1887

Abstract

Pathogenic strains of mycobacteria produce copious amounts of glutamine synthetase (GS) in the culture medium. The enzyme activity is linked to synthesis of poly-α--glutamine (PLG) in the cell walls. This study describes a mutant of that produces reduced levels of GS. The mutant was able to grow in enriched 7H9 medium without glutamine supplementation. The strain contained no detectable PLG in the cell walls and showed marked sensitivity to different chemical and physical stresses such as lysozyme, SDS and sonication. The sensitivity of the mutant to two antitubercular drugs, rifampicin and -cycloserine, was also increased. The strain infected THP-1 cells with reduced efficiency and was also attenuated for growth in macrophages. A strain containing the gene survived longer in THP-1 cells than the wild-type strain and also produced cell wall-associated PLG. The mutant was not able to replicate in the organs of BALB/c mice and was cleared within 4–6 weeks of infection. Disruption of the gene adversely affected biofilm formation on polystyrene surfaces. The results of this study demonstrate that the absence of not only attenuates the pathogen but also affects cell surface properties by altering the cell wall chemistry of the organism via the synthesis of PLG; this may be a target for drug development.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.043828-0
2010-12-01
2019-10-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/12/3669.html?itemId=/content/journal/micro/10.1099/mic.0.043828-0&mimeType=html&fmt=ahah

References

  1. Amon, J., Titgemeyer, F. & Burkovski, A. ( 2009; ). A genomic view on nitrogen metabolism and nitrogen control in mycobacteria. J Mol Microbiol Biotechnol 17, 20–29.[CrossRef]
    [Google Scholar]
  2. Candela, T. & Fouet, A. ( 2006; ). Poly-gamma-glutamate in bacteria. Mol Microbiol 60, 1091–1098.[CrossRef]
    [Google Scholar]
  3. 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]
  4. Fisher, S. H. ( 1999; ). Regulation of nitrogen metabolism in Bacillus subtilis: vive la difference! Mol Microbiol 32, 223–232.[CrossRef]
    [Google Scholar]
  5. Flynn, J. L. & Chan, J. ( 2001; ). Tuberculosis: latency and reactivation. Infect Immun 69, 4195–4201.[CrossRef]
    [Google Scholar]
  6. Garnier, T., Eiglmeier, K., Camus, J. C., Medina, N., Mansoor, H., Pryor, M., Duthoy, S., Grondin, S., Lacroix, C. & other authors ( 2003; ). The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U S A 100, 7877–7882.[CrossRef]
    [Google Scholar]
  7. Hanson, C. W. & Martin, W. J. ( 1978; ). Modified agar dilution method for rapid antibiotic susceptibility testing of anaerobic bacteria. Antimicrob Agents Chemother 13, 383–388.[CrossRef]
    [Google Scholar]
  8. Harper, C., Hayward, D., Wild, I. & Helden, P. V. ( 2008; ). Regulation of nitrogen metabolism in Mycobacterium tuberculosis: a comparision with mechanisms in Corynebacterium glutamicum and Streptomyces coelicolor. IUBMB Life 60, 643–650.[CrossRef]
    [Google Scholar]
  9. Harth, G. & Horwitz, M. A. ( 1999; ). An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic Mycobacterium in axenic culture and in human monocytes: extracellular proteins as potential novel drug targets. J Exp Med 189, 1425–1436.[CrossRef]
    [Google Scholar]
  10. Harth, G., Clemens, D. L. & Horwitz, M. A. ( 1994; ). Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity. Proc Natl Acad Sci U S A 91, 9342–9346.[CrossRef]
    [Google Scholar]
  11. Harth, G., Zamecnik, P. C., Tang, J. Y., Tabatadze, D. & Horwitz, M. A. ( 2000; ). Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication. Proc Natl Acad Sci U S A 97, 418–423.[CrossRef]
    [Google Scholar]
  12. Harth, G., Masleša-Galić, S., Tullius, M. V. & Horwitz, M. A. ( 2005; ). All four Mycobacterium tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly expressed and essential for bacterial homeostasis. Mol Microbiol 58, 1157–1172.[CrossRef]
    [Google Scholar]
  13. Hett, E. C. & Rubin, E. J. ( 2008; ). Bacterial growth and cell division: a mycobacterial perspective. Microbiol Mol Biol Rev 72, 126–156.[CrossRef]
    [Google Scholar]
  14. Hirschfield, G. R., McNeil, M. & Brennan, P. J. ( 1990; ). Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis. J Bacteriol 172, 1005–1013.
    [Google Scholar]
  15. Howard, N. S., Gomez, J. E., Ko, C. & Bishai, W. R. ( 1995; ). Color selection with a hygromycin-resistance-based Escherchia coli-mycobacterial shuttle vector. Gene 166, 181–182.[CrossRef]
    [Google Scholar]
  16. Krajewski, W. W., Collins, R., Holmberg-Schiavone, L., Jones, A., Karlberg, T. & Mowbray, S. L. ( 2008; ). Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design. J Mol Biol 375, 217–228.[CrossRef]
    [Google Scholar]
  17. MacKenzie, S. L. & Hogge, L. R. ( 1977; ). Gas chromatography mass spectrometry of the N(O)-heptafluorobutyryl isobutyl esters of the protein amino acids using electron impact ionization. J Chromatogr 132, 485–493.[CrossRef]
    [Google Scholar]
  18. Merrick, M. J. & Edwards, R. A. ( 1995; ). Nitrogen control in bacteria. Microbiol Rev 59, 604–622.
    [Google Scholar]
  19. Ojha, A., Anand, M., Bhatt, A., Kremer, L., Jacobs, W. R., Jr & Hatfull, G. F. ( 2005; ). GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell 123, 861–873.[CrossRef]
    [Google Scholar]
  20. Ojha, A. K., Baughn, A. D., Sambandan, D., Hsu, T., Trivelli, X., Guerardel, Y., Alahari, A., Kremer, L., Jacobs, W. R., Jr & Hatfull, G. F. ( 2008; ). Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol Microbiol 69, 164–174.[CrossRef]
    [Google Scholar]
  21. O'Toole, G. A., Pratt, L. A., Watnick, P. I., Newman, D. K., Weaver, V. B. & Kolter, R. ( 1999; ). Genetic approaches to study of biofilms. Methods Enzymol 310, 91–109.
    [Google Scholar]
  22. 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]
  23. Pavelka, M. S., Jr & Jacobs, W. R., Jr ( 1999; ). Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J Bacteriol 181, 4780–4789.
    [Google Scholar]
  24. 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]
  25. Reitzer, L. J. ( 1996; ). Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, l-alanine, and d-alanine. In Escherichia coli and Salmonella typhimurium, 2nd edn, pp. 391–407. Edited by Neidhardt, F. C. & Curtiss, R.. Washington, DC. : American Society for Microbiology.
    [Google Scholar]
  26. Theus, S. A., Cave, M. D. & Eisenach, K. D. ( 2004; ). Activated THP-1 cells: an attractive model for the assessment of intracellular growth rates of Mycobacterium tuberculosis isolates. Infect Immun 72, 1169–1173.[CrossRef]
    [Google Scholar]
  27. Tullius, M. V., Harth, G. & Horwitz, M. A. ( 2003; ). Glutamine synthetase GlnA1 is essential for growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs. Infect Immun 71, 3927–3936.[CrossRef]
    [Google Scholar]
  28. Vandal, O. H., Roberts, J. A., Odaira, T., Schnappinger, D., Nathan, C. F. & Ehrt, S. ( 2009; ). Acid-susceptible mutants of Mycobacterium tuberculosis share hypersusceptibility to cell wall and oxidative stress and to the host environment. J Bacteriol 191, 625–631.[CrossRef]
    [Google Scholar]
  29. Wietzerbin, J., Lederer, F. & Petit, J. F. ( 1975; ). Structural study of the poly-l-glutamic acid of the cell wall of Mycobacterium tuberculosis var hominis, strain Brevannes. Biochem Biophys Res Commun 62, 246–252.[CrossRef]
    [Google Scholar]
  30. Woolfolk, C. A., Shapiro, B. & Stadtman, E. R. ( 1966; ). Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch Biochem Biophys 116, 177–192.[CrossRef]
    [Google Scholar]
  31. Yamazaki, Y., Danelishvili, L., Wu, M., MacNab, M. & Bermudez, L. E. ( 2006; ). Mycobacterium avium genes associated with the ability to form a biofilm. Appl Environ Microbiol 72, 819–825.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.043828-0
Loading
/content/journal/micro/10.1099/mic.0.043828-0
Loading

Data & Media loading...

[PDF](92 KB)

PDF

[PDF](8 KB)

PDF

Construction of the mutant [PDF](71 KB)

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error