Intact molecular characterization of cord factor (trehalose 6,6'-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry

• Published in: Microbiology (Society for General Microbiology) Vol. 151; no. 10; pp. 3403 - 3416
• Main Authors: Fujita, Yukiko, Naka, Takashi, McNeil, Michael R, Yano, Ikuya
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.10.2005; Society for General Microbiology
• Subjects: Animals; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cattle; Cord Factors - analysis; Cord Factors - genetics; Cord Factors - isolation & purification; Fundamental and applied biological sciences. Psychology; Genetics; Humans; Microbiology; Molecular Structure; Mycobacterium - chemistry; Mycobacterium - classification; Mycobacterium - growth & development; Mycobacterium avium Complex - chemistry; Mycobacterium avium-intracellulare; Mycobacterium bovis; Mycobacterium bovis - chemistry; Mycobacterium kansasii; Mycobacterium phlei; Mycobacterium phlei - chemistry; Mycobacterium tuberculosis; Mycobacterium tuberculosis - chemistry; Nontuberculous Mycobacteria - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Characterization; Thin layer chromatography; Mycobacterium; Data analysis; Mycobacterium tuberculosis; Trehalose; Mycobacteriales; Mycobacteriaceae; Bacteria; Actinomycetes; Mycobacterium intracellulare; Mass spectrometry
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.28158-0

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 Cord factor (trehalose 6,6'-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 µg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [ M +Na] + . Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H 37 Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with -, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of -, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens , the mass ion distribution due to polar mycolates was shifted lower than that from the Mycobacterium avium–intracellulare group. Since the physico-chemical properties and antigenic structure of mycolic acid of TDM affect the host immune responses profoundly, the molecular characterization of TDM by MALDI-TOF mass analysis may give very useful information on the relationship of glycolipid structure to its biological activity. Abbreviations: 2,5-DHB, 2,5-dihydroxybenzoic acid; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; TBAH, tetrabutylammonium hydroxide; TDM, trehalose 6,6'-dimycolate; Mycolic acid designations: (0), saturated; (1), monocylcopropanoic or monoenoic; k, keto; (k+H 2 ), reduced keto; m, methoxy; w, wax ester