Advertisement

Analytical and Bioanalytical Chemistry

, Volume 405, Issue 23, pp 7415–7426 | Cite as

Characterization of mycobacterial triacylglycerols and monomeromycolyl diacylglycerols from Mycobacterium smegmatis biofilm by electrospray ionization multiple-stage and high-resolution mass spectrometry

  • Georgiana E. Purdy
  • Sophia Pacheco
  • John Turk
  • Fong-Fu HsuEmail author
Research Paper

Abstract

The storage of triacylglycerols (TAGs) is essential for non-replicating persistence relevant to survival and the re-growth of mycobacteria during their exit from non-replicating state stress conditions. However, the detailed structures of this lipid family in mycobacteria largely remain unexplored. In this contribution, we describe a multiple-stage linear ion-trap mass spectrometric approach with high resolution mass spectrometry toward direct structural analysis of the TAGs, including a novel lipid subclass previously defined as monomeromycolyl diacylglycerol (MMDAG) isolated from biofilm of Mycobacterium smegmatis, a rapidly growing, non-pathogenic mycobacterium that has been used as a tool for molecular analysis of mycobacteria. Our results demonstrate that the major isomer in each of the molecular species of TAGs and MMDAGs consists of the common structure in which Δ918:1- and 16:0-fatty acyl substituents are exclusively located at sn-1 and sn-2, respectively. Several isomers were found for most of the molecular species, and thus hundreds of structures are present in this lipid family. More importantly, this study revealed the structures of MMDAG, a novel subclass of TAG that has not been previously reported by direct mass spectrometric approaches.

Keywords

Apolar lipid Triacylglycerol Mycobacteria smegmatis Meromycolyl chain Mass spectrometry 

Abbreviations

ESI-MS

Electrospray ionization-MS

FA

Fatty acid

HRMS

High-resolution mass spectrometry

LIT

Linear ion-trap

MMDAG

Monomeromycolyl diacylglycerol

TAG

Triacylglycerol

Notes

Acknowledgments

This research is supported by US Public Health Service Grants P41-RR-00954, P60-DK-20579, and P30-DK56341 (mass spectrometry facility) and AI087840 (GEP). We acknowledge the technical assistance from Meei-Hua Lin and Alan Bohrer.

Supplementary material

216_2013_7179_MOESM1_ESM.pdf (276 kb)
ESM 1 (PDF 275 KB)

References

  1. 1.
    Sirakova TD, Dubey VS, Deb C, Daniel J, Korotkova TA, Abomoelak B, Kolattukudy PE (2006) Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress. Microbiology 152(9):2717–2725. doi: 10.1099/mic.0.28993-0 CrossRefGoogle Scholar
  2. 2.
    Low KL, Rao PSS, Shui G, Bendt AK, Pethe K, Dick T, Wenk MR (2009) Triacylglycerol utilization is required for regrowth of in vitro hypoxic nonreplicating Mycobacterium bovis Bacillus Calmette-Guerin. J Bacteriol 191(16):5037–5043. doi: 10.1128/jb.00530-09 CrossRefGoogle Scholar
  3. 3.
    Garton NJ, Christensen H, Minnikin DE, Adegbola RA, Barer MR (2002) Intracellular lipophilic inclusions of mycobacteria in vitro and in sputum. Microbiology 148(10):2951–2958Google Scholar
  4. 4.
    Daniel J, Maamar H, Deb C, Sirakova TD, Kolattukudy PE (2011) Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog 7(6):e1002093. doi: 10.1371/journal.ppat.1002093 CrossRefGoogle Scholar
  5. 5.
    Nakagawa H, Kashiwabara Y, Matsuki G (1976) Metabolism of triacylglycerol in Mycobacterium smegmatis. J Biochem 80(5):923–928Google Scholar
  6. 6.
    Ortalo-Magné A, Lemassu A, Lanéelle MA, Bardou F, Silve G, Gounon P, Marchal G, Daffé M (1996) Identification of the surface-exposed lipids on the cell envelopes of Mycobacterium tuberculosis and other mycobacterial species. J Bacteriol 178(2):456–461Google Scholar
  7. 7.
    Etienne G, Laval F, Villeneuve C, Dinadayala P, Abouwarda A, Zerbib D, Galamba A, Daffé M (2005) The cell envelope structure and properties of Mycobacterium smegmatis mc2155: is there a clue for the unique transformability of the strain? Microbiology 151(6):2075–2086. doi: 10.1099/mic.0.27869-0 CrossRefGoogle Scholar
  8. 8.
    Kremer L, De Chastellier C, Dobson G, Gibson KJC, Bifani P, Balor S, Gorvel J-P, Locht C, Minnikin DE, Besra GS (2005) Identification and structural characterization of an unusual mycobacterial monomeromycolyl-diacylglycerol. Mol Microbiol 57(4):1113–1126. doi: 10.1111/j.1365-2958.2005.04717.x CrossRefGoogle Scholar
  9. 9.
    Kim M-J, Wainwright HC, Locketz M, Bekker L-G, Walther GB, Dittrich C, Visser A, Wang W, Hsu F-F, Wiehart U, Tsenova L, Kaplan G, Russell DG (2010) Caseation of human tuberculosis granulomas correlates with elevated host lipid metabolism. EMBO Mol Med 2(7):258–274. doi: 10.1002/emmm.201000079 CrossRefGoogle Scholar
  10. 10.
    Daniel J, Maamar H, Deb C, Sirakova TD, Kolattukudy PE (2011) Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog 7(6):e1002093CrossRefGoogle Scholar
  11. 11.
    Reed MB, Gagneux S, DeRiemer K, Small PM, Barry CE (2007) The W-Beijing lineage of Mycobacterium tuberculosis overproduces triglycerides and has the DosR dormancy regulon constitutively upregulated. J Bacteriol 189(7):2583–2589. doi: 10.1128/jb.01670-06 CrossRefGoogle Scholar
  12. 12.
    Hsu F-F, Turk J (1999) Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisionally activated dissociation on a triple stage quadrupole instrument. J Am Soc Mass Spectrom 10(7):587–599. doi: 10.1016/s1044-0305(99)00035-5 CrossRefGoogle Scholar
  13. 13.
    Hsu F-F, Turk J (2010) Electrospray ionization multiple-stage linear ion-trap mass spectrometry for structural elucidation of triacylglycerols: assignment of fatty acyl groups on the glycerol backbone and location of double bonds. J Am Soc Mass Spectrom 21(4):657–669. doi: 10.1016/j.jasms.2010.01.007 CrossRefGoogle Scholar
  14. 14.
    Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR Jr (1990) Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4(11):1911–1919CrossRefGoogle Scholar
  15. 15.
    Ojha AK, Baughn AD, Sambandan D, Hsu T, Trivelli X, Guerardel Y, Alahari A, Kremer L, Jacobs WR, Hatfull GF (2008) Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol Microbiol 69(1):164–174. doi: 10.1111/j.1365-2958.2008.06274.x CrossRefGoogle Scholar
  16. 16.
    Hsu FF, Turk J (2008) Elucidation of the double-bond position of long-chain unsaturated fatty acids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization. J Am Soc Mass Spectrom 19(11):1673–1680CrossRefGoogle Scholar
  17. 17.
    Walker RW, Barakat H, Hung JG (1970) The positional distribution of fatty acids in the phospholipids and triglycerides of Mycobacterium smegmatis and M. bovis BCG. Lipids 5(8):684–691CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Georgiana E. Purdy
    • 2
  • Sophia Pacheco
    • 2
  • John Turk
    • 1
  • Fong-Fu Hsu
    • 1
    Email author
  1. 1.Mass Spectrometry Resource, Division of Endocrinology, Diabetes, Metabolism, and Lipid research, Department of Internal MedicineWashington University School of MedicineSt. LouisUSA
  2. 2.Department of Molecular Microbiology & ImmunologyOregon Health & Science UniversityPortlandUSA

Personalised recommendations