1887

Abstract

The identification of genes expressed within host cells would contribute greatly to the development of new strategies to combat tuberculosis. By combining the natural fluorescence of the green fluorescent protein (GFP) with the counterselectable property of the SacB protein, promoters displaying enhanced activity have been isolated. Macrophages were infected with recombinant bacille Calmette–Guérin containing a library of promoters controlling and expression, and fluorescent bacteria recovered by fluorescence-activated cell sorting. The expression of was used to eliminate clones with strong promoter activity outside the macrophage, resulting in the isolation of seven clones containing promoters with greater activity intracellularly. The gene products identified displayed similarity to proteins from other organisms whose functions include nutrient utilization, protection from oxidative stress and defence against xenobiotics. These proposed functions are consistent with conditions encountered within the host cell and thus suggest that the augmented activity of the isolated promoters/genes may represent strategies employed by to enhance intracellular survival and promote infection.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-10-2923
1999-10-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/10/1452923a.html?itemId=/content/journal/micro/10.1099/00221287-145-10-2923&mimeType=html&fmt=ahah

References

  1. Barker, L. P., Brooks, D. M. & Small, P. L. (1998). The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein. Mol Microbiol 29, 1167-1177.[CrossRef] [Google Scholar]
  2. Clemens, D. L. (1996). Characterization of the Mycobacterium tuberculosis phagosome. Trends Microbiol 4, 113-118.[CrossRef] [Google Scholar]
  3. Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef] [Google Scholar]
  4. Cormack, B. P., Valdivia, R. H. & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33-38.[CrossRef] [Google Scholar]
  5. Dhandayuthapani, S., Via, L. E., Thomas, C. A., Horowitz, P. M., Deretic, D. & Deretic, V. (1995). Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages. Mol Microbiol 17, 901-912.[CrossRef] [Google Scholar]
  6. Gercken, J., Pryjma, J., Ernst, M. & Flad, H. D. (1994). Defective antigen presentation by Mycobacterium tuberculosis-infected monocytes. Infect Immun 62, 3472-3478. [Google Scholar]
  7. Itoh, K., Chiba, T., Takahashi, S. & 12 other authors (1997). An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 236, 313–322.[CrossRef] [Google Scholar]
  8. Kremer, L., Baulard, A., Estaquier, J., Poulain-Godefroy, O. & Locht, C. (1995). Green fluorescent protein as a new expression marker in mycobacteria. Mol Microbiol 17, 913-922.[CrossRef] [Google Scholar]
  9. Miller, R. A. & Britigan, B. E. (1997). Role of oxidants in microbial pathophysiology, Clin Microbiol Rev 10, 1-18. [Google Scholar]
  10. Moors, M. A. & Portnoy, D. A. (1995). Identification of bacterial genes that contribute to survival and growth in an intracellular environment. Trends Microbiol 3, 83-85.[CrossRef] [Google Scholar]
  11. Otal, I., Martin, C., Vincent-Levy-Frebault, V., Thierry, D. & Gicquel, B. (1991). Restriction fragment length polymorphism analysis using IS6110 as an epidemiological marker in tuberculosis. J Clin Microbiol 29, 1252-1254. [Google Scholar]
  12. Pelicic, V., Reyrat, J.-M. & Gicquel, B. (1996a). Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J Bacteriol 178, 1197-1199. [Google Scholar]
  13. Pelicic, V., Reyrat, J.-M. & Gicquel, B. (1996b). Generation of unmarked directed mutations in mycobacteria using sucrose counterselectable suicide vectors. Mol Microbiol 20, 919-925.[CrossRef] [Google Scholar]
  14. 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 USA 94, 10955-10960.[CrossRef] [Google Scholar]
  15. Quandt, J. & Hynes, M. F. (1993). Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. J Bacteriol 127, 15-21. [Google Scholar]
  16. Ranes, M. G., Rauzier, J., Lagranderie, M., Gheroghiu, M. & Gicquel, B. (1990). Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a ‘Mini’ mycobacterium–Escherichia coli shuttle vector. J Bacteriol 172, 2793-2797. [Google Scholar]
  17. Raviglione, M. C., Snider, D. E. & Kochi, A. (1995). Global epidemiology of tuberculosis. Morbidity and mortality of a worldwide epidemic. JAMA 273, 220-226.[CrossRef] [Google Scholar]
  18. Sabaty, M. & Kaplan, S. (1996).MgpS, a complex regulatory locus involved in the transcriptional control of the puc and puf operons in Rhodobacter sphaeroides. J Bacteriol 178, 35-45. [Google Scholar]
  19. Salamon, C., Chervenak, M., Piatigorsky, J. & Sax, C. M. (1998). The mouse transketolase (TKT) gene: cloning, characterization and functional promoter analysis. Genomics 48, 209-220.[CrossRef] [Google Scholar]
  20. Slekar, K. H., Kosman, D. J. & Culotta, V. C. (1996). The yeast copper/zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection. J Biol Chem 271, 28831-28836.[CrossRef] [Google Scholar]
  21. Timm, J., Perilli, M. G., Duez, C. & 9 other authors (1994). Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum β-lactamase genes cloned from a natural isolate and a high-level β-lactamase producer. Mol Microbiol 12, 491–504.[CrossRef] [Google Scholar]
  22. Tumminia, S. J., Rao, P. V., Zigler, J. S.Jr & Russell, P. (1993). Xenobiotic induction of quinone oxidoreductase activity in lens epithelial cells. Biochim Biophys Acta 1203, 251-259.[CrossRef] [Google Scholar]
  23. Valdivia, R. H. & Falkow, S. (1996). Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol Microbiol 22, 367-378.[CrossRef] [Google Scholar]
  24. Valdivia, R. H. & Falkow, S. (1997). Fluorescence-based isolation of bacterial genes expressed within host-cells. Science 277, 2007-2011.[CrossRef] [Google Scholar]
  25. Yildiz, F. H., Davies, J. P. & Grossman, A. (1996). Sulfur availability and the SAC1 gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii. Plant Physiol 112, 669-675. [Google Scholar]
  26. Yuan, Y., Crane, D. G., Simpson, R. M., Zhu, Y. Q., Hickey, M. J., Sherman, D. R. & Barry, C. E. (1998). The 16-kDa α-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages. Proc Natl Acad Sci USA 95, 9578-9583.[CrossRef] [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-10-2923
Loading
/content/journal/micro/10.1099/00221287-145-10-2923
Loading

Data & Media loading...

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