Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells

被引:63
作者
Triccas, JA [1 ]
Berthet, FX [1 ]
Pelicic, V [1 ]
Gicquel, B [1 ]
机构
[1] Inst Pasteur, Unite Genet Mycobacterienne, F-75724 Paris 15, France
来源
MICROBIOLOGY-SGM | 1999年 / 145卷
关键词
Mycobacterium tuberculosis; macrophage; gene induction; fluorescence;
D O I
10.1099/00221287-145-10-2923
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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-Guerin 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.
引用
收藏
页码:2923 / 2930
页数:8
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