title: Double recombinant Mycobacterium bovis BCG strain for screening of primary and rationale-based antimycobacterial compounds.
abstract: Conventional antimycobacterial screening involves CFU analysis, which poses a great challenge due to slow growth of mycobacteria. 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 inhibiting 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 mirrors the selective induction of the reporter gene expression as a result of pathway-specific inhibition. Thus, the double recombinant 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-throughput mode.
authors.affiliation: Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, India.
authors.forename: Vandana
authors.initials: V
authors.lastname: Singh
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Biswas
authors.forename: Bhupendra N
authors.initials: BN
authors.lastname: Singh
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology* / Q000627:therapeutic use; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D049409:Luciferases, Firefly / Q000235:genetics; D049410:Luciferases, Renilla / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009163:Mycobacterium bovis / Q000187:drug effects* / Q000235:genetics; D030781:Organisms, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D000995:Antitubercular Agents; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly