Journal of Microbiology

, Volume 51, Issue 5, pp 619–626 | Cite as

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

  • Sally A. Cantrell
  • Michael D. Leavell
  • Olivera MarjanovicEmail author
  • Anthony T. Iavarone
  • Julie A. Leary
  • Lee W. Riley
Microbial Physiology and Biochemistry


The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen’s persistence.


mce α-mycolates methoxymycolates ketomycolates mass spectrometry thin layer chromatography 


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  1. Asselineau, C., Asselineau, J., Laneelle, G., and Laneelle, M.A. 2002. The biosynthesis of mycolic acids by Mycobacteria: current and alternative hypotheses. Prog. Lipid Res.41, 501–523.PubMedCrossRefGoogle Scholar
  2. Asselineau, J. and Lederer, E. 1950. Structure of the mycolic acids of Mycobacteria. Nature166, 782–783.PubMedCrossRefGoogle Scholar
  3. Barry, C.E., 3rd, Lee, R.E., Mdluli, K., Sampson, A.E., Schroeder, B.G., Slayden, R.A., and Yuan, Y. 1998. Mycolic acids: structure, biosynthesis and physiological functions. Prog. Lipid Res.37, 143–179.PubMedCrossRefGoogle Scholar
  4. Brennan, P.J. 1989. Structure of mycobacteria: recent developments in defining cell wall carbohydrates and proteins. Rev. Infect. Dis.11Suppl 2, S420–430.PubMedCrossRefGoogle Scholar
  5. Brennan, P.J. and Nikaido, H. 1995. The envelope of mycobacteria. Annu. Rev. Biochem.64, 29–63.PubMedCrossRefGoogle Scholar
  6. Casali, N. and Riley, L.W. 2007. A phylogenomic analysis of the Actinomycetales mce operons. BMC Genomics8, 60.PubMedCrossRefGoogle Scholar
  7. Casali, N., White, A.M., and Riley, L.W. 2006. Regulation of the Mycobacterium tuberculosis mce1 operon. J. Bacteriol.188, 441–449.PubMedCrossRefGoogle Scholar
  8. Chitale, S., Ehrt, S., Kawamura, I., Fujimura, T., Shimono, N., Anand, N., Lu, S., Cohen-Gould, L., and Riley, L.W. 2001. Recombinant Mycobacterium tuberculosis protein associated with mammalian cell entry. Cell Microbiol.3, 247–254.PubMedCrossRefGoogle Scholar
  9. Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry, C.E., 3rd, andet al. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature393, 537–544.PubMedCrossRefGoogle Scholar
  10. Converse, S.E., Mougous, J.D., Leavell, M.D., Leary, J.A., Bertozzi, C.R., and Cox, J.S. 2003. MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence. Proc. Natl. Acad. Sci. USA100, 6121–6126.PubMedCrossRefGoogle Scholar
  11. De Koning, L.J., Nibbering, N.M.M., van Orden, S.L., and Laukien, F.H. 1997. Mass selection of ions in a Fourier ion cyclotron resonance trap using correlated harmonic excitation fields (CHEF). Int. J. Mass Spec. Ion. Proc.165/166, 209–219.CrossRefGoogle Scholar
  12. Dobson, G.M.D., Minnikin, S.M., Parlett, J.H., Goodfellow, M., Ridell, M., and Magnusson, M. 1985. Analysis of complex mycobacterial lipids, pp. 237–265. In Minnikin, D.E. (ed.), Chemical Methods in Bacterial Systematics, Academic Press, London, UK.Google Scholar
  13. Dubnau, E., Chan, J., Raynaud, C., Mohan, V.P., Laneelle, M.A., Yu, K., Quemard, A., Smith, I., and Daffe, M. 2000. Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol. Microbiol.36, 630–637.PubMedCrossRefGoogle Scholar
  14. Dunphy, K.Y., Senaratne, R.H., Masuzawa, M., Kendall, L.V., and Riley, L.W. 2010. Attenuation of Mycobacterium tuberculosis functionally disrupted in a fatty acyl-coenzyme A synthetase gene fadD5. J. Infect. Dis.201, 1232–1239.PubMedCrossRefGoogle Scholar
  15. Gauthier, J.W., Trautman, T.R., and Jacobson, D.B. 1991. Sustained off-resonance irradiation for collision-activated dissociation involving Fourier transform mass spectrometry. Collision-activated dissociation technique that emulates infrared multiphoton dissociation. Anal. Chim. Acta.246, 211–225.CrossRefGoogle Scholar
  16. Glickman, M.S., Cox, J.S., and Jacobs, W.R. Jr. 2000. A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol. Cell.5, 717–727.PubMedCrossRefGoogle Scholar
  17. Goren, M.B. 1972. Mycobacterial lipids: selected topics. Bacteriol. Rev.36, 33–64.PubMedGoogle Scholar
  18. Harboe, M., Christensen, A., Ahmad, S., Ulvund, G., Harkness, R.E., Mustafa, A.S., and Wiker, H.G. 2002. Cross-reaction between mammalian cell entry (Mce) proteins of Mycobacterium tuberculosis. Scand. J. Immunol.56, 580–587.PubMedCrossRefGoogle Scholar
  19. Harris, D. 1995. Quantitative Chemical Analysis, 4th ed. W.H. Freeman and Co., New York, N.Y., USA.Google Scholar
  20. Jackson, M., Raynaud, C., Laneelle, M.A., Guilhot, C., Laurent-Winter, C., Ensergueix, D., Gicquel, B., and Daffe, M. 1999. Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol. Microbiol.31, 1573–1587.PubMedCrossRefGoogle Scholar
  21. Karakousis, P.C., Bishai, W.R., and Dorman, S.E. 2004. Mycobacterium tuberculosis cell envelope lipids and the host immune response. Cell. Microbiol.6, 105–116.PubMedCrossRefGoogle Scholar
  22. Liu, J., Barry, C.E., 3rd, Besra, G.S., and Nikaido, H. 1996. Mycolic acid structure determines the fluidity of the mycobacterial cell wall. J. Biol. Chem.271, 29545–29551.PubMedCrossRefGoogle Scholar
  23. McNeil, M., Daffe, M., and Brennan, P.J. 1991. Location of the mycolyl ester substituents in the cell walls of mycobacteria. J. Biol. Chem.266, 13217–13223.PubMedGoogle Scholar
  24. Minnikin, D.E. 1982. Lipids: Complex lipids, their chemistry, biosynthesis and role, pp. 95–184. In R. C. a. S. J. (ed.), The Biology of Mycobacteria, vol. 1. Academic Press, London, UK.Google Scholar
  25. Noll, H., Bloch, H., Asselineau, J., and Lederer, E. 1956. The chemical structure of the cord factor of Mycobacterium tuberculosis. Biochim. Biophys. Acta.20, 299–309.PubMedCrossRefGoogle Scholar
  26. Ojha, A.K., Baughn, A.D., Sambandan, D., Hsu, T., Trivelli, X., Guerardel, Y., Alahari, A., Kremer, L., Jacobs, W.R.Jr., and Hatfull, G.F. 2008. Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol. Microbiol.69, 164–174.PubMedCrossRefGoogle Scholar
  27. Ojha, A.K., Trivelli, X., Guerardel, Y., Kremer, L., and Hatfull, G.F. 2010. Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms. J. Biol. Chem.285, 17380–17389.PubMedCrossRefGoogle Scholar
  28. Pandey, A.K. and Sassetti, C.M. 2008. Mycobacterial persistence requires the utilization of host cholesterol. Proc. Natl. Acad. Sci. USA105, 4376–4380.PubMedCrossRefGoogle Scholar
  29. Parish, T. and Stoker, N.G. 1998. Mycobacteria Protocols. Chapter 8, pp. 98–99, vol. 101. Humana Press, Totowa, New Jersey, USA.CrossRefGoogle Scholar
  30. Parish, T. and Stoker, N.G. 2000. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology146, 1969–1975.PubMedGoogle Scholar
  31. Payne, K., Sun, Q., Sacchettini, J., and Hatfull, G.F. 2009. Mycobacteriophage lysin B is a novel mycolylarabinogalactan esterase. Mol. Microbiol.73, 367–381.PubMedCrossRefGoogle Scholar
  32. Rao, V., Fujiwara, N., Porcelli, S.A., and Glickman, M.S. 2005. Mycobacterium tuberculosis controls host innate immune activation through cyclopropane modification of a glycolipid effector molecule. J. Exp. Med.201, 535–543.PubMedCrossRefGoogle Scholar
  33. Rao, V., Gao, F., Chen, B., Jacobs, W.R.Jr., and Glickman, M.S. 2006. Trans-cyclopropanation of mycolic acids on trehalose dimycolate suppresses Mycobacterium tuberculosis-induced inflammation and virulence. J. Clin. Invest.116, 1660–1667.PubMedCrossRefGoogle Scholar
  34. Shimono, N., Morici, L., Casali, N., Cantrell, S., Sidders, B., Ehrt, S., and Riley, L.W. 2003. Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon. Proc. Natl. Acad. Sci. USA100, 15918–15923.PubMedCrossRefGoogle Scholar
  35. Singh, A., Crossman, D.K., Mai, D., Guidry, L., Voskuil, M.I., Renfrow, M.B., and Steyn, A.J. 2009. Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response. PLoS Pathog.5, e1000545.PubMedCrossRefGoogle Scholar
  36. Song, H., Sandie, R., Wang, Y., Andrade-Navarro, M.A., and Niederweis, M. 2008. Identification of outer membrane proteins of Mycobacterium tuberculosis. Tuberculosis (Edinb).88, 526–544.PubMedCrossRefGoogle Scholar
  37. Takayama, K., Wang, C., and Besra, G.S. 2005. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev.18, 81–101.PubMedCrossRefGoogle Scholar
  38. Tekaia, F., Gordon, S.V., Garnier, T., Brosch, R., Barrell, B.G., and Cole, S.T. 1999. Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber. Lung Dis.79, 329–342.PubMedCrossRefGoogle Scholar
  39. Trivedi, O.A., Arora, P., Sridharan, V., Tickoo, R., Mohanty, D., and Gokhale, R.S. 2004. Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria. Nature428, 441–445.PubMedCrossRefGoogle Scholar
  40. Uchida, Y., Casali, N., White, A., Morici, L., Kendall, L.V., and Riley, L.W. 2007. Accelerated immunopathological response of mice infected with Mycobacterium tuberculosis disrupted in the mce1 operon negative transcriptional regulator. Cell. Microbiol.9, 1275–1283.PubMedCrossRefGoogle Scholar
  41. WHO. 2009. Global tuberculosis control: a short update to the 2009 report. Geneva.Google Scholar
  42. Yang, Y., Bhatti, A., Ke, D., Gonzalez-Juarrero, M., Lenaerts, A., Kremer, L., Guerardel, Y., Zhang, P., and Ojha, A.K. 2013. Exposure to a cutinase-like serine esterase triggers rapid lysis of multiple mycobacterial species. J. Biol. Chem.288, 382–392.PubMedCrossRefGoogle Scholar
  43. Yuan, Y., Zhu, Y., Crane, D.D., and Barry, C.E.3rd. 1998. The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis. Mol. Microbiol.29, 1449–1458.PubMedCrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Sally A. Cantrell
    • 1
  • Michael D. Leavell
    • 2
    • 3
  • Olivera Marjanovic
    • 1
    Email author
  • Anthony T. Iavarone
    • 4
  • Julie A. Leary
    • 2
  • Lee W. Riley
    • 1
  1. 1.Division of Infectious Diseases and Vaccinology, School of Public HealthUniversity of CaliforniaBerkeleyUSA
  2. 2.Genome and Biomedical Sciences FacilityUniversity of CaliforniaDavisUSA
  3. 3.Amyris, Inc.EmeryvilleUSA
  4. 4.QB3/Chemistry Mass Spectrometry FacilityUniversity of CaliforniaBerkeleyUSA

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