Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter.
The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc(2)155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.
Changsen
Chartchai
C
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Franzblau
Scott G
SG
Palittapongarnpim
Prasit
P
D000581:Amidohydrolases / Q000096:biosynthesis / Q000235:genetics*; D000995:Antitubercular Agents / Q000494:pharmacology*; D018834:Chaperonin 60 / Q000235:genetics; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D018274:Electroporation; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D020102:Mycobacterium smegmatis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*
D000995:Antitubercular Agents; D018834:Chaperonin 60; D004269:DNA, Bacterial; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000581:Amidohydrolases; C021445:acetamidase