title: Green fluorescent protein as a reporter in rapid screening of antituberculosis compounds in vitro and in macrophages.
abstract: The development of new drugs against Mycobacterium tuberculosis is impeded by slow growth and highly infectious nature of the organism that warrants the need to work under highly stringent biosafety conditions. These problems can be overcome by use of reporter genes and surrogate strains. A strain of rapidly growing M. aurum has been recommended as test organism to screen inhibitors of mycobacteria to preselect compounds for progression into testing against M. tuberculosis. We have investigated the application of recombinant M. aurum expressing green fluorescent protein in rapid screening of antituberculosis compounds in vitro and in infected macrophages. Recombinant M. aurum[pGFM-11] expressing green fluorescent protein was constructed. The assay is based on measurement of fluorescent intensity at 509 nm. A good correlation was found between fluorescence and growth. Fluorescence of recombinant M. aurum was inhibited in vitro within 8 to 24 h by frontline antimycobacterial drugs at their reported MICs whereas inhibition in infected macrophages was observed in 72 h. Therefore green fluorescent reporter system provides a convenient screen to test antimycobacterial compounds that are active in vitro and within infected macrophages.
authors.affiliation: Division of Microbiology, Central Drug Research Institute, Lucknow, India.
authors.forename: R
authors.initials: R
authors.lastname: Srivastava
authors.forename: D K
authors.initials: DK
authors.lastname: Deb
authors.forename: K K
authors.initials: KK
authors.lastname: Srivastava
authors.forename: C
authors.initials: C
authors.lastname: Locht
authors.forename: B S
authors.initials: BS
authors.lastname: Srivastava
mesh_terms: D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D003470:Culture Media; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006131:Growth Inhibitors / Q000494:pharmacology; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D009161:Mycobacterium / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D037401:Scyphozoa; D014170:Transformation, Genetic
chemical_list: D000995:Antitubercular Agents; D003470:Culture Media; D006131:Growth Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins