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Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

Nature Chemical Biology volume 8, pages 334–341 (2012)Cite this article

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Abstract

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.

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Figure 1: Structure and bactericidal activity of AU1235.
Figure 2: Effect of AU1235 on mycolic acid biosynthesis and transfer in M. tuberculosis.
Figure 3: mmpL3 is an essential gene of M. smegmatis.
Figure 4: Effect of repressing mmpL3 expression on the mycolic acid content of M. smegmatis.
Figure 5: A model for mycolic acid biosynthesis and transport across the inner membrane.

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References

  1. World Health Organization. Global Tuberculosis Control – Epidemiology, Strategy, Financing (World Health Organization, 2009).

  2. Global Alliance for TB Drug Development. Scientific blueprint for TB drug development. Tuberculosis (Edinb.) 81, 1–52 (2001).

  3. Center for Disease Control and Prevention (CDC). Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs worldwide, 2000–2004. MMWR Morb. Mortal. Wkly. Rep. 55, 301–305 (2006).

  4. Schroeder, E.K., de Souza, O.N., Santos, D.S., Blanchard, J.S. & Basso, L.A. Drugs that inhibit mycolic acid biosynthesis in Mycobacterium tuberculosis. Curr. Pharm. Biotechnol. 3, 197–225 (2002).

    Article  CAS  Google Scholar 

  5. Barry, C.E., Crick, D.C. & McNeil, M.R. Targeting the formation of the cell wall core of Mycobacterium tuberculosis. Infect. Disord. Drug Targets 7, 182–202 (2007).

    Article  CAS  Google Scholar 

  6. Alahari, A. et al. Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria. PLoS ONE 2, e1343 (2007).

    Article  Google Scholar 

  7. Daffé, M. & Draper, P. The envelope layers of mycobacteria with reference to their pathogenicity. Adv. Microb. Physiol. 39, 131–203 (1998).

    Article  Google Scholar 

  8. Barry, C.E. III et al. Mycolic acids: structure, biosynthesis and physiological functions. Prog. Lipid Res. 37, 143–179 (1998).

    Article  CAS  Google Scholar 

  9. Dubnau, E. et al. Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol. Microbiol. 36, 630–637 (2000).

    Article  CAS  Google Scholar 

  10. Glickman, M.S. Cording, cord factors and trehalose dimycolate. in The Mycobacterial Cell Envelope (eds. Daffé, M. & Reyrat, J.-M.) 63–73 (ASM Press, 2008).

  11. Hoffmann, C., Leis, A., Niederweis, M., Plitzko, J.M. & Engelhardt, H. Disclosure of the mycobacterial outer membrane: cryo-electron tomography and vitreous sections reveal the lipid bilayer structure. Proc. Natl. Acad. Sci. USA 105, 3963–3967 (2008).

    Article  CAS  Google Scholar 

  12. Zuber, B. et al. Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state. J. Bacteriol. 190, 5672–5680 (2008).

    Article  CAS  Google Scholar 

  13. Vander Beken, S. et al. Molecular structure of the Mycobacterium tuberculosis virulence factor, mycolic acid, determines the elicited inflammatory pattern. Eur. J. Immunol. 41, 450–460 (2011).

    Article  CAS  Google Scholar 

  14. Takayama, K., Wang, C. & Besra, G.S. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev. 18, 81–101 (2005).

    Article  CAS  Google Scholar 

  15. Gavalda, S. et al. The Pks13/FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium tuberculosis. J. Biol. Chem. 284, 19255–19264 (2009).

    Article  CAS  Google Scholar 

  16. Lea-Smith, D.J. et al. The reductase that catalyzes mycolic motif synthesis is required for efficient attachment of mycolic acids to arabinogalactan. J. Biol. Chem. 282, 11000–11008 (2007).

    Article  CAS  Google Scholar 

  17. Bhatt, A., Brown, A.K., Singh, A., Minnikin, D.E. & Besra, G.S. Loss of a mycobacterial gene encoding a reductase leads to an altered cell wall containing beta-oxo-mycolic acid analogs and accumulation of ketones. Chem. Biol. 15, 930–939 (2008).

    Article  CAS  Google Scholar 

  18. Woodruff, P.J. et al. Trehalose is required for growth of Mycobacterium smegmatis. J. Biol. Chem. 279, 28835–28843 (2004).

    Article  CAS  Google Scholar 

  19. Murphy, H.N. et al. The OtsAB pathway is essential for trehalose biosynthesis in Mycobacterium tuberculosis. J. Biol. Chem. 280, 14524–14529 (2005).

    Article  CAS  Google Scholar 

  20. Tropis, M. et al. The crucial role of trehalose and structurally related oligosaccharides in the biosynthesis and transfer of mycolic acids in Corynebacterineae. J. Biol. Chem. 280, 26573–26585 (2005).

    Article  CAS  Google Scholar 

  21. Kalscheuer, R., Weinrick, B., Veeraraghavan, U., Besra, G.S. & Jacobs, W.R. Jr. Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 107, 21761–21766 (2010).

    Article  CAS  Google Scholar 

  22. Besra, G.S. et al. Identification of the apparent carrier in mycolic acid synthesis. Proc. Natl. Acad. Sci. USA 91, 12735–12739 (1994).

    Article  CAS  Google Scholar 

  23. Sathyamoorthy, N. & Takayama, K. Purification and characterization of a novel mycolic acid exchange enzyme from Mycobacterium tuberculosis. J. Biol. Chem. 262, 13417–13423 (1987).

    CAS  PubMed  Google Scholar 

  24. Belisle, J.T. et al. Role of the major antigen of Mycobacterium tuberculosis in the cell wall biogenesis. Science 276, 1420–1422 (1997).

    Article  CAS  Google Scholar 

  25. Jackson, M. et al. 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 (1999).

    Article  CAS  Google Scholar 

  26. Puech, V. et al. Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis. Mol. Microbiol. 44, 1109–1122 (2002).

    Article  CAS  Google Scholar 

  27. Sanki, A.K., Boucau, J., Ronning, D.R. & Sucheck, S.J. Antigen 85C-mediated acyl-transfer between synthetic acyl donors and fragments of the arabinan. Glycoconj. J. 26, 589–596 (2009).

    Article  CAS  Google Scholar 

  28. Brown, J.R. et al. The structure–activity relationship of urea derivatives as anti-tuberculosis agents. Bioorg. Med. Chem. 19, 5585–5595 (2011).

    Article  CAS  Google Scholar 

  29. Camacho, L.R. et al. Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier. J. Biol. Chem. 276, 19845–19854 (2001).

    Article  CAS  Google Scholar 

  30. Morisseau, C. & Hammock, B.D. Epoxide hydrolases: Mechanisms, inhibitor designs, and biological roles. Annu. Rev. Pharmacol. Toxicol. 45, 311–333 (2005).

    Article  CAS  Google Scholar 

  31. Jain, M., Chow, E.D. & Cox, J.S. The MmpL protein family. in The Mycobacterial Cell Envelope (eds. Daffé, M. & Reyrat, J.-M.) 201–210 (ASM Press, 2008).

  32. Domenech, P., Reed, M.B. & Barry, C.E. III. Contribution of the Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance. Infect. Immun. 73, 3492–3501 (2005).

    Article  CAS  Google Scholar 

  33. Tekaia, F. et al. Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber. Lung Dis. 79, 329–342 (1999).

    Article  CAS  Google Scholar 

  34. De Rossi, E., Ainsa, J.A. & Riccardi, G. Role of mycobacterial efflux transporters in drug resistance: an unsolved question. FEMS Microbiol. Rev. 30, 36–52 (2006).

    Article  CAS  Google Scholar 

  35. Saier, M.H. Jr. & Paulsen, I.T. Phylogeny of multidrug transporters. Semin. Cell Dev. Biol. 12, 205–213 (2001).

    Article  CAS  Google Scholar 

  36. Murakami, S. Multidrug efflux transporter, AcrB–the pumping mechanism. Curr. Opin. Struct. Biol. 18, 459–465 (2008).

    Article  CAS  Google Scholar 

  37. Sennhauser, G., Bukowska, M.A., Briand, C. & Grutter, M.G. Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa. J. Mol. Biol. 389, 134–145 (2009).

    Article  CAS  Google Scholar 

  38. Cox, J.S., Chen, B., McNeil, M. & Jacobs, W.R. Jr. Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402, 79–83 (1999).

    Article  CAS  Google Scholar 

  39. Converse, S.E. et al. MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence. Proc. Natl. Acad. Sci. USA 100, 6121–6126 (2003).

    Article  CAS  Google Scholar 

  40. Domenech, P. et al. The role of MmpL8 in sulfatide biogenesis and virulence of Mycobacterium tuberculosis. J. Biol. Chem. 279, 21257–21265 (2004).

    Article  CAS  Google Scholar 

  41. Gilleron, M. et al. Diacylated sulfoglycolipids are novel mycobacterial antigens stimulating CD1-restricted T cells during infection with Mycobacterium tuberculosis. J. Exp. Med. 199, 649–659 (2004).

    Article  CAS  Google Scholar 

  42. Tullius, M.V. et al. Discovery and characterization of a unique mycobacterial heme acquisition system. Proc. Natl. Acad. Sci. USA 108, 5051–5056 (2011).

    Article  CAS  Google Scholar 

  43. Jackson, M., Stadthagen, G. & Gicquel, B. Long-chain multiple methyl-branched fatty acid-containing lipids of Mycobacterium tuberculosis: biosynthesis, transport, regulation and biological activities. Tuberculosis (Edinb.) 87, 78–86 (2007).

    Article  CAS  Google Scholar 

  44. Jain, M. & Cox, J.S. Interaction between polyketide synthase and transporter suggests coupled synthesis and export of virulence lipid in M. tuberculosis. PLoS Pathog. 1, e2 (2005).

    Article  Google Scholar 

  45. Jackson, M., Camacho, L.R., Gicquel, B. & Guilhot, C. Gene replacement and transposon delivery using the negative selection marker sacB. Methods Mol. Med. 54, 59–75 (2001).

    CAS  PubMed  Google Scholar 

  46. Guo, X.V. et al. Silencing essential protein secretion in Mycobacterium smegmatis by using tetracyclin repressors. J. Bacteriol. 189, 4614–4623 (2007).

    Article  CAS  Google Scholar 

  47. Korduláková, J. et al. Definition of the first mannosylation step in phosphatidylinositol synthesis: PimA is essential for growth of mycobacteria. J. Biol. Chem. 277, 31335–31344 (2002).

    Article  Google Scholar 

  48. Stadthagen, G. et al. p-hydroxybenzoic acid synthesis in Mycobacterium tuberculosis. J. Biol. Chem. 280, 40699–40706 (2005).

    Article  CAS  Google Scholar 

  49. Leistikow, R.L. et al. The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy. J. Bacteriol. 192, 1662–1670 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the NIAID and NIH grants AI085992, AI063054 and AI057836; the American Lebanese Syrian Associated Charities; and the Slovak Research and Development Agency under contract no. APVV-0441-10. We thank R. Dhiman for helpful scientific discussions and D. Dick for LC/MS analyses. Purified recombinant FbpC (Ag85C) was kindly provided by K. Dobos (Colorado State University).

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Authors and Affiliations

  1. Department of Microbiology, Mycobacteria Research Laboratories, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA

    Anna E Grzegorzewicz, Ha Pham, Vijay A K B Gundi, Michael S Scherman, Tamara Hess, Victoria Jones, Veronica Gruppo, Sarah E M Born, Sivagami Sundaram Chavadi, Anne J Lenaerts, Michael R McNeil & Mary Jackson

  2. Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Elton J North & Richard E Lee

  3. Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia

    Jana Korduláková

  4. Department of Entomology, University of California–Davis, Davis, California, USA

    Christophe Morisseau

  5. University of California Davis Cancer Center, University of California, Davis, California, USA

    Christophe Morisseau

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  1. Anna E Grzegorzewicz
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Contributions

A.E.G., A.J.L., J.K., R.E.L., M.R.M. and M.J. designed experiments. E.J.N. and C.M. synthesized compounds. A.E.G., H.P., V.A.K.B.G., M.S.S., V.J., T.H., S.S.C., J.K., S.E.M.B., V.G. and C.M. selected resistant mutants, constructed recombinant mycobacterial strains and performed MIC testing, enzymatic assays, metabolic labeling, lipid and mycolic acid analyses and genomic analyses. T.H. performed compound accumulation studies in M. tuberculosis. M.R.M., R.E.L. and M.J. wrote the paper.

Corresponding authors

Correspondence to Michael R McNeil or Mary Jackson.

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Grzegorzewicz, A., Pham, H., Gundi, V. et al. Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane. Nat Chem Biol 8, 334–341 (2012). https://doi.org/10.1038/nchembio.794

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