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Antimicrobial Agents and Chemotherapy, December 2003, p. 3682-3687, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3682-3687.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter

Chartchai Changsen,1,2 Scott G. Franzblau,2* and Prasit Palittapongarnpim1,3

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400,1 National Center for Genetic Engineering and Biotechnology, Pathumthani 12120, Thailand,3 Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 606122

Received 19 June 2003/ Returned for modification 23 July 2003/ Accepted 3 September 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and METHODS
 Results
 Discussion
 References
 
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 mc2155. 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and METHODS
 Results
 Discussion
 References
 
Tuberculosis continues to be a major global public health problem. In addition, the increasing prevalence of multidrug-resistant Mycobacterium tuberculosis is of concern (20) and has fostered a sense of urgency with regard to the need to acquire new drugs. Several rapid methods such as the microplate Alamar Blue assay (MABA) (3, 5), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based assay (9), and the luciferase assay (1, 2, 7, 21) have been established for the screening of the antimycobacterial activities of compounds. However, each has one or more drawbacks for high-throughput screening, including the need to add a dye or a substrate to test the viability of the bacteria, a step which both increases labor and decreases safety.

Fluorometric assays based on the Aequorea victoria gfp gene encoding a green fluorescent protein (GFP) or its mutants have been used widely in prokaryotes (8, 10, 11, 24) due to several properties of gfp that are advantageous for use as a reporter for bacterial viability and growth, including the low level of toxicity, continuous production during replication, and easy imaging and quantification (11, 25). Previous studies with mycobacteria exclusively used the hsp60 promoter, which has been fused to gfp in a variety of investigations (8, 11, 26).

Collins et al. (6) demonstrated that a recombinant M. tuberculosis strain carrying pFPV2, an hsp60::gfp construct, could be used to assess the MICs of known antimycobacterial compounds. However, the low level of the fluorescence signal and the relatively high background autofluorescence of the medium precluded the establishment of a robust assay that could be used with confidence for high-throughput screening.

Previous investigators (6, 13, 17, 18, 23) have identified a highly inducible promoter sequence which regulates the expression of the acetamidase gene in M. smegmatis NCTC 8159 and which demonstrated a high level of gene expression in the presence of amide inducers such as acetamide.

In this study we demonstrate that an acetamidase promoter from M. smegmatis strain mc2155 permits constitutive, high-level gfp expression in M. tuberculosis strains H37Ra, H37Rv, and Erdman, resulting in a fluorescence signal significantly higher than that which is achievable with the hsp60 promoter and facilitating the determination of MICs of antimicrobial agents without the addition of reagents postincubation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and METHODS
Results
 Discussion
 References
 
Bacterial strains. M. tuberculosis H37Rv (ATCC 27294), M. tuberculosis H37Ra (ATCC 25177), and M. tuberculosis Erdman (ATCC 35801) were obtained from the American Type Culture Collection (ATCC; Manassas, Va.). pFPV2 was originally obtained from R. H. Valdivia (25).

DNA manipulation, recombinant DNA methods, and analyses. The chromosomal DNA of M. smegmatis mc2155 was purified as described previously (16). The acetamidase promoter of M. smegmatis mc2155 was amplified by PCR with primers AmiP1 (5'-CGTCTAGACGAGTA CGGCGCCCTGCTGACG-3') and AmiP2 (5'-GAGGATCCTCCGCCGACGAAGAAGTTCAGC-3'), which annealed to sequences 0.6 and 2.9 kb upstream from the start codon of the acetamidase gene, respectively. The amplified product was subcloned into the BamHI and XbaI sites of plasmid pPFV2 to remove the hsp60 promoter, which was upstream of gfp. Colonies were selected on the basis of kanamycin resistance and detection of the acetamidase promoter by using primers AmiP1 and AmiP2. The plasmids were then purified and digested with BamHI and XbaI. A plasmid containing the expected fragment of 2.3 kb was selected for confirmatory sequencing and was designated pFPCA1.

Electroporation, plasmid transformation, and clone selection. Plasmid DNA was isolated from Escherichia coli DH5{alpha}. Strains H37Rv, H37Ra, and Erdman were cultivated in 7H9 broth supplemented with glycerol and Tween 80 (7H9GTw), pelleted, and then suspended in 10% sterile glycerol. Electroporation and selection of transformants were performed as described previously (12). GFP-associated fluorescence was determined with a Victor2 D multilabel counter fluorometer (Perkin-Elmer Life Sciences Inc., Boston, Mass.) in the top-reading mode with excitation at 485 nm and emission at 535 nm. The selected transformants were cultured in 200 ml of 7H9GTw with kanamycin. The bacterial suspensions were then washed once, suspended in 20 ml of phosphate-buffered saline (PBS), and passaged through an 8-mm-pore-size filter; and aliquots were stored at -80°C.

Antimicrobial agents. Capreomycin, chloramphenicol, clindamycin, clofazimine, cycloserine, ethambutol HCl, ethionamide, fusidic acid, isoniazid, minocycline, ofloxacin, p-aminosalicylic acid, streptomycin sulfate, and thiacetazone were purchased from Sigma Chemical Company. Amoxicillin, clavulanate lithium (2:2; wt/wt), and clarithromycin were purchased from the U.S. Pharmacopeia. Rifampin was purchased from Fisher Chemical Company. Ciprofloxacin HCl was purchased from Serologicals Corporation.

GFP microplate assay (GFPMA). Antimycobacterial susceptibility testing was performed in black, clear-bottom, 96-well microplates (black view plates; Packard Instrument Company, Meriden, Conn.). Frozen H37Rv gfp, H37Ra gfp, and Erdman gfp were thawed, sonicated for 15 s, and cultured at 37°C with shaking in 200 ml of 7H9GTw-kanamycin until the turbidity reached 50 to 70 Klett units. The cells were pelleted, washed with PBS, and then suspended in 20 ml of PBS buffer. Aliquots were stored at -80°C for up to 2 to 3 months. Drug stock solutions were prepared in either dimethyl sulfoxide or distilled deionized water and were further diluted in 7H12 broth (7H9 broth supplemented with 4 µg of catalase per ml, 5 mg of bovine serum albumin per ml, 5.6 µg of palmitic acid per ml, and 1 mg of Casitone per ml) supplemented with 0.2% vol/vol glycerol (7H12G). Frozen inocula were diluted in 7H12G to make final bacterial densities of 5 x 104 to 5 x 105 CFU/ml. The remaining steps were done as previously reported by Collins et al. (6).

MABA. Cell aliquots and serial dilution of drugs were prepared as described above for GFPMA. Frozen inocula were initially diluted in 7H12G to make final bacterial titers of 3.0 x 105 to 5.0 x 105 CFU/ml. The remaining steps were done as described previously (5).


   RESULTS
Top
 Abstract
 Introduction
 Materials and METHODS
 Results
Discussion
 References
 
GFP expression directed by the acetamidase promoter was continually detectable during cultivation without addition of acetamide, which is known to be an inducer of the acetamidase promoter (4, 13-15, 17, 19, 23). This was observed not only for H37Ra gfp but also for H37Rv gfp and Erdman gfp, whereas this construct lost promoter activity in M. bovis BCG (data not shown).

H37Rv harboring pFPCA1 produced a significantly higher net fluorescence signal (over that of the background) than the same strain harboring pFPV2 (Fig. 1). In addition, the fluorescence curve derived from the former construct (pFPCA1) was better correlated to cell growth (numbers of CFU) (Fig. 2), as determined by a better correlation (Pearson product-moment correlation coefficient; r value) between the fluorescence reading and cell growth [r values for Erdman(pFPCA1), H37Ra(pFPCA1), and H37Rv(pFPCA1), 0.9613, 0.9823, and 0.9661, respectively; r value for H37Rv(pFPV2), 0.9356]. Thus, the fluorescence reading could be used to monitor more efficiently the growth of the bacteria in culture. At day 7 the relative fluorescence units per CFU for M. tuberculosis Pacetamidase(Pace)::gfp strains H37Ra, Erdman, and H37Rv were approximately 1.56 x 10-2, 0.47 x 10-2, and 0.37 x 10-2, respectively, whereas that for M. tuberculosis Phsp60::gfp strain H37Rv was approximately 2.58 x 10-4.



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  		FIG. 1. Ratio of fluorescence signal to medium background signal during incubation of M. tuberculosis H37Rv gfp containing either pFPV2 (•; Phsp60::gfp) or pFPCA1 ({circ}; Pace::gfp) in 7H12G. Data are means ± standard deviations for two independent experiments.

 


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  		FIG. 2. GFP expression (solid lines) and CFU (dotted lines) during growth of M. tuberculosis H37Ra Pace::gfp (•), H37Rv Pace::gfp ({blacksquare}), Erdman Pace::gfp ({blacktriangledown}), and H37Rv Phsp60::gfp ({diamondsuit}) in 7H12G. Data are from one representative experiment of two experiments.

 
The utilities of these gfp strains for screening for antituberculosis activity were investigated by determining MICs in tests with a 96-well format. The kinetic response to serial twofold dilutions of antimycobacterial agents was easily detected by fluorescence reading of the microplate cultures for 1 week, an example of which is shown in Fig. 3 for H37Rv gfp and rifampin.



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  		FIG. 3. Rifampin dose-response curves for M. tuberculosis H37Rv gfp determined by fluorescence.

 
The MICs of 18 antimicrobial agents for H37Rv (Table 1), Erdman (Table 2), and H37Ra (Table 3) were determined by GFPMA and were compared with those determined by MABA after day 6 to 8 days of incubation. At least two independent experiments were conducted with each strain. The MICs of all drugs by GFPMA differed by <=2 twofold dilutions with respect to the MICs obtained by both the fluorometric and the visual MABA, with most differences being <=1 twofold dilution. For all three strains, no significant differences in MICs determined by the fluorometric or the visual MABA were found between the corresponding parent and gfp strains of M. tuberculosis.


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  		TABLE 1. Comparative MICs of 18 established drugs for M. tuberculosis H37Rva

 

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  		TABLE 2. Comparative MICs of 18 established drugs for M. tuberculosis Erdmana

 

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  		TABLE 3. Comparative MICs of 18 established drugs for M. tuberculosis H37Raa

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and METHODS
 Results
 Discussion
References
 
Previously, GFP coupled with a widely used hsp60 promoter has been used to screen drugs for their activities against M. aurum both in vitro and in macrophages (22), as well as to screen germicides for their activities against M. terrae (27). The hsp60 promoter was also used to regulate GFP expression in M. tuberculosis H37Ra to determine the MICs of established anti-TB drugs (6). However, neither the level of the absolute fluorescence signal nor the signal-to-noise (background) ratio was sufficiently high to make this construct a candidate for routine high-throughput screening.

In this study we replaced the hsp60 promoter in pFPV2 (25) with the 2.3-kb DNA segment containing the acetamidase promoter from M. smegmatis mc2155. The resulting recombinant M. tuberculosis strain produced strong fluorescence regardless of the presence of acetamide.

The genetic variation within the promoter region between two different strains of M. smegmatis (NCTC 8159 versus mc2155) might account for the different promoter activities.

Evidently, the resulting constitutive property of pFPCA1 is more favorable for a general viability reporter. It gave a higher relative fluorescence unit per CFU compared with that obtained with the previous gfp construct (pFPV2) as well as emitted a fluorescence signal that correlated well with the increase in cell growth.

We demonstrated here that by using an improved gfp recombinant M. tuberculosis strain with a high signal-to-noise ratio, a good correlation between cell growth and fluorescence activity during incubation can be obtained and that the MICs of antimycobacterial agents determined by GFPMA are consistent with those obtained by MABA. The GFPMA described here appears to be sufficiently robust for use for routine high-throughput screening for antituberculosis agents.


   ACKNOWLEDGMENTS
 
We thank Chanpen Wiwat, Saradee Warit, Sanghyun Cho, Fangqiu Zhang, Yuehong Wang, Kamolchanok Rakseree, Arunee Thong-On, and Nantawan Thong-On for technical assistance.

This study was supported by the Thailand Research Fund, the National Center for Genetic Engineering and Biotechnology, and contract NIH/NIAID/DAIDS-01-13 from the National Institutes of Health. Chartchai Changsen was supported by the Mahidol University Medical Scholar Program.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Tuberculosis Research, MC 964, Rm. 412, 833 S. Wood St., College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612-7231. Phone: (312) 355-1715. Fax: (312) 355-2693. E-mail: sgf{at}uic.edu. Back


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Antimicrobial Agents and Chemotherapy, December 2003, p. 3682-3687, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3682-3687.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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