Antimycobacterial screening is done primarily at three levels
in vitro, ex vivo and in vivo. In earlier studies, we had generated a double recombinant Mycobacterium bovis BCG strain carrying firefly and Renilla luciferase genes as two reporters under the control of a constitutive and an inducible mycobacterial promoter. The presence of dual reporters allows simultaneous expression and analysis of two reporter enzymes within a single system. The expression profile of the firefly luciferase gene, rendered by a constitutive mycobacterial promoter, corroborates with the decline in bacterial growth in response to a wide range of antimycobacterial drugs, while the enhanced expression of Renilla luciferase mirrors the selective induction of the reporter gene expression as a result of FAS-II pathway-specific inhibition. Thus, the double recombinant strain allows the screening of both primary and rationally synthesized FAS-II pathway inhibitors in a single assay. While this was successfully used for in vitro screening, ex vivo adaptation of this screen-system posed several challenges. The constitutive hsp60pr showed appreciable expression inside macrophages, but the expression of the inducible kas operon promoter was found to be meager. This became a limiting factor as more number of bacilli needed per screening sample and with continued treatment the decline in CFU level worsens the detection limit of the luciferase assay. To develop a screen-system that compensate the lower level expression of a given mycobacterial promoter inside macrophages we introduced Nano luciferase reporter in recombinant mycobacteria. Nano luciferase emits several-fold brighter luminescence than firefly and Renilla luciferases and duly compensates the lower level expression of the kas operon promoter inside macrophages. The newly engineered double recombinant strain stays stable inside macrophages and serves as a model screen-system for general and pathway specific anti-mycobacterial ex vivo screening. The turnaround time is significantly reduced and the outcomes are similar and more consistent with those attained using conventional CFU based procedures.