Assessing the essentiality of the decaprenyl-phospho-D-arabinofuranose pathway in Mycobacterium tuberculosis using conditional mutants
Assessing the essentiality of the decaprenyl-phospho-D-arabinofuranose pathway in Mycobacterium tuberculosis using conditional mutants
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DOI：
10.1111/mmi.12546
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发表时间：
2014-04-01
影响因子：
3.6
通讯作者：
Cole, Stewart T.
中科院分区：
文献类型：
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作者：
Kolly, Gaelle S.;Boldrin, Francesca;Cole, Stewart T.
在结核分枝杆菌中，十异戊二烯基-磷酸-d-阿拉伯呋喃糖 (DPA) 途径是乙胺丁醇和苯并噻嗪酮类药物的有效靶点。为了识别该途径中的其他潜在药物靶点，我们使用 TET-PIP OFF 系统生成了每个相关基因的条件敲低突变体。 dprE1、dprE2、ubiA、prsA、rv2361c、tkt 和 rpiB 被证实在非许可条件下是必需的，而 rv3807c 不是生存所必需的。在最脆弱的群体中，DprE1耗尽的细胞在体外和细胞内的死亡速度比那些缺乏UbiA和PrsA的细胞更快。 DprE1 和 UbiA 的下调导致相似的表型，即通过实时显微镜和电子显微镜在单细胞水平观察到的细菌肿胀、细胞壁损伤和裂解。相比之下，PrsA 的消耗导致细胞伸长和内爆，这表明更具多效性。使用已知 DPA 抑制剂进行的药物敏感性测定支持使用条件敲低菌株进行基于靶标的全细胞筛选。总之，我们的工作为 DPA 途径中除一种酶外的所有酶的脆弱性提供了强有力的证据，并为识别针对不同生物合成步骤的先导化合物提供了有价值的工具。 PrsA（磷酸核糖焦磷酸合成酶）似乎是药物发现中特别有吸引力的新靶标。
In Mycobacterium tuberculosis the decaprenyl-phospho-d-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1-depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA-inhibitors supported the use of conditional knock-down strains for target-based whole-cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl-pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.