title: Identification and Validation of Aspartic Acid Semialdehyde Dehydrogenase as a New Anti-Mycobacterium Tuberculosis Target.
abstract: 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.
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. mengjianzhou@126.com.
authors.forename: Jianzhou
authors.initials: J
authors.lastname: Meng
authors.affiliation: The School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, China. yyhysf@163.com.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. xiaocl318@163.com.
authors.forename: Chunling
authors.initials: C
authors.lastname: Xiao
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. guanyan20@163.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Guan
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. haoxueqin@hotmail.com.
authors.forename: Xueqin
authors.initials: X
authors.lastname: Hao
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. dengqi1992@163.com.
authors.forename: Qi
authors.initials: Q
authors.lastname: Deng
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. m18500395543@163.com.
authors.forename: Zhongyang
authors.initials: Z
authors.lastname: Lu
mesh_terms: D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D051379:Mice; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D000995:Antitubercular Agents; D001223:Aspartate-Semialdehyde Dehydrogenase
keywords: Mycobacterium tuberculosis; aspartic acid semialdehyde dehydrogenase; drug resistance; pathogenicity; pristinamycin; promotor-replacement mutant