E2泛素结合酶关键分子调控胞内布鲁氏菌存活的分子机制研究

Brucellosis can cause serious harm to public health and safety. The main target cells of brucella are the macrophages. Brucella could persist and replicate within the host through inducing cell autophagy. Our previous studies have shown that ubiquitin-like protein SUMO-1 is essential for Brucella survival, E2 ubiquitin-conjugating enzyme is key in the process of SUMO-1 modification and play an important role in host resistance against this pathogen. Therefore, it is assumed that E2 ubiquitin-conjugating enzyme may regulate Brucella survival. This study is about: ①RAW264.7 macrophages will be transfected with E2 enzymes siRNA library. Then we will establish the macrophages model of Brucella melitensis infection.The key molecules of E2 ubiquitin-conjugating enzyme will be screened by automated fluorescent microscope and CFU count; ②The effect of key molecules on Brucella-dependent cell autophagy will be detected by Flow cytometry, Electron microscopy, RT-PCR, and Western blot. Therefore, the regulation mechanism of key molecules will be elaborated. This study will lay the foundation for revealing the pathogenic mechanism of intracellular Brucella.

布鲁氏菌病是严重危害公共卫生安全的人畜共患病。巨噬细胞是布鲁氏菌侵袭的主要靶细胞，布鲁氏菌诱导细胞自噬是逃避宿主杀伤在胞内生存繁殖的机制之一。我们前期研究表明：类泛素蛋白SUMO-1在胞内布鲁氏菌存活起着重要作用，由于E2泛素结合酶参与类泛素蛋白SUMO-1的修饰过程，并且在宿主抵抗病原菌感染中发挥重要作用，因此假设：E2泛素结合酶可能调控胞内布鲁氏菌的存活。本研究拟通过①将E2泛素结合酶siRNA文库转染巨噬细胞，建立布鲁氏菌侵染巨噬细胞模型，通过自动荧光显微镜和菌斑计数，筛选出影响胞内布鲁氏菌存活的E2泛素结合酶关键分子；②通过电镜、RT-PCR、Western blot检测关键分子对布鲁氏菌介导的细胞自噬的影响，阐述关键分子的调控机制。本研究为揭示布鲁氏菌在胞内致病机制提供科学依据。

将E2泛素结合酶siRNA文库转染细胞，利用带GFP的布鲁氏菌感染细胞，利用Hoechst33258将细胞核染为蓝色，通过灰度值分析绿色荧光与蓝色荧光强度比值筛选出细胞内影响布鲁氏菌繁殖的关键基因Ube2g2和Ube2t。利用siRNA片段和慢病毒重组载体方法分别沉默和过表达细胞Ube2g2和Ube2t基因。结果发现沉默细胞Ube2g2和Ube2t基因后，抑制M5-90介导的细胞自噬水平，抑制M5-90在RAW264.7细胞内的生存能力，抑制M5-90介导的细胞PI3K/Akt信号通路的激活，提高M5-90介导IFN-γ、IL-6和TNF-α水平。过表达细胞Ube2g2和Ube2t基因后，可增加M5-90在细胞内的生存能力；过表达Ube2g2基因后，提高M5-90介导的细胞自噬水平，提高M5-90对细胞PI3K/Akt信号通路的激活程度；而过表达Ube2t基因，与M5-90介导的细胞自噬不相关，对M5-90感染的细胞PI3K/Akt信号通路的激活无影响。