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

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.