The application of tetracyclineregulated gene expression systems in the validation of novel drug targets in Mycobacterium tuberculosis

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.

Keywords: Mycobacterium tuberculosis; drug discovery; hypomorphs; regulated gene expression; target validation; target-based whole cell screening.

FIGURE 1

Schematic representation of tetracycline (Tet) export in Gram-negative bacteria. In the absence of Tet, Tet repressor (TetR) dimers bind to Tet operators (tetO) located upstream of tetA, encoding the Tet-exporting protein, repressing its transcription. Upon introduction of anhydrotetracycline (ATc) and its subsequent binding to TetR, a conformational change occurs that results in dissociation of TetR from tetO, enabling Tet-mediated transcription of tetA to occur. Tet is then transported across the cytoplasmic membrane by TetA.

FIGURE 2

Tetracycline-mediated gene regulation in Mycobacterium tuberculosis (Mtb). (A) In the Tet-ON configuration, TetR dimers bind to Tet operators (tetO) in the absence of ATc, repressing transcription. Upon introduction of ATc and its subsequent binding to TetR, a conformational change occurs that results in dissociation of TetR from tetO, enabling Tet-mediated transcription from P_myc1tetO to occur. (B) In the Tet-OFF configuration, introduction of ATc results in binding of revTetR to P_myc1tetO, causing repression of transcription. TetR, Tet repressor; ATc, anhydrotetracycline; tetO, Tet operator; P_myc1tetO, mycobacterial promoter with tetOs inserted; revTetR, reverse Tet repressor (Adapted from Guo et al., 2007).

FIGURE 3

The application of hypomorphs in (A) assessing target vulnerability and (B) expanding antitubercular chemical space. (A) Dose-dependent silencing of a target gene enables determination of the extent to which that target must be depleted in order to result in growth inhibition. A target that requires only 50% depletion in order to reduce 50% of bacterial growth (black line) is considered more vulnerable that one that requires 75% depletion to achieve the same effect (gray line). (B) Reduction of the absolute concentration of a target by conditional knockdown (gray line) relative to wildtype levels (black line) facilitates the identification of on-target inhibitors by lowering the minimum inhibitory concentration (MIC).

FIGURE 4

Tetracycline-mediated regulated protein degradation in Msm. (A) Degradation of SsrA-tagged proteins by ClpXP is enhanced by the adapter protein, SspB. (B) Mutation of the wildtype LAA ClpX recognition site of SsrA to generate a modified DAS tag weakens the interaction of ClpXP with the aberrant protein, resulting in SspB-dependent proteolysis of the tagged protein. (C) By replacement of the native sspB promoter with a Tet-inducible promoter, SspB-mediated degradation of proteins that have been engineered to carry a modified DAS tag can be achieved upon induction with ATc.

FIGURE 5

Dual-control (DUC) genetic switch for simultaneous transcriptional silencing and degradation of the encoded protein in Mtb. (A) In the absence of ATc sspB expression is repressed by TetR, while expression of the target gene under control of revTetR proceeds. (B) Upon introduction of ATc, sspB transcription proceeds, enabling ClpXP-mediated proteolysis of the target protein and simultaneous transcriptional repression of the target gene due to binding of revTet to the Tet-regulated promoter. tetR, Tet repressor; GOI, gene of interest; rev tetR, reverse Tet repressor; ATc, anhydrotetracycline; ClpXP, protease complex mediating controlled proteolysis of the target protein (Adapted from Kim et al., 2013).