Meng Jianzhou, Yang Yanhui, Xiao Chunling, Guan Yan, Hao Xueqin, Deng Qi, Lu Zhongyang
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
The School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, China.
Int J Mol Sci. 2015 Sep 30;16(10):23572-86. doi: 10.3390/ijms161023572.
Aspartic acid semialdehyde dehydrogenase (ASADH) lies at the first branch point in the essential aspartic acid biosynthetic pathway that is found in bacteria and plants but is absent from animals. Mutations in the asadh gene encoding ASADH produce an inactive enzyme, which is lethal. Therefore, in this study, we investigated the hypothesis that ASADH represents a new anti-Mycobacterium tuberculosis (MTB) target. An asadh promoter-replacement mutant MTB, designated MTB::asadh, in which asadh gene expression is regulated by pristinamycin, was constructed to investigate the physiological functions of ASADH in the host bacteria. Bacterial growth was evaluated by monitoring OD600 and ASADH expression was analyzed by Western blotting. The results showed that the growth and survival of MTB::asadh was completely inhibited in the absence of the inducer pristinamycin. Furthermore, the growth of the mutant was rigorously dependent on the presence of the inducer in the medium. The starved mutant exhibited a marked reduction (approximately 80%) in the cell wall materials compared to the wild-type, in addition to obvious morphological differences that were apparent in scanning electron microscopy studies; however, with the addition of pristinamycin, the cell wall contents and morphology similar to those of the wild-type strain were recovered. The starved mutant also exhibited almost no pathogenicity in an in vitro model of infection using mouse macrophage J774A.1 cells. The mutant showed a concentration-dependent recovery of pathogenicity with the addition of the inducer. These findings implicate ASADH as a promising target for the development of novel anti-MTB drugs.
天冬氨酸半醛脱氢酶（ASADH）处于细菌和植物中存在但动物中不存在的必需天冬氨酸生物合成途径的第一个分支点。编码ASADH的asadh基因突变会产生无活性的酶，这是致命的。因此，在本研究中，我们研究了ASADH代表一种新的抗结核分枝杆菌（MTB）靶点的假说。构建了一个asadh启动子替换突变型MTB，命名为MTB::asadh，其中asadh基因表达受 pristinamycin调控，以研究ASADH在宿主细菌中的生理功能。通过监测OD600评估细菌生长，并通过蛋白质印迹分析ASADH表达。结果表明，在没有诱导剂pristinamycin的情况下，MTB::asadh的生长和存活完全受到抑制。此外，突变体的生长严格依赖于培养基中诱导剂的存在。与野生型相比，饥饿的突变体细胞壁物质显著减少（约80%），此外在扫描电子显微镜研究中明显可见明显的形态差异；然而，添加pristinamycin后，细胞壁成分和形态恢复到与野生型菌株相似。在使用小鼠巨噬细胞J774A.1细胞的体外感染模型中，饥饿的突变体也几乎没有致病性。添加诱导剂后，突变体的致病性呈浓度依赖性恢复。这些发现表明ASADH是开发新型抗MTB药物的一个有前景的靶点。
