Abstract
Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of β-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression strain.
Highlights
The process of target based drug discovery and development is laborious, expensive, and time consuming [1]
For growing E. coli, Luria Bertani (LB) broth and LB agar was used and supplemented with appropriate antibiotic as required. 7H9 broth supplemented with 0.2% glycerol (v/v), 0.05% tween 80 (w/v) and 7H11 were used for the growth of mycobacteria with the addition of appropriate antibiotics and IPTG as required
Previous studies on the successful use of IPTG in animal models to induce gene expression [10, 45] positively influenced our decision to work with an IPTG inducible system as one of the objectives of our work was to use this system in M. tuberculosis to assess gene essentiality both under in vitro and in vivo growth conditions
Summary
The process of target based drug discovery and development is laborious, expensive, and time consuming [1]. The ability to modulate gene expression by varying the inducer concentration in the growth environment allows the use of conditional expression strains to study the effect of target depletion under a variety of physiological conditions and the target’s relevance to disease biology. There are several reports of the successful application of a tet system for target evaluation in Mycobacterium tuberculosis under both in vitro and in vivo conditions [6,7, 14,15,16,17,18,19], there is always a distinct possibility that the inducer concentration required to achieve sufficient expression for growth could reach non-permissible levels resulting in growth arrest. Except for a few, most of the studies involving these systems have investigated target validation only under in vitro growth conditions [6,7,8,9, 11,12,13,14, 15,16,17,18]
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