Use of an arrayed promoter-probe library for the identification of macrophage-regulated genes in Free

Abstract

The survival of within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes. To identify such genes, a promoter-probe library was constructed in which fragments of H37Rv DNA were inserted upstream of a reporter gene, using an –mycobacterial shuttle vector. Bacille Calmette–Guérin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions. From preliminary screening, 41 clones were selected that exhibited upregulation of expression when subjected to acidified sodium nitrite. Subsequent sequence analyses identified 26 of these clones as containing potential promoters. After measuring expression in BCG clones recovered from a THP-1 macrophage cell line, three genes were selected for assessment of their expression in during macrophage infection, by real-time RT-PCR. Two of these genes, Rv1265 (with unknown function) and Rv2711 (encoding the iron-dependent repressor protein IdeR), showed evidence of being upregulated within macrophages.

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2002-05-01
2024-03-29
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References

  1. Arruda S., Bomfim G., Knights R., Huima-Byron T., Riley L. W. 1993; Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells. Science 261:1454–1457 [CrossRef]
    [Google Scholar]
  2. Barker L. P., Brooks D. M., Small P. L. C. 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]
  3. Batoni G., Maisetta G., Florio W., Freer G., Campa M., Senesi S. 1998; Analysis of the Mycobacterium bovis hsp60 promoter activity in recombinant Mycobacterium avium . FEMS Microbiol Lett 169:117–124 [CrossRef]
    [Google Scholar]
  4. Berthet F. X., Lagranderie M., Gounon P. 9 other authors 1998; Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene. Science 282:759–762 [CrossRef]
    [Google Scholar]
  5. 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]
  6. Collins D. M., Gicquel B. 2000; Genetics of mycobacterial virulence. In Molecular Genetics of Mycobacteria pp 265–278 Edited by Hatfull G. F. Jacobs W. R. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  7. Collins D. M., Kawakami R. P., Delisle G. W., Pascopella L., Bloom B. R., Jacobs W. R. 1995; Mutation of the principal σ factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex. Proc Natl Acad Sci USA 92:8036–8040 [CrossRef]
    [Google Scholar]
  8. Dellagostin O. A., Esposito G., Eales L.-J., Dale J. W., McFadden J. 1995; Activity of mycobacterial promoters during intracellular and extracellular growth. Microbiology 141:1785–1792 [CrossRef]
    [Google Scholar]
  9. Dussurget O., Rodriguez M., Smith I. 1996; An ideR mutant of Mycobacterium smegmatis has derepressed siderophore production and an altered oxidative-stress response. Mol Microbiol 22:535–544 [CrossRef]
    [Google Scholar]
  10. Gold B., Rodriguez G. M., Marras S. A. E., Pentecost M., Smith I. 2001; The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages. Mol Microbiol 42:851–865
    [Google Scholar]
  11. Gomez M., Smith I. 2000; Determinants of mycobacterial gene expression. In Molecular Genetics of Mycobacteria pp 111–129 Edited by Hatfull G. F. Jacobs W. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  12. Graham J. E., Clark-Curtiss J. E. 1999; Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci USA 96:11554–11559 [CrossRef]
    [Google Scholar]
  13. Lee B.-Y., Horwitz M. A. 1995; Identification of macrophage and stress-induced proteins of Mycobacterium tuberculosis . J Clin Invest 96:245–249 [CrossRef]
    [Google Scholar]
  14. Manabe Y. C., Saviola B. J., Sun L., Murphy J. R., Bishai W. R. 1999; Attenuation of virulence in Mycobacterium tuberculosis expressing a constitutively active iron repressor. Proc Natl Acad Sci USA 96:12844–12848 [CrossRef]
    [Google Scholar]
  15. Manganelli R., Dubnau E., Tyagi S., Kramer F. R., Smith I. 1999; Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis . Mol Microbiol 31:715–724 [CrossRef]
    [Google Scholar]
  16. McKinney J. D.7 other authors Honer zu Bentrup K., Muñoz-Elias E. J. 2000; Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735–738 [CrossRef]
    [Google Scholar]
  17. Plum G., Clark-Curtiss J. E. 1994; Induction of Mycobacterium avium gene expression following phagocytosis by human macrophages. Infect Immun 62:476–483
    [Google Scholar]
  18. Ranes M. G., Rauzier J., Lagranderie M., Gheorghiu 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]
  19. Rindi L., Lari N., Garzelli C. A. 1999; Search for genes potentially involved in Mycobacterium tuberculosis virulence by mRNA differential display. Biochem Biophys Res Commun 258:94–101 [CrossRef]
    [Google Scholar]
  20. Schmitt M. P., Predich M., Doukhan L., Smith I., Holmes R. K. 1995; Characterization of an iron-dependent regulatory protein (IdeR) of Mycobacterium tuberculosis as a functional homolog of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae . Infect Immun 63:4284–4289
    [Google Scholar]
  21. Stover C. K., Fuerst T. R. 11 other authors de la Cruz V. F. 1991; New use of BCG for recombinant vaccines. Nature 351:456–460 [CrossRef]
    [Google Scholar]
  22. Sun L., vanderSpek J., Murphy J. R. 1998; Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR. Proc Natl Acad Sci USA 95:14985–14990 [CrossRef]
    [Google Scholar]
  23. Timm J., Gomez M., Smith I. 1999; Gene expression and regulation. In Mycobacteria: Molecular Biology and Virulence pp 59–92 Edited by Ratledge C. Dale J. W. Oxford: Blackwell Science;
    [Google Scholar]
  24. Triccas J. A., Berthet F.-X., Pelicic V., Gicquel B. 1999; Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells. Microbiology 145:2923–2930
    [Google Scholar]
  25. Triccas J. A., Britton W. J., Gicquel B. 2001; Isolation of strong expression signals of Mycobacterium tuberculosis . Microbiology 147:1253–1258
    [Google Scholar]
  26. Tyagi A. K., Das Gupta S. K., Jain S. 2000; Gene expression: reporter gene technologies. In Molecular Genetics of Mycobacteria pp 131–147 Edited by Hatfull G. F. Jacobs W. R. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  27. Via L. E., Curcic R., Mudd M. H., Dhandayuthapani S., Ulmer R. J., Deretic V. 1996; Elements of signal transduction in Mycobacterium tuberculosis : in vitro phosphorylation and in vivo expression of the response regulator MtrA. J Bacteriol 178:3314–3321
    [Google Scholar]
  28. Zahrt T. C., Deretic V. 2000; An essential two-component signal transduction system in Mycobacterium tuberculosis . J Bacteriol 182:3832–3838 [CrossRef]
    [Google Scholar]
  29. Zhang Y., Young D. B. 1993; Molecular mechanisms of isoniazid: a drug at the front line of tuberculosis control. Trends Microbiol 1:109–113 [CrossRef]
    [Google Scholar]
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