1887

Abstract

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the genome contains four genes (, , and ) coding for the large catalytic subunit of AHAS, whereas only one gene () coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism and , we inactivated the gene of . The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C or C carbon sources, suggesting that the gene product is the major AHAS in . Depletion of these branched chain amino acids in the medium led to loss of viability of the Δ strain , resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the Δ strain in mice indicated that although this mutant strain showed defective growth , it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

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2009-09-01
2024-12-05
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References

  1. Alexander J. E., Andrew P. W., Jones D., Roberts I. S. 1993; Characterization of an aromatic amino acid-dependent Listeria monocytogenes mutant: attenuation, persistence, and ability to produce protective immunity in mice. Infect Immun 61:2245–2248
    [Google Scholar]
  2. Atkins T., Prior R. G., Mack K., Russell P., Nelson M., Oyston P. C., Dougan G., Titball R. W. 2002; A mutant of Burkholderia pseudomallei, auxotrophic in the branched chain amino acid biosynthetic pathway, is attenuated and protective in a murine model of melioidosis. Infect Immun 70:5290–5294
    [Google Scholar]
  3. Barak Z., Chipman D. M., Gollop N. 1987; Physiological implications of the specificity of acetohydroxy acid synthase isozymes of enteric bacteria. J Bacteriol 169:3750–3756
    [Google Scholar]
  4. Boigegrain R. A., Liautard J. P., Köhler S. 2005; Targeting of the virulence factor acetohydroxyacid synthase by sulfonylureas results in inhibition of intramacrophagic multiplication of Brucella suis . Antimicrob Agents Chemother 49:3922–3925
    [Google Scholar]
  5. Chan E. D., Iseman M. D. 2008; Multidrug-resistant and extensively drug-resistant tuberculosis: a review. Curr Opin Infect Dis 21:587–595
    [Google Scholar]
  6. Choi K. J., Yu Y. G., Hahn H. G., Choi J. D., Yoon M. Y. 2005; Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library. FEBS Lett 579:4903–4910
    [Google Scholar]
  7. 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
    [Google Scholar]
  8. Cole S. T., Eiglmeier K., Parkhill J., James K. D., Thomson N. R., Wheeler P. R., Honoré N., Garnier T., Churcher C. other authors 2001; Massive gene decay in the leprosy bacillus. Nature 409:1007–1011
    [Google Scholar]
  9. Dailey F. E., Cronan J. E. Jr, Maloy S. R. 1987; Acetohydroxy acid synthase I is required for isoleucine and valine biosynthesis by Salmonella typhimurium LT2 during growth on acetate or long-chain fatty acids. J Bacteriol 169:917–919
    [Google Scholar]
  10. Ewann F., Jackson M., Pethe K., Cooper A., Mielcarek N., Ensergueix D., Gicquel B., Locht C., Supply P. 2002; Transient requirement of the PrrA-PrrB two-component system for early intracellular multiplication of Mycobacterium tuberculosis . Infect Immun 70:2256–2263
    [Google Scholar]
  11. Filho J. C., Bergström J., Stehle P., Fürst P. 1997; Simultaneous measurements of free amino acid patterns of plasma, muscle and erythrocytes in healthy human subjects. Clin Nutr 16:299–305
    [Google Scholar]
  12. Grandoni J. A., Marta P. T., Schloss J. V. 1998; Inhibitors of branched-chain amino acid biosynthesis as potential antituberculosis agents. J Antimicrob Chemother 42:475–482
    [Google Scholar]
  13. Guardiola J., De Felice M., Iaccarino M. 1974; Mutant of Escherichia coli K-12 missing acetolactate synthase activity. J Bacteriol 120:536–538
    [Google Scholar]
  14. Hoft D. F. 2008; Tuberculosis vaccine development: goals, immunological design, and evaluation. Lancet 372:164–175
    [Google Scholar]
  15. Hoiseth S. K., Stocker B. A. 1981; Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291:238–239
    [Google Scholar]
  16. Hondalus M. K., Bardarov S., Russell R., Chan J., Jacobs W. R., Bloom B. R. 2000; Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis . Infect Immun 68:2888–2898
    [Google Scholar]
  17. Jackson M., Phalen S. W., Lagranderie M., Ensergueix D., Chavarot P., Marchal G., McMurray D. N., Gicquel B., Guilhot C. 1999; Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine. Infect Immun 67:2867–2873
    [Google Scholar]
  18. Keilhauer C., Eggeling L., Sahm H. 1993; Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175:5595–5603
    [Google Scholar]
  19. LaRossa R. A., Schloss J. V. 1984; The sulfonylurea herbicide sulfometuron methyl is an extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium . J Biol Chem 259:8753–8757
    [Google Scholar]
  20. LaRossa R. A., Smulski D. R. 1984; ilvB-encoded acetolactate synthase is resistant to the herbicide sulfometuron methyl. J Bacteriol 160:391–394
    [Google Scholar]
  21. Martin C. 2006; Tuberculosis vaccines: past, present and future. Curr Opin Pulm Med 12:186–191
    [Google Scholar]
  22. Muñoz-Elías E. J., McKinney J. D. 2005; Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med 11:638–644
    [Google Scholar]
  23. Muñoz-Elias E. J., McKinney J. D. 2006; Carbon metabolism of intracellular bacteria. Cell Microbiol 8:10–22
    [Google Scholar]
  24. Parish T., Stoker N. G. 2000; Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146:1969–1975
    [Google Scholar]
  25. Pascopella L., Collins F. M., Martin J. M., Lee M. H., Hatfull G. F., Stover C. K., Bloom B. R., Jacobs W. R. Jr 1994; Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence. Infect Immun 62:1313–1319
    [Google Scholar]
  26. Porat I., Vinogradov M., Vyazmensky M., Lu C. D., Chipman D. M., Abdelal A. T., Barak Z. 2004; Cloning and characterization of acetohydroxyacid synthase from Bacillus stearothermophilus . J Bacteriol 186:570–574
    [Google Scholar]
  27. Rivers E. C., Mancera R. L. 2008; New anti-tuberculosis drugs in clinical trials with novel mechanisms of action. Drug Discov Today 13:1090–1098
    [Google Scholar]
  28. Sambandamurthy V. K., Jacobs W. R. Jr 2005; Live attenuated mutants of Mycobacterium tuberculosis as candidate vaccines against tuberculosis. Microbes Infect 7:955–961
    [Google Scholar]
  29. Shaw K. J., Berg C. M., Sobol T. J. 1980; Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II. J Bacteriol 141:1258–1263
    [Google Scholar]
  30. Smith D. A., Parish T., Stoker N. G., Bancroft G. J. 2001; Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates. Infect Immun 69:1142–1150
    [Google Scholar]
  31. Sohn H., Lee K. S., Ko Y. K., Ryu J. W., Woo J. C., Koo D. W., Shin S. J., Ahn S. J., Shin A. R. other authors 2008; In vitro and ex vivo activity of new derivatives of acetohydroxyacid synthase inhibitors against Mycobacterium tuberculosis and non-tuberculous mycobacteria. Int J Antimicrob Agents 31:567–571
    [Google Scholar]
  32. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H. other authors 1991; New use of BCG for recombinant vaccines. Nature 351:456–460
    [Google Scholar]
  33. Sun R., Converse P. J., Ko C., Tyagi S., Morrison N. E., Bishai W. R. 2004; Mycobacterium tuberculosis ECF sigma factor sigC is required for lethality in mice and for the conditional expression of a defined gene set. Mol Microbiol 52:25–38
    [Google Scholar]
  34. Tamburini E., Mastromei G. 2000; Do bacterial cryptic genes really exist?. Res Microbiol 151:179–182
    [Google Scholar]
  35. Wards B. J., Collins D. M. 1996; Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Microbiol Lett 145:101–105
    [Google Scholar]
  36. Zohar Y., Einav M., Chipman D. M., Barak Z. 2003; Acetohydroxyacid synthase from Mycobacterium avium and its inhibition by sulfonylureas and imidazolinones. Biochim Biophys Acta 164997–105
    [Google Scholar]
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