IncX3型耐药质粒中新型毒素-抗毒素系统的功能研究

IncX3 type plasmid harboring blaNDM played an important role in the dissemination of Carbapenem-resistant Enterobacteriaceae(CRE). Stabilization of plasmids in the bacterial population can be achieved by the action of toxin-antoxin systems (TAs), which promotes the spread of the plasmids among various species of bacterial pathogens. Our recent studies reveal that there is a novel TAs (orf55、orf56) in the IncX3 type plasmids. The screen by PCR in the clinical samples and in silico analysis reveals that the novel TAs is popular and conserved in the IncX3 plasmids. But their roles in plasmid stability and the transmission of antibiotic resistance genes are still not well defined. Based on the previous studies, this project will evaluate the influence of TAs on the cell growth of host cell and identify the effect on plasmid stability by gene knockout and overexpression technique of TAs. In addition, we will try to further investigate the role of the novel TAs in plasmid stability and the transmission of blaNDM mediated by IncX3 type plasmids among bacterial population.This subject reveals the role of novel TAs in the stability of IncX3 type plasmids which might by crucial for the dissemination of blaNDM, and provides new therapeutic targets and strategies for the prevention and treatment of CRE.

IncX3型质粒水平传播blaNDM是碳青霉烯类耐药的肠杆菌科细菌（CRE）的重要耐药传播机制。质粒中固有的毒素-抗毒素系统（TAs）通过维持质粒稳定性协助质粒在菌株间传播、流行。前期研究中，课题组首次在携带blaNDM的IncX3型野生质粒中发现了新型的TAs（orf55、orf56）。临床分离株筛查及比较基因组学证实了该新型TAs在IncX3型质粒中普遍存在且序列保守，提示其对于维持IncX3型质粒稳定性具有重要功能，但机制不明。本课题拟采用基因敲除及调控过表达技术手段，研究该新型TAs的缺失及过表达对宿主菌生长的影响，及其对于维持IncX3型质粒稳定性的作用，探讨新型TA通过维持IncX3型质粒稳定性来增加宿主菌株间blaNDM传播的作用。该课题将在新型TAs系统维持质粒稳定性的角度阐述IncX3型耐药质粒流行的机制，为CRE的防控、抗菌药物的研发提供新的靶点和策略。

证实IncX3型质粒中存在新型的TAs，暂时命名为XpcR（IncX3 plasmid control repressor）和XpcA（IncX3 plasmid control Anti-repressor），XpcR能够发挥毒素的作用，抑制宿主菌株的生长，XpcR/XpcA组合能够对质粒稳定性产生影响。但课题尚未解决的问题是XpcR/XpcA组合不能以经典II型毒素-抗毒素形成蛋白复合物的方式发挥功能，课题组将继续围绕XpcR/XpcA组合具体的作用机制开展深入研究。研究过程中，课题组发现XpcR/XpcA基因敲除菌株质粒拷贝数和适应性代价大大增加，因此推测XpcR/XpcA参与了质粒拷贝数和适应性代价的调控。分析IncX3质粒复制子RepB蛋白构象，推测其在IncX3型质粒θ复制过程中，通过与Ori区域重复子Iteron结合调控质粒拷贝数。分析IncX3型质粒复制子RepB上游序列，存在四个串联“5’-TGAGnG-3’”重复子序列。综上，我们推测XpcA通过与Iteron作用，阻断质粒复制子RepB与Iteron结合从而降低质粒拷贝数。进而为解释IncX3的广泛传播提供新的理论依据，为blaNDM耐药基因传播防控提供新的靶点和策略。