Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter
Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter
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DOI：
10.1128/aac.47.12.3682-3687.2003
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发表时间：
2003-12-01
影响因子：
4.9
通讯作者：
Palittapongarnpim, P
中科院分区：
文献类型：
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作者：
Changsen, C;Franzblau, SG;Palittapongarnpim, P
绿色荧光蛋白（GFP）基因作为高通量抗菌药物筛选的活性报告基因具有许多优势。然而，利用热休克启动子hsp60驱动的GFP筛选抗结核化合物，由于信噪比低，效用有限。因此，利用绿色荧光蛋白微孔板实验（GFPMA）评估了一种替代启动子在微孔板培养过程中的荧光增强效果，以及对18种已建立的抗菌剂的反应。用pFPCA1转化结核分枝杆菌H37Rv、H37Ra和Erdman菌株，pFPCA1含有一个红移的gfp基因，该基因由耻垢分枝杆菌mc(2)155的乙酰氨基酶启动子驱动。与hsp60构建体相比，pFPCA1转化体获得了更高水平的gfp介导荧光，信噪比分别为20.6 - 27.8和3.8 - 4.5。在GFPMA中，所有携带pFPCA1的菌株的18种抗菌药物的mic值均在荧光法或视觉微孔板Alamar Blue assay （MABA）测定值的1至2倍稀释范围内。经MABA检测，野生型和pFPCA1转化子的mic无显著差异。优化后的GFPMA足够简单、可靠和廉价（无试剂成本），可用于常规的抗结核化合物高通量筛选。
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.