Published on March 2014 | Antimycobacterial, Drug screening, Dual recombinant system, Antibiotic sensitivity, Mycobacterium bovis BCG, Luminescence, Luciferase assay, Firefly luciferase, Renilla luciferase,

Double Recombinant Mycobacterium bovis BCG Strain for Screening of Primary and Rationale-Based Antimycobacterial Compounds

Conventional antimycobacterial screening involves CFU analysis, which poses a great challenge due to slow growth of mycobac- teria. Recombinant strains carrying reporter genes under the influence of constitutive promoters allow rapid and wide screening of compounds but without revealing their modes of action. Reporter strains using pathway-specific promoters provide a better alternative but allow a limited screening of compounds interfering with only a particular metabolic pathway. This reduces these strains to merely a second-line screening system, as they fail to identify even the more potent compounds if they are not inhibit- ing the pathway of interest. In this study, we have 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, coincides with the decline in bacterial growth in response to a wide range of antimycobacterial drugs, while the enhanced expression of Renilla luciferase mir- rors the selective induction of the reporter gene expression as a result of pathway-specific inhibition. Thus, the double recombi- nant strain allows the screening of both primary and rationally synthesized antimycobacterial compounds in a single assay. The inhibiting response of drugs was monitored with a dual-luciferase reporter assay which can be easily adapted in high-through- put mode