分枝杆菌丝氨酸/苏氨酸蛋白激酶PknK的功能研究  

Characterization of Serine/Threonine protein kinases K(PknK) in Mycobacterium

作　　者：周怡璇 周心童[1] 杨健 胡新玲[1] 米凯霞[1,2] Yixuan Zhou;Xintong Zhou;Jian Yang;Xinling Hu;Kaixia Mi(CAS Key Laboratory of Pathogenic Microbiology and Immunology,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;Savaid Medical School,University of Chinese Academy of Sciences,Beijing 101408,China)

机构地区：[1]中国科学院微生物研究所,中国科学院病原微生物与免疫学重点实验室,北京100101 [2]中国科学院大学存济医学院,北京101408

出　　处：《微生物学报》2019年第12期2378-2389,共12页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2018YFC1603900,2017YFA0505901);国家自然科学基金(31600114,31700128)~~

摘　　要：【目的】丝氨酸/苏氨酸蛋白激酶K(Serine/Threonine protein kinases K)是分枝杆菌类似真核样的蛋白激酶,预测在分枝杆菌的生长和新陈代谢等生理过程中起着重要的作用,解析PknK的生物功能及作用机制,将为结核病的防治提供一定的理论基础。【方法】通过基因敲除等遗传方法获得结核分枝杆菌疫苗株BCG的pknK敲除菌株△pknK、回补菌株pMV361-pknK/△pknK和过表达菌株pMV261-pknK/BCG;对获得的菌株进行生长曲线测定和抗药性分析;通过pulldown-MS方法及生物信息学方法鉴定了PknK相互作用蛋白。【结果】监测各种分枝杆菌△pknK、pMV361-pknK/△pknK和pMV261-pknK/BCG生长,确定PknK负调控BCG生长;抗药性分析显示PknK降低BCG的耐药性;pulldown-MS方法显示PknK与丝氨酸/苏氨酸蛋白激酶PknA和双组分系统中的反应调节因子MtrA、TrcR、MoxR等蛋白相互作用。【结论】研究发现PknK调控分枝杆菌的生长和耐药性,我们的研究为深入研究PknK在结核分枝杆菌中的功能奠定了基础。[Objective] Serine/Threonine Protein Kinases K(PknK) is a eukaryotic-like Ser/Thr protein kinase in Mycobacterium, with important roles in cell growth and signaling transduction. However, its underlying mechanism of action is not completed. [Methods] The pknK knockout strain pknK was obtained by phage specialized transduction meanwhile the complement strain pMV361-pknK/ΔpknK and the overexpressing strain with pMV261-pknK/BCG was construed for further analysis. The growth curve and resistance of the obtained strains were determined. PknK-interacting proteins were identified by pulldown-MS. [Results] PknK affected Mycobacterium growth, and pknK had growth advantage over BCG and pMV261-pknK/BCG. Knockout pknK led to increased multiple antibiotic susceptibility. We identified the binding proteins for PnK in BCG using in pulldown combined with Mass Spectrometry. [Conclusion] PknK negatively regulates BCG growth and increases antibiotic susceptibility in mycobacteria. The PknK binding proteins include a Ser/Thr protein kinase PknA, and two component system regulators such as MtrA, MoxR1 and TrcR, which is expected to be a resource for understanding the PknK-mediated signaling pathways in Mycobacterium, thus facilitating new therapeutic strategies for antibiotic-resistant infections.

关 键 词：丝氨酸/苏氨酸蛋白激酶 PknK 生长 耐药性 

分 类 号：R44[医药卫生—诊断学]