Abstract
Although efforts to identify novel therapies for the treatment of tuberculosis have led to the identification of several promising drug candidates, the identification of high-quality hits from conventional whole-cell screens remains disappointingly low. The elucidation of the genome sequence of Mycobacterium tuberculosis (Mtb) facilitated a shift to target-based approaches to drug design but these efforts have proven largely unsuccessful. More recently, regulated gene expression systems that enable dose-dependent modulation of gene expression have been applied in target validation to evaluate the requirement of individual genes for the growth of Mtb both in vitro and in vivo. Notably, these systems can also provide a measure of the extent to which putative targets must be depleted in order to manifest a growth inhibitory phenotype. Additionally, the successful implementation of Mtb strains engineered to under-express specific molecular targets in whole-cell screens has enabled the simultaneous identification of cell-permeant inhibitors with defined mechanisms of action. Here, we review the application of tetracycline-regulated gene expression systems in the validation of novel drug targets in Mtb, highlighting both the strengths and limitations associated with this approach to target validation.
Highlights
We review the application of tetracycline-regulated gene expression systems in the validation of novel drug targets in Mycobacterium tuberculosis (Mtb), highlighting both the strengths and limitations associated with this approach to target validation
We focus on the use of tetracycline (Tet)-regulated gene expression systems in Mtb, in the context of target validation
We focus on the Tet-regulated gene expression systems (Table 1B; Blokpoel et al, 2005; Carroll et al, 2005; Ehrt et al, 2005), which have been widely applied, and whose utility in TB drug discovery is enhanced by the fact that they can be used to regulate gene expression in animal models of infection
Summary
Tet-inducible promoter, PfurA102tetO (B) Tetracycline (Tet)-regulated gene expression systems optimized for use in mycobacterial gene regulation. Since the removal of ATc can be difficult to achieve in some experimental settings, the manipulation of Tet-dependent hypomorphs was greatly simplified by the development of a modified “Tet-OFF” system, which utilizes a mutated, “reverse” TetR (revTetR) that binds to tetO only in the presence of ATc (Guo et al, 2007) This system enables the generation of mutants in which target gene expression is repressed upon addition of the ATc inducer (Figure 2B). The regulatory capacity of both Tet-ON and TetOFF systems in mycobacteria has been enhanced by codon optimization of the genes encoding TetR and revTetR to allow increased expression in Mtb (Klotzsche et al, 2009) Together, these modifications have provided a means of evaluating the effects of transcriptional silencing of target genes on growth and viability of the organism.
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