Singh Vandana, Biswas Rajesh Kumar, Singh Bhupendra N
Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, India.
Antimicrob Agents Chemother. 2014;58(3):1389-96. doi: 10.1128/AAC.01301-13. Epub 2013 Dec 16.
Conventional antimycobacterial screening involves CFU analysis, which poses a great challenge due to slow growth of mycobacteria. Recombinant strains carrying reporter genes under the influence of constitutive promoters allow rapid and wide screening of compounds but without revealing their modes of action. Reporter strains using pathway-specific promoters provide a better alternative but allow a limited screening of compounds interfering with only a particular metabolic pathway. This reduces these strains to merely a second-line screening system, as they fail to identify even the more potent compounds if they are not inhibiting the pathway of interest. In this study, we have generated a double recombinant Mycobacterium bovis BCG strain carrying firefly and Renilla luciferase genes as two reporters under the control of a constitutive and an inducible mycobacterial promoter. The presence of dual reporters allows simultaneous expression and analysis of two reporter enzymes within a single system. The expression profile of the firefly luciferase gene, rendered by a constitutive mycobacterial promoter, coincides with the decline in bacterial growth in response to a wide range of antimycobacterial drugs, while the enhanced expression of Renilla luciferase mirrors the selective induction of the reporter gene expression as a result of pathway-specific inhibition. Thus, the double recombinant strain allows the screening of both primary and rationally synthesized antimycobacterial compounds in a single assay. The inhibiting response of drugs was monitored with a dual-luciferase reporter assay which can be easily adapted in high-throughput mode.
传统的抗分枝杆菌筛选涉及菌落形成单位（CFU）分析，由于分枝杆菌生长缓慢，这带来了巨大挑战。携带在组成型启动子影响下的报告基因的重组菌株能够快速且广泛地筛选化合物，但无法揭示其作用方式。使用途径特异性启动子的报告菌株提供了更好的选择，但只能对仅干扰特定代谢途径的化合物进行有限的筛选。如果它们不抑制感兴趣的途径，即使是更有效的化合物也无法被识别，这使得这些菌株沦为仅作为二线筛选系统。在本研究中，我们构建了一种双重组牛分枝杆菌卡介苗（Mycobacterium bovis BCG）菌株，其携带萤火虫荧光素酶基因和海肾荧光素酶基因作为两个报告基因，分别受一个组成型和一个诱导型分枝杆菌启动子的控制。双报告基因的存在使得在单个系统中能够同时表达和分析两种报告酶。由组成型分枝杆菌启动子驱动的萤火虫荧光素酶基因的表达谱，与对多种抗分枝杆菌药物反应时细菌生长的下降情况一致，而海肾荧光素酶表达的增强反映了由于途径特异性抑制导致的报告基因表达的选择性诱导。因此，这种双重组菌株能够在单一测定中筛选一线和合理合成的抗分枝杆菌化合物。通过双荧光素酶报告基因测定法监测药物的抑制反应，该方法可以很容易地适应高通量模式。
