1887

Abstract

Mycolic acids are essential for the survival, virulence and antibiotic resistance of the human pathogen . Inhibitors of mycolic acid biosynthesis, such as isoniazid and ethionamide, have been used as efficient drugs for the treatment of tuberculosis. However, the increase in cases of multidrug-resistant tuberculosis has prompted a search for new targets and agents that could also affect synthesis of mycolic acids. In mycobacteria, the acyl-CoA carboxylases (ACCases) provide the building blocks for fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I products by the FAS II complex to produce meromycolic acids. By generating a conditional mutant in the gene of we demonstrated that AccD6 is the essential carboxyltransferase component of the ACCase 6 enzyme complex implicated in the biosynthesis of malonyl-CoA, the substrate of the two FAS enzymes of species. Based on the conserved structure of the AccD5 and AccD6 active sites we screened several inhibitors of AccD5 as potential inhibitors of AccD6 and found that the ligand NCI-172033 was capable of inhibiting AccD6 with an IC of 8 μM. The compound showed bactericidal activity against several pathogenic species by producing a strong inhibition of both fatty acid and mycolic acid biosynthesis at minimal inhibitory concentrations. Overexpression of in conferred resistance to NCI-172033, confirming AccD6 as the main target of the inhibitor. These results define the biological role of a key ACCase in the biosynthesis of membrane and cell envelope fatty acids, and provide a new target, AccD6, for rational development of novel anti-mycobacterial drugs.

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2009-08-01
2019-10-22
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References

  1. Bhatt, A., Fujiwara, N., Bhatt, K., Gurcha, S. S., Kremer, L., Chen, B., Chan, J., Porcelli, S. A., Kobayashi, K. & other authors ( 2007; ). Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. Proc Natl Acad Sci U S A 104, 5157–5162.[CrossRef]
    [Google Scholar]
  2. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  3. Bramwell, H., Hunter, I. S., Coggins, J. R. & Nimmo, H. G. ( 1996; ). Propionyl-CoA carboxylase from Streptomyces coelicolor A3(2): cloning of the gene encoding the biotin-containing subunit. Microbiology 142, 649–655.[CrossRef]
    [Google Scholar]
  4. Brennan, P. J. & Nikaido, H. ( 1995; ). The envelope of mycobacteria. Annu Rev Biochem 64, 29–63.[CrossRef]
    [Google Scholar]
  5. Chopra, K. ( 1996; ). Multi-drug resistant tuberculosis. Indian J Pediatr 63, 159–162.[CrossRef]
    [Google Scholar]
  6. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S. & other authors ( 1998; ). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef]
    [Google Scholar]
  7. Connell, N. D. ( 1994; ). Mycobacterium: isolation, maintenance, transformation, and mutant selection. Methods Cell Biol 45, 107–125.
    [Google Scholar]
  8. Corbett, E. L. & De Cock, K. M. ( 1996; ). Tuberculosis in the HIV-positive patient. Br J Hosp Med 56, 200–204.
    [Google Scholar]
  9. Cronan, J. E., Jr & Waldrop, G. L. ( 2002; ). Multi-subunit acetyl-CoA carboxylases. Prog Lipid Res 41, 407–435.[CrossRef]
    [Google Scholar]
  10. Daniel, J., Oh, T. J., Lee, C. M. & Kolattukudy, P. E. ( 2007; ). AccD6, a member of the Fas II locus, is a functional carboxyltransferase subunit of the acyl-coenzyme A carboxylase in Mycobacterium tuberculosis. J Bacteriol 189, 911–917.[CrossRef]
    [Google Scholar]
  11. Dhiman, R. K., Schulbach, M. C., Mahapatra, S., Baulard, A. R., Vissa, V., Brennan, P. J. & Crick, D. C. ( 2004; ). Identification of a novel class of ω,E,E-farnesyl diphosphate synthase from Mycobacterium tuberculosis. J Lipid Res 45, 1140–1147.[CrossRef]
    [Google Scholar]
  12. Diacovich, L., Peiru, S., Kurth, D., Rodriguez, E., Podesta, F., Khosla, C. & Gramajo, H. ( 2002; ). Kinetic and structural analysis of a new group of acyl-CoA carboxylases found in Streptomyces coelicolor A3(2). J Biol Chem 277, 31228–31236.[CrossRef]
    [Google Scholar]
  13. Erfle, J. D. ( 1973; ). Acetyl-CoA and propionyl-CoA carboxylation by Mycobacterium phlei. Partial purification and some properties of the enzyme. Biochim Biophys Acta 316, 143–155.[CrossRef]
    [Google Scholar]
  14. Freiberg, C., Brunner, N. A., Schiffer, G., Lampe, T., Pohlmann, J., Brands, M., Raabe, M., Habich, D. & Ziegelbauer, K. ( 2004; ). Identification and characterization of the first class of potent bacterial acetyl-CoA carboxylase inhibitors with antibacterial activity. J Biol Chem 279, 26066–26073.[CrossRef]
    [Google Scholar]
  15. Gago, G., Kurth, D., Diacovich, L., Tsai, S. C. & Gramajo, H. ( 2006; ). Biochemical and structural characterization of an essential acyl coenzyme A carboxylase from Mycobacterium tuberculosis. J Bacteriol 188, 477–486.[CrossRef]
    [Google Scholar]
  16. Gande, R., Gibson, K. J., Brown, A. K., Krumbach, K., Dover, L. G., Sahm, H., Shioyama, S., Oikawa, T., Besra, G. S. & Eggeling, L. ( 2004; ). Acyl-CoA carboxylases (accD2 and accD3), together with a unique polyketide synthase (Cg-pks), are key to mycolic acid biosynthesis in Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis. J Biol Chem 279, 44847–44857.[CrossRef]
    [Google Scholar]
  17. Gandhi, N. R., Moll, A., Sturm, A. W., Pawinski, R., Govender, T., Lalloo, U., Zeller, K., Andrews, J. & Friedland, G. ( 2006; ). Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368, 1575–1580.[CrossRef]
    [Google Scholar]
  18. Guilhot, C., Gicquel, B. & Martin, C. ( 1992; ). Temperature-sensitive mutants of the Mycobacterium plasmid pAL5000. FEMS Microbiol Lett 77, 181–186.
    [Google Scholar]
  19. Guilhot, C., Otal, I., Van, R. I., Martin, C. & Gicquel, B. ( 1994; ). Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol 176, 535–539.
    [Google Scholar]
  20. Hanahan, D. ( 1983; ). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[CrossRef]
    [Google Scholar]
  21. Henrikson, K. P. & Allen, S. H. ( 1979; ). Purification and subunit structure of propionyl coenzyme A carboxylase of Mycobacterium smegmatis. J Biol Chem 254, 5888–5891.
    [Google Scholar]
  22. Hunaiti, A. R. & Kolattukudy, P. E. ( 1982; ). Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus. Arch Biochem Biophys 216, 362–371.[CrossRef]
    [Google Scholar]
  23. Jackson, M., Stadthagen, G. & Gicquel, B. ( 2007; ). Long-chain multiple methyl-branched fatty acid-containing lipids of Mycobacterium tuberculosis: biosynthesis, transport, regulation and biological activities. Tuberculosis (Edinb) 87, 78–86.[CrossRef]
    [Google Scholar]
  24. Janiyani, K., Bordelon, T., Waldrop, G. L. & Cronan, J. E., Jr ( 2001; ). Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer. J Biol Chem 276, 29864–29870.[CrossRef]
    [Google Scholar]
  25. Karakousis, P. C., Bishai, W. R. & Dorman, S. E. ( 2004; ). Mycobacterium tuberculosis cell envelope lipids and the host immune response. Cell Microbiol 6, 105–116.[CrossRef]
    [Google Scholar]
  26. Kremer, L., Douglas, J. D., Baulard, A. R., Morehouse, C., Guy, M. R., Alland, D., Dover, L. G., Lakey, J. H., Jacobs, W. R., Jr & other authors ( 2000; ). Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis. J Biol Chem 275, 16857–16864.[CrossRef]
    [Google Scholar]
  27. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  28. Lin, T. W., Melgar, M. M., Kurth, D., Swamidass, S. J., Purdon, J., Tseng, T., Gago, G., Baldi, P., Gramajo, H. & Tsai, S. C. ( 2006; ). Structure-based inhibitor design of AccD5, an essential acyl-CoA carboxylase carboxyltransferase domain of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 103, 3072–3077.[CrossRef]
    [Google Scholar]
  29. Oh, T. J., Daniel, J., Kim, H. J., Sirakova, T. D. & Kolattukudy, P. E. ( 2006; ). Identification and characterization of Rv3281 as a novel subunit of a biotin-dependent acyl-CoA carboxylase in Mycobacterium tuberculosis H37Rv. J Biol Chem 281, 3899–3908.[CrossRef]
    [Google Scholar]
  30. Palomino, J. C., Martin, A., Camacho, M., Guerra, H., Swings, J. & Portaels, F. ( 2002; ). Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 46, 2720–2722.[CrossRef]
    [Google Scholar]
  31. Pelicic, V., Reyrat, J. M. & Gicquel, B. ( 1996; ). Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors. Mol Microbiol 20, 919–925.[CrossRef]
    [Google Scholar]
  32. Pelicic, V., Jackson, M., Reyrat, J. M., Jacobs, W. R., Jr, Gicquel, B. & Guilhot, C. ( 1997; ). Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 94, 10955–10960.[CrossRef]
    [Google Scholar]
  33. Pohlmann, J., Lampe, T., Shimada, M., Nell, P. G., Pernerstorfer, J., Svenstrup, N., Brunner, N. A., Schiffer, G. & Freiberg, C. ( 2005; ). Pyrrolidinedione derivatives as antibacterial agents with a novel mode of action. Bioorg Med Chem Lett 15, 1189–1192.[CrossRef]
    [Google Scholar]
  34. Rodriguez, E. & Gramajo, H. ( 1999; ). Genetic and biochemical characterization of the alpha and beta components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2). Microbiology 145, 3109–3119.
    [Google Scholar]
  35. Rodriguez, E., Banchio, C., Diacovich, L., Bibb, M. J. & Gramajo, H. ( 2001; ). Role of an essential acyl coenzyme A carboxylase in the primary and secondary metabolism of Streptomyces coelicolor A3(2). Appl Environ Microbiol 67, 4166–4176.[CrossRef]
    [Google Scholar]
  36. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  37. Sassetti, C. M., Boyd, D. H. & Rubin, E. J. ( 2003; ). Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48, 77–84.[CrossRef]
    [Google Scholar]
  38. Schweizer, E. & Hofmann, J. ( 2004; ). Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems. Microbiol Mol Biol Rev 68, 501–517.[CrossRef]
    [Google Scholar]
  39. Slayden, R. A., Lee, R. E., Armour, J. W., Cooper, A. M., Orme, I. M., Brennan, P. J. & Besra, G. S. ( 1996; ). Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis. Antimicrob Agents Chemother 40, 2813–2819.
    [Google Scholar]
  40. Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs, W. R., Jr ( 1990; ). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4, 1911–1919.[CrossRef]
    [Google Scholar]
  41. Studier, F. W. & Moffatt, B. A. ( 1986; ). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113–130.[CrossRef]
    [Google Scholar]
  42. Tong, L. ( 2005; ). Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery. Cell Mol Life Sci 62, 1784–1803.[CrossRef]
    [Google Scholar]
  43. Trivedi, O. A., Arora, P., Vats, A., Ansari, M. Z., Tickoo, R., Sridharan, V., Mohanty, D. & Gokhale, R. S. ( 2005; ). Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid. Mol Cell 17, 631–643.[CrossRef]
    [Google Scholar]
  44. Vieira, J. & Messing, J. ( 1982; ). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259–268.[CrossRef]
    [Google Scholar]
  45. Walsh, C. T. ( 2003; ). Antibiotics. Actions, Origins, Resistance. Washington, DC: American Society for Microbiology.
  46. Zhang, Y. ( 2005; ). The magic bullets and tuberculosis drug targets. Annu Rev Pharmacol Toxicol 45, 529–564.[CrossRef]
    [Google Scholar]
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vol. , part 8, pp. 2664 - 2675

[ PDF. 428 kb], including: (PCR analysis for the allelic replacement of , and assay of NADP-dependent malic enzyme activity in cell-free extracts) and : Agarose gel electrophoresis of PCR products amplified from genomic DNA from :: mutants, strain D6SCO1 and (wt) mc2155. NADP malic enzyme activity in the D6DCO2 mutant under restrictive growth conditions. Effect of NCI-172033 on growth of and H37Rv.



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