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Antimicrobial Agents and Chemotherapy, February 1998, p. 344-347, Vol. 42, No. 2
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Green Fluorescent Protein Reporter Microplate Assay for High-Throughput Screening of Compounds against Mycobacterium tuberculosis

L. A. Collins, M. N. Torrero, and S. G. Franzblau*

Pharmacology Research Department, Laboratory Research Branch, Gillis W. Long Hansen's Disease Center, Baton Rouge, Louisiana 70894

Received 7 July 1997/Returned for modification 20 August 1997/Accepted 17 November 1997

An optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation into Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log2 dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response of M. tuberculosis to the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.

* Corresponding author. Mailing address: Laboratory Research Branch, GWL Hansen's Disease Center, P.O. Box 25072, Baton Rouge, LA 70894. Phone: (504) 346-5773. Fax: (504) 346-5786. E-mail: franzblau{at}vt8200.vetmed.lsu.edu.

Antimicrobial Agents and Chemotherapy, February 1998, p. 344-347, Vol. 42, No. 2
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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