典型毒素-抗毒素系统介导细菌程序性死亡的分子机理研究

Despite of success in controlling or eliminating bacterial infections, widespread use of antibiotics has led to drug resistance in pathogenic bacteria. Development of new anti-microbial drugs and finding their targets are imperative. Toxin-antitoxin systems are operons that are widespread in prokaryotes genomes. Because of its regulation on bacterial growth inhibition and programmed cell death, TAs have been proposed as an antibacterial strategy and has considerable potential, while the mechanism underlying the TAs induced PCD remains obscure. Previous research revealed that the regulation of growth inhibition was switched to PCD with the enhanced toxin activity. The typical toxin of TAs, MazF, activated a small amount of protein synthesis cooperating with global inhibition on the translation level. These proteins may play important roles in the TAs induced PCD pathways. In this project, RNA sequencing, qPCR, dimensional electrophoresis and gene-knockout will be used to investigate the key genes and molecular mechanism involved in the induced bacterial cell death.Additionally, the stress conditions and toxin activities will be exploited to optimize the anti-microbial strategy based on TAs. Our research will investigate the molecular mechanism of TAs function and pave the way for screening of anti-microbial drugs based on TAs.

抗生素的广泛使用导致大量耐药菌的出现，对新型抗耐药菌药物和靶点的开发迫在眉睫。毒素-抗毒素系统（TAs）是原核系统特有的一对基因操纵元件，由于具有调控细菌生长抑制和程序性死亡的功能，可作为一种安全有效的新型抗耐药菌策略，TAs诱发细菌程序性死亡的分子机理是研究该抗菌策略的关键科学问题。前期研究发现，随着毒素作用的加强，TAs的调节机制由生长抑制通路向程序性死亡通路转变。MazF毒素抑制整体蛋白质合成的同时，保留和促进了小部分蛋白的生成，可能与其介导的程序性死亡相关。本项目以此为切入点，通过转录组测序、qPCR、双向电泳、基因突变等手段研究TAs调节介导程序性死亡的关键基因和分子机理；通过摸索干预条件和评估典型TAs的抗菌活性，优化TAs的抗菌策略。本研究不仅有助于进一步阐明TAs的分子调控机理，也为后续基于TAs系统的抗菌药筛选奠定基础。

本课题围绕毒素-抗毒素系统（TAs）在细菌程序性死亡中的调控机制开展研究工作，通过解析TAs参与细菌严谨反应的分子机制，揭示其抗菌原理及潜在应用。本课题主要以典型TAs中的MazEF为对象，从毒素蛋白的构效关系、致毒机制及毒素-抗毒素系统对大肠杆菌严谨反应的调控模式三个层次开展工作，取得以下进展：1、构建了一系列大肠杆菌TAs基因敲除和遗传改造菌株，验证了不同基因背景下多种胁迫环境、EDF小分子、毒蛋白等对大肠杆菌程序性死亡的影响；2、系统研究了MazF毒素蛋白的构效关系，研究发现MazF（C48A）突变体具有高活性高稳定性的特性。3、对典型毒素MazF在不同基因背景下过表达对细菌的影响进行了两轮转录组测序与相关的分析验证，结果表明MazG基因存在对MazF对细菌的致死作用有显著影响；4、系统研究了MazG蛋白参与mazEF介导的大肠杆菌严谨反应调控的作用机制。本研究初步解决了课题提出的TAs调节细菌程序性死亡机制的科学问题，创新性的发现了第三基因（MazG）在毒素-抗毒素介导细菌严谨反应中的重要作用，对相关领域研究提供了新的思路。