Ja. Triccas et al., Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells, MICROBIO-UK, 145, 1999, pp. 2923-2930
The identification of Mycobacterium tuberculosis genes expressed within hos
t cells would contribute greatly to the development of new strategies to co
mbat tuberculosis. By combining the natural fluorescence of the Aequoria vi
ctoria 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 wi
th recombinant Mycobacterium bovis bacille Calmette-Guerin containing a lib
rary 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 activi
ty outside the macrophage, resulting in the isolation of seven clones conta
ining M. tuberculosis promoters with greater activity intracellularly, The
gene products identified displayed similarity to proteins from other organi
sms whose functions include nutrient utilization, protection from oxidative
stress and defence against xenobiotics. These proposed functions are consi
stent with conditions encountered within the host cell and thus suggest tha
t the augmented activity of the isolated promoters/genes may represent stra
tegies employed by M. tuberculosis to enhance intracellular survival and pr
omote infection.