大肠杆菌原噬菌体e14、rac和CPS-53中毒素-抗毒素系统的功能性研究

Bacteria Toxin-antitoxin systems have recerived worldwide attention due to its role in persister cell formation in microbilogy and in clinical studies. However, the mechanism remains to be elucidated. In many genomes including pathogenic bacteria, toxin-antitoxin systems have been identified and variations are found in term of structure and regulation; however, their role in cell physiology has been mostly unclear. This project focuses on the uncharacterized Toxin-antitoxin systems on the three prophages of Escherichia coli e14, rac and CPS-53. Prophage CPS-53 increases resistance to oxidative stress while rac and e14 increase resistance to antibiotics.After screening of the functional genes in three prophages based on structural features, the putative toxin-antitoxin pairs will be further verified by the toxicity of the toxins, the attenuation of the antitoxin and the co-transcription of toxin and antitoxin genes. Next, we will study the stress responses of these toxin-antitoxin pairs under oxidative stress, acid stress and heat stress, from the levels of transcription, translation and post-translational regulation. Specific efforts will be put into the functional study of these toxin-antitoxin pairs in persister cell formation. Furthermore, we will explore whether toxin or antitoxin can influence the integration and excision of the three prophages as well as the consequences of such genome loss of prophages. The proposed study will help us to gain more knowledge about the role of toxin-antitoxin systems in the formation of persister cells;in addition, it will help us to understand the co-evolution between toxin-antitoxin systems and the prophages. Moreover, the study will also provide new strategies to cope with persister cells in clinical treatments.

细菌基因组上的毒素-抗毒素系统影响细菌的顽固耐药性已引起微生物学界和医学界的高度关注。目前毒素-抗毒素的作用机理尚未阐明。毒素-抗毒素系统广泛地存在于原核生物包括很多病原菌上，结构和功能上具有多样性,但被深入研究的毒素-抗毒素系统非常有限。本项目围绕大肠杆菌原噬菌体中与氧化胁迫和抗生素的耐受性密切相关的3个原始菌体e14、rac 和CPS-53，从中筛选出符合遗传结构条件并经过实验验证的毒素-抗毒素系统为研究对象，综合运用传统微生物学和现代分子生物学的技术，在环境胁迫条件下，研究毒素-抗毒素系统在转录和翻译水平的响应机制；聚焦原噬菌体中的毒素-抗毒素在顽固耐药性形成过程中的调节；并结合原噬菌体自身的可诱导性，探讨毒素-抗毒素系统对于原噬菌体在基因组的整合和切离过程。此项研究有助于进一步认识毒素-抗毒素系统对于细菌顽固耐药性形成的影响，揭示毒素-抗毒素系统和原噬菌体的协同进化。

细菌基因组上的毒素-抗毒素系统已引起微生物学界和医学界的高度关注。目前毒素-抗毒素的作用机理和调控机理尚未阐明。毒素-抗毒素系统广泛地存在于原核生物包括很多病原菌上，结构和功能上具有多样性，但被深入研究的毒素-抗毒素系统尤其是原噬菌体上的毒素-抗毒素系统非常有限。基于本项目的研究，（1）揭示大肠杆菌原噬菌体rac生理功能及切离的调控过程。rac原噬菌体抑制细菌的游动性及对卡苄西林的抗性，rac在稳定期切离率的提高是通过RpoS诱导剪切酶XisR的表达实现的；（2）首次发现原噬菌体rac中2个相邻的基因ralR和ralA组成一对新型的I型毒素-抗毒素系统，并阐明毒素RalR毒力作用的分子机制，为DNA水解酶。抗毒素RalA为sRNA，其抗毒素功能受RNA分子伴侣Hfq严格调控；（3）揭示原噬菌体rac中毒素以及毒素-抗毒素分子增强细胞对广谱性抗生素磷霉素的抗性；（4）探讨了CP4-6，CP4-57和CP4-44中毒素-抗毒素系统间的相互作用；（5）综述了隐匿型原噬菌体如何作为相对永久的基因库参与宿主生理与代谢过程，指出隐匿性原噬菌体是非常具有潜力的药物靶点。（6）发现不同类型毒素-抗毒素系统间的Cross-talk，首次揭示了毒素-抗毒素系统之间的调控关系；（7）发现HEPN-MNT家族毒素-抗毒素系统，这是目前分布最广泛的II型毒素-抗毒素系统。基于本项目的资助，我们在2012年-2016年期间在微生物的权威杂志共发表SCI 收录论文6篇，培养博士后1名，培养博士生和硕士共 4 名， 完成了预期的研究目标。综合上述研究，这些研究有助于进一步认识毒素-抗毒素系统对于细菌多重耐药性形成的影响，揭示毒素-抗毒素系统和原噬菌体的协同进化。