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

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。

Abstract:

[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.