Transport Assays and Permeability in Pathogenic Mycobacteria

  • Marie-Antoinette Lanéelle
  • Mamadou Daffé
Part of the Methods in Molecular Biology book series (MIMB, volume 465)


Mycobacteria produce an effective permeability layer that consists of a mycolic acid–containing cell wall. This protection confers a natural resistance to many chemical agents and results in a low permeability toward both hydrophilic and lipophilic agents. The permeability of cells is classically measured using methods that generally need cell suspensions and are hazardous with pathogens (e.g., nutrient and antibiotic uptake). A major problem encountered with mycobacteria is their propensity to form aggregates; the addition of detergent to the cell suspension is not recommended as this disorganizes the cell envelope, rendering it more permeable to antibiotics. To circumvent this problem, growing cells are uniformly labeled with [3H]-uracil, allowing a quantification of the aliquots; then, the uptake of [14C]-labeled probes is followed during the first minutes. To avoid the generation of aerosols associated with the commonly used filtration methods, centrifugation through an oil mixture is the preferred alternative technique for use with Mycobacterium tuberculosis.


chenodeoxycholate uptake glycerol uptake Mycobacterium tuberculosis permeability barrier transport assays 


  1. 1.
    Daffé, M., and Draper, P. (1998) The envelope layers of mycobacteria with reference to their pathogenicity. Adv. Microbiol. Physiol. 39, 131–203.CrossRefGoogle Scholar
  2. 2.
    Goren, M. B. and Brennan P. J. (1979) Mycobacterial lipids: chemistry and biological activities, in Tuberculosis (Youmans, G. P., ed.) W.B. Saunders, Philadelphia, pp 63–193.Google Scholar
  3. 3.
    Brennan, P., and Nikaido, H. (1995) The envelope of mycobacteria. Annu. Rev. Biochem. 64, 29–63.PubMedCrossRefGoogle Scholar
  4. 4.
    Draper, P. (1998) The outer parts of the mycobacterial envelope as permeability barriers. Frontiers Biosci. 3:d1253–d1261.Google Scholar
  5. 5.
    Draper, P. and Daffé, M. (2005) The cell envelope of M. tuberculosis with special reference to the capsule and the outer permeability barrier, in Tuberculosis (Cole, S. T. et al., eds.) ASM Press, Washington, DC, pp 261–273.Google Scholar
  6. 6.
    Jarlier, V., Gutman L. and Nikaido, H. (1991) Interplay of cell wall barrier and beta-lactamase activity determines high resistance to beta-lactam antibiotics in Mycobacterium chelonae. Antimicrob. Agents Chemother. 35, 1937–1939.PubMedCrossRefGoogle Scholar
  7. 7.
    Trias, J., Jarlier, V., Benz, R. (1992) Porins in the cell wall of Mycobacterium chelonae. Science 258, 1479–1481.Google Scholar
  8. 8.
    Niederweis M. (2003) Mycobacterial porins—new channel proteins in unique outer membranes. Mol. Microbiol. 49, 1167–1177.PubMedCrossRefGoogle Scholar
  9. 9.
    Nikaido, H., Kim S. H., and Rosenberg E.Y. (1993) Physical organization of lipids in the cell wall of Mycobacterium chelonae. Mol. Microbiol. 8, 1025–1030.PubMedCrossRefGoogle Scholar
  10. 10.
    Jarlier, V. and Nikaido, H. (1994) Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiol. Lett. 123, 11–18.PubMedCrossRefGoogle Scholar
  11. 11.
    Liu, J., Barry C. E. III, 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
  12. 12.
    Nikaido, H., (2001) Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria. Semin. Cell Dev. Biol. 12, 215–223.PubMedCrossRefGoogle Scholar
  13. 13.
    Ortalo-Magné, A., Lemassu, A., Lanéelle, M.-A., Bardou, F., Silve, G., Gounon P., Marchal, G., and Daffé, M. (1996) Identification of the surface-exposed lipids on the cell envelopes of Mycobacterium tuberculosis and other mycobacterial species. J. Bacteriol. 178, 456–461.Google Scholar
  14. 14.
    Camacho, L. R., Constant, P., Raynaud, C., Lanéelle, M. A., Triccas, J., A., Gicquel, B., Daffé, M., and Guilhot, C. (2001) Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. J. Biol. Chem. 276, 19845–19854.PubMedCrossRefGoogle Scholar
  15. 15.
    Jarlier, V. and Nikaido, H. (1990) Permeability barrier to hydrophilic solutes in Mycobacterium chelonei. J. Bacteriol. 172, 1418–1423.PubMedGoogle Scholar
  16. 16.
    Jackson, M., Raynaud, C., Lanéelle, M.-A., Guilhot, C., Laurent-Winter, C., Ensergueix, D., Gicquel, B., and Daffé, M. (1999) Inactivation of the antigen 85C gene profoundly affects and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol. Microbiol. 31, 1573–1587.PubMedCrossRefGoogle Scholar
  17. 17.
    Liu, J., and Nikaido, H. (1999) A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates. Proc. Natl. Acad. Sci. U. S. A. 96, 4011–4016.PubMedCrossRefGoogle Scholar
  18. 18.
    Wang, L., Slayden, R. A., Barry, C. E. III and Liu, J. (2000) Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids. J. Biol. Chem. 10, 7224–7229.CrossRefGoogle Scholar
  19. 19.
    Liu, J., Rosenberg, Y. and Nikaido, H. (1995) Fluidity of the lipid domain of cell wall from Mycobacterium chelonae. Proc. Natl. Acad. Sci. U. S. A. 92, 11254–11258.PubMedCrossRefGoogle Scholar
  20. 20.
    Yuan, Y., Crane D. C., Musser, J. M., Sreevatsan, S. and Barry, C. E. III (1997) MMAS-1, the branch point between cis- and trans- cyclopropane-containing oxygenated mycolates in Mycobacterium tuberculosis. J. Biol. Chem. 272, 10041–10049.PubMedCrossRefGoogle Scholar
  21. 21.
    Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle, M.A., Yu, K., Quémard, A., Smith, I., and Daffé, M. (2000) Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol. Microbiol. 36, 630–637.PubMedCrossRefGoogle Scholar
  22. 22.
    Stephan, J., Mailaender, C., Etienne, G., Daffé, M. and Niederweis, M. (2004) Multidrug resistance of a porin deletion mutant of Mycobacterium smegmatis. Antimicrob. Agents Chemother. 48, 4163–4170.CrossRefGoogle Scholar
  23. 23.
    Bardou, F., Raynaud, C., Ramos, C., Lanéelle, M.A., and Lanéelle, G. (1998) Mechanism of isoniazid uptake in Mycobacterium tuberculosis. Microbiology 144, 2539–2544.PubMedCrossRefGoogle Scholar
  24. 24.
    Raynaud, C., Papavinasasundaram, K.G., Speight, R.A., Springer, B., Sander, P., Böttger, E.C., Colston, J.C. and Draper, P. (2002) The function of OmpATb, a pore- forming protein of Mycobacterium tuberculosis. Mol. Microbiol. 46, 191–201.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Molecular Mechanisms of Mycobacterial InfectionsInstitut of Pharmacology and Structural Biology, UMR 5089 of the National Centre for Scientific Research (CNRS) and University Paul Sabatier (Toulouse III)Toulouse IIIFrance
  2. 2.Department of ‘Molecular Mechanisms of Mycobacterial Infections’Institut de Pharmacologie et Biologie Structurale du Centre National de la Recherche Scientifique and Université Paul SabatierToulouse cedex 04France

Personalised recommendations