IncI2型黏菌素抗性质粒上毒素-抗毒素系统的功能及调控机理研究

近几年，mcr-1基因介导的粘菌素耐药问题受到全球各领域的高度关注。mcr-1基因最早发现于IncI2型质粒（pHNSHP45）且分布广泛，但这些质粒的稳定和传播机制不清楚。毒素-抗毒素系统（TA）最早发现于质粒且维持其稳定性，主流机制为“分裂致死效应”。该理论主要基于TA的过表达，缺乏生理状态下毒素杀死细菌的直接证据。我们发现pHNSHP45编码三对TA，均符合“分裂致死效应”条件，但维持质粒的功能差异巨大。本项目主要以pHNSHP45为切入点，基于TA敲除和回补菌株，综合运用微生物和分子遗传学技术，结合小鼠模型，阐明这三对TA的类型和分子特征，揭示其在细菌宿主和动物宿主体内稳定质粒作用差异的分子机制和调控机理，阐明环境因子通过TA对IncI2型粘菌素抗性质粒稳定和传播的调控机理，探索靶向TA控制粘菌素耐药菌的新途径，研究结果对开发治疗多重耐药菌的“新型抗生素”具有一定的理论和现实意义。

Colistin is a last-resort parenteral antimicrobial drug, and the global spread of mobilized colistin resistance determinants (mcr-1) by conjugation or through mobilizable plasmids has triggered extensive concerns worldwide. However, the molecular mechanisms that promote the segregational stability of colistin resistance plasmids remain unclear. Bacterial toxin-antitoxin (TA) systems are mobile genetic elements, and TA systems encoded by plasmids have been proposed to mediate plasmid stability through a mechanism known as “post-segregational killing (PSK)”. Although PSK has been proposed over 30 years, direct evidences supporting this mechanism is still lacking, especially the experimental evidences showing plasmid-free cells are indeed killed by the toxins in a physiological relevant condition. Megaplasmid pHNSHP45 is the first reported mcr-1 carrying plasmid belonging to the IncI2 type. On this megaplasmid, we identified three different TA systems including HicA/HicB, TsxA/TsxB and StbE/StbD. Although homologs of all of the three TA were predicted to maintain plasmid through PSK, by constructing the TA deletion mutant strains, we found clear differences of the three TA pairs in maintaining pHNSHP45 stability. The aim of the project is to reveal the function and regulation of these three TA pairs on pHNSHP45. Knockout and complementation of each TA genes will be conducted instead of only using overexpressing vectors to explore the function of TA in physiological relevant conditions. Combined with the technologies of microbiology, molecular genetics and mouse infection model, specific efforts will be put into the mechanistic study of the TA systems that can maintain plasmid stability, not only in the bacterial host but also in the mice host. We will also explore the function of these TAs in the other five IncI2 type colistin resistance plasmids. The proposed study will help us gain more knowledge on the physiological roles of TA systems. Given this ubiquity of TA in IncI2 type colistin resistance plasmids and their roles in plasmid maintenance, disruption of TA with pharmacological agents might be an attractive strategy for sanitizing colistin resistance during clinical treatment.