T., Doi, T. & Yano, I. (2005). Direct molecular mass determination of trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry. Microbiology 151, 14431452.[CrossRef][Medline]
Gotoh, K., Mitsuyama, M., Imaizumi, S., Kawamura, I. & Yano, I. (1991). Mycolic acid-containing glycolipid as a possible virulence factor of Rhodococcus equi for mice. Microbiol Immunol 35, 175185.[Medline]
Liu, J., Barry, C. E., 3rd, Besra, G. S. & Nikaido, H. (1996). Mycolic acid structure determines the fluidity of the mycobacterial cell wall. J Biol Chem 271, 2954529551.
Minnikin, D. E. (1982). Lipids: complex lipids, their chemistry, biosynthesis and roles. Part I: physiology of the mycobacteria. In The Biology of the Mycobacteria, pp. 95184. Edited by C. Ratledge & J. Stanford. London: Academic Press.
Minnikin, D. E. (1988). Isolation and purification of mycobacterial wall lipids. In Bacterial Cell Surface Techniques, pp. 125135. Edited by I. C. Hancock & I. R. Poxton. Chichester: Wiley.
Minnikin, D. E., Parlett, J. H., Magnusson, M., Ridell, M. & Lind, A. (1984). Mycolic acid patterns of representatives of Mycobacterium bovis BCG. J Gen Microbiol 130, 27332736.[Medline]
Moody, D. B., Reinhold, B. B., Guy, M. R. & 9 other authors (1997). Structural requirements for glycolipid antigen recognition by CD1b-restricted T cells. Science 278, 283286.
Moody, D. B., Reinhold, B. B., Reinhold, V. N., Besra, G. S. & Porcelli, S. A. (1999). Uptake and processing of glycosylated mycolates for presentation to CD1b-restricted T cells. Immunol Lett 65, 8591.[CrossRef][Medline]
Niazi, K., Chiu, M., Mendoza, R. & 8 other authors (2001). The A' and F' pockets of human CD1b are both required for optimal presentation of lipid antigens to T cells. J Immunol 166, 25622570.
Noll, H., Bloch, H., Asselineau, J. & Lederer, E. (1956). The chemical structure of the cord factor of Mycobacterium tuberculosis. Biochim Biophys Acta 20, 299309.[CrossRef][Medline]
Oswald, I. P., Dozois, C. M., Petit, J. F. & Lemaire, G. (1997). Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages. Infect Immun 65, 13641369.[Abstract]
Pan, J., Fujiwara, N., Oka, S., Maekura, R., Ogura, T. & Yano, I. (1999). Anti-cord factor (trehalose 6,6'-dimycolate) IgG antibody in tuberculosis patients recognizes mycolic acid subclasses. Microbiol Immunol 43, 863869.[Medline]
Rastogi, N., Legrand, E. & Sola, C. (2001). The mycobacteria: an introduction to nomenclature and pathogenesis. Rev Sci Tech 20, 2154.[Medline]
Ryll, R., Kumazawa, Y. & Yano, I. (2001a). Immunological properties of trehalose dimycolate (cord factor) and other mycolic acid-containing glycolipids a review. Microbiol Immunol 45, 801811.[Medline]
Ryll, R., Watanabe, K., Fujiwara, N., Takimoto, H., Hasunuma, R., Kumazawa, Y., Okada, M. & Yano, I. (2001b). Mycobacterial cord factor, but not sulfolipid, causes depletion of NKT cells and upregulation of CD1d1 on murine macrophages. Microbes Infect 3, 611619.[CrossRef][Medline]
Spargo, B. J., Crowe, L. M., Ioneda, T., Beaman, B. L. & Crowe, J. H. (1991). Cord factor (,
-trehalose 6,6'-dimycolate) inhibits fusion between phospholipid vesicles. Proc Natl Acad Sci U S A 88, 737740.
Toriyama, S., Imaizumi, S., Tomiyasu, I., Masui, M. & Yano, I. (1982). Incorporation of 18O into long-chain, secondary alcohols derived from ester mycolic acids in Mycobacterium phlei. Biochim Biophys Acta 712, 427429.
Ueda, S., Fujiwara, N., Naka, T., Sakaguchi, I., Ozeki, Y., Yano, I., Kasama, T. & Kobayashi, K. (2001). Structure-activity relationship of mycoloyl glycolipids derived from Rhodococcus sp. 4306. Microb Pathog 30, 9199.[CrossRef][Medline]
Received 27 April 2005;
revised 15 June 2005;
accepted 12 July 2005.
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