Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is responsible for the deaths of millions of individuals each year. One obstacle in the crusade against this pathogen is the ability of the organism to persist in an asymptomatic state, referred to as latency. Another rapidly increasing trend in tuberculosis is the emergence of multiply drug resistant strains of M. tuberculosis. In order to better understand the biology of M. tuberculosis during active or latent disease, and to identify new potential drug targets and candidate vaccine strains, a powerful technique known as differential fluorescence induction will be used to identify M. tuberculosis genes induced specifically during host infection. In this approach, a plasmid library of M. tuberculosis genomic DNA (gDNA) fragments fused to a promoterless gfp, encoding the green fluorescent protein, will be transferred into M. tuberculosis. The resulting clones will be used to infect Balb/c mice. At specific time points mice will be euthanized, and specific organs will be harvested and homogenized. The resulting homogenates will be analyzed in a fluorescence-activated cell sorter (FACS). In this way, fluorescent bacilli will be identified and collected. .These bacilli harbor plasmids in which the M. tuberculosis gDNA fragments upstream of gfp contain promoters that are expressed in vivo. The collected bacilli will be cultured in vitro and then re-analyzed by FACS. During this round of analysis, those bacilli that exhibit low levels of fluorescence will be collected and used to infect a second set of mice. By successive rounds of in vivo and in vitro propagation, a set of M. tuberculosis promoters will be identified that are specifically induced in the animal host.
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| Saviola, Beatrice; Woolwine, Samuel C; Bishai, William R (2003) Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology. Infect Immun 71:1379-88 |
