毒素-抗毒素系统LsoA/RnlA-Dmd介导的细菌拮抗噬菌体侵染的分子机制研究

Bacterial toxin-antitoxin (TA) systems are closely associated with their physiological processes, such as the protection from bacteriophage (phage) infection and antibiotic resistance. LsoA-LsoB and RnlA-RnlB complexes are homologous TA systems involved in phage resistance, derived from enterohaemorrhagic Escherichia coli (E. coli) O157:H7 and E. coli K-12, respectively. The antitoxin Dmd from T4 phage can substitute the role of the antitoxins LsoB and RnlB, and directly interact with both LsoA and RnlA and against their toxicities which are activated during T4 infection. Therefore, LsoA/RnlA-Dmd complex represents a novel heterogenous TA system. In this study, we aim to determine the crystal structures and solution conformations of RnlA-RnlB, LsoA-LsoB and LsoA/RnlA-Dmd complexes using protein crystallography and small-angle X-ray scattering (SAXS) method, respectively. On this basis, we will further study the in vivo and in vitro interactions between the toxins and antitoxins in these TA systems and the resulting effects on host physiological processes. Our studies will reveal the bacteria defense mechanism involved in T4 phage resistance by RnlA-RnlB and LsoA-LsoB systems, and also reveal the unique counter-defense mechanism of phage infection in bacteria by LsoA/RnlA-Dmd systems. This project will suggest new strategies to deal with the phage infection in large-scale culture of engineering bacteria in food and antibiotic industries. Moreover, our findings will provide novel insights into antibacterial therapies by phage.

细菌的毒素-抗毒素系统与其拮抗噬菌体侵染和耐药性等生理功能密切相关。LsoA-LsoB和RnlA-RnlB复合物是分别来自大肠杆菌O157:H7和K-12的同源毒素-抗毒素系统，与T4噬菌体抗性相关。T4噬菌体的抗毒素蛋白Dmd在感染大肠杆菌时能取代抗毒素蛋白LsoB/RnlB，抑制并抵消被激活的毒素LsoA/RnlA的RNA酶活性，LsoA/RnlA-Dmd复合物是第一个抗毒素能拮抗多种毒素的系统。本课题采用结构生物学和微生物学相结合的方法，解析这些复合物的三维结构，并在此基础上研究这些毒素和抗毒素在体内和体外的相互作用及对宿主生理状态影响，揭示细菌通过RnlA-RnlB和LsoA-LsoB拮抗噬菌体侵染，以及噬菌体通过Dmd抑制毒素LsoA/RnlA活性来侵染宿主菌的分子机制。本研究将为防治食品和抗生素等产业中的噬菌体污染提供新策略，也为噬菌体代替抗生素治疗病原菌感染的研究提供新思路。

细菌的毒素-抗毒素系统与其耐药性和程序性细胞死亡等多项重要生理过程密切相关，对它们的三维结构和功能深入研究将有助于开发以这些系统作为作用靶点的新型抗菌肽或药物。本课题开展了致病性和模式大肠杆菌的毒素-抗毒素系统LsoA-LsoB、RnlA-RnlB，以及毒素蛋白LsoA/RnlA与来自T4噬菌体的抗毒素蛋白Dmd组成的新型毒素-抗毒素系统的结构和功能研究。我们成功解析了LsoA-Dmd复合物和RnlA单体的晶体结构，并进行了结构分析和相应的生化功能及体内体外蛋白相互作用研究。这些工作揭示了T4噬菌体通过LsoA-Dmd系统侵染大肠杆菌的独特分子机制，即Dmd通过模拟底物RNA来占据LsoA/RnlA的活性位点，达到抑制毒素的核酸酶活性目的。在这一过程中LsoA/RnlA的NRD (N端重复结构域)-DBD (Dmd结合结构域)的构象由“闭合”到“开放”的变化实现了Dmd的结合和自身毒性的抑制。通过进一步的蛋白相互作用研究，我们发现LsoB/RnlB可能采取与Dmd不同的机制来抑制LsoA/RnlA毒素的活性。在本课题资助下我们还对几个与细菌耐药性相关的毒素-抗毒素系统开展了结构和功能研究。通过解析绿脓杆菌的抗毒素蛋白HigA与启动子DNA复合物的结构和蛋白-DNA相互作用研究，揭示了HigA调控毒素-抗毒素系统HigA-HigB转录以及多个毒力因子合成的新颖分子机制。通过对希瓦氏菌新型毒素-抗毒素系统SO_3165/SO_3166复合物的结构和功能研究，揭示了该复合物的独特结构及新颖的毒素中和机制。以上这些重要的毒素-抗毒素结构和功能研究为开发新型抗菌药物提供了新靶点和重要参考。

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