A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren.
A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren.
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DOI：
10.1371/journal.pone.0141658
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Matsumoto S
中科院分区：
文献类型：
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作者：
Ozeki Y;Igarashi M;Doe M;Tamaru A;Kinoshita N;Ogura Y;Iwamoto T;Sawa R;Umekita M;Enany S;Nishiuchi Y;Osada-Oka M;Hayashi T;Niki M;Tateishi Y;Hatano M;Matsumoto S
结核病（TB）是由细菌病原体引起的严重传染病。据估计，2013年全世界结核病死亡人数为150万。不仅要缩短用药周期，而且要治疗耐多药和广泛耐药结核病，需要开发新的结核病药物。结核分枝杆菌（Mycobacterium tuberculosis，Mtb）生长缓慢，每天仅繁殖一到两次。因此，常规药物筛查需要3周以上。此外，还需要一个生物安全3级（BSL-3）设施。因此，我们开发了一种新的筛选方法来确定结核病候选药物，通过利用表达重组牛分枝杆菌卡介苗（rBCG）。使用这种方法，我们确定了几个候选人在4天内在非BSL-3设施。我们筛选了10，080个来自放线菌和链霉菌的粗提物，并鉴定了137个具有rBCG荧光素酶抑制活性的提取物。其中，41个化合物抑制Mtb H37 Rv和广泛耐药Mtb（XDR-Mtb）菌株的生长。我们纯化了1904-1提取物的活性物质，其对rBCG、Mtb H37 Rv和XDR-Mtb具有强活性，但对宿主真核细胞无害。该物质对rBCG、H37 Rv和2株XDR的MIC分别为0.13 μg/ml、0.5 μ g/ml和2.0-7.5 μg/ml。除对鸟分枝杆菌胞内复合体外，对耐酸菌均有特异性作用。质谱和核磁共振分析表明，1904-1的活性物质为cyclomarin A。为了证实1904-1衍生化合物的作用模式，使用了抗性BCG克隆。全基因组DNA序列分析表明，这些克隆包含clpc基因中的突变，该基因编码酪蛋白溶解蛋白，酪蛋白溶解蛋白是ATP依赖性蛋白酶的基本组分，并且可能是1904-1活性物质的靶标。我们的方法提供了一个快速和方便的筛选，以确定抗分枝杆菌药物。
Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904–1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0–7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904–1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904–1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.