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Genetics and Molecular Biology |
Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Brigitte Gicquel. Tel: +33 1 45 68 88 28. Fax: +33 1 45 68 88 43. e-mail: bgicquel{at}pasteur.fr
The identification of Mycobacterium tuberculosis genes expressed within host cells would contribute greatly to the development of new strategies to combat tuberculosis. By combining the natural fluorescence of the Aequoria victoria green fluorescent protein (GFP) with the counterselectable property of the Bacillus subtilis SacB protein, M. tuberculosis promoters displaying enhanced in vivo activity have been isolated. Macrophages were infected with recombinant Mycobacterium bovis bacille Calmette–Guérin containing a library of M. tuberculosis promoters controlling gfp and sacB expression, and fluorescent bacteria recovered by fluorescence-activated cell sorting. The expression of sacB was used to eliminate clones with strong promoter activity outside the macrophage, resulting in the isolation of seven clones containing M. tuberculosis 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 M. tuberculosis to enhance intracellular survival and promote infection.
Keywords: Mycobacterium tuberculosis, macrophage, gene induction, fluorescence
Abbreviations: GFP, green fluorescent protein; (r)BCG, (recombinant) bacille Calmette–Guérin; FACS, fluorescence-activated cell sorting
a Present address: SmithKline Beecham Biologicals, Laboratory of Molecular Bacteriology, Rue de l’Institut 89, B-1330 Rixensart, Belgium.
b Present address: INSERM U411, Faculté de Médecine Necker-Enfants Malades, 156, Rue de Vaugirard, 75730 Paris Cedex 15, France.
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