肺炎克雷伯菌替加环素耐药分子调控机制复杂，调控子之间的互作机制尚未完全阐明。根据前期研究基础和文献报道，我们提出肺炎克雷伯菌中Lon蛋白与RamRA存在互作机制，共同参与调控替加环素耐药的科学假说。为验证此假说，拟采用等位基因替换、同源重组构建相应菌株的Lon回补株和RarA敲除株，研究有/无RamA时，Lon替代通路在调控替加环素耐药中的作用；采用转录组测序找到受Lon和RarA调控的下游靶基因并验证基因表达量变化的普遍性；采用顺序肽亲和纯化和细菌双杂交实验筛选并验证与Lon有相互作用的蛋白，Lon与RamRA和RarA的交互作用。最终勾画以Lon蛋白酶和RamRA为核心的介导替加环素耐药的分子调控通路，为防控替加环素耐药菌的播散及延缓耐药趋势提供重要基础。

The molecular regulation mechanism of tigecycline-resistant Klebsiella pneumoniae is complex, and the mechanism of interaction between regulators has not been fully elucidated. Based on our previous research and literature reports, we propose a scientific hypothesis that Lon and RamRA have an interaction mechanism to participate in tigecycline resistance in Klebsiella pneumoniae. To validate this hypothesis, it is proposed to construct Lon complementary strains and RarA knockout strains of the corresponding parent by allelic substitution and homologous recombination, respectively, to study the role of Lon alternative pathway in regulating tigecycline resistance of Klebsiella pneumoniae with or without RamA. RNA-Sequencing and qRT-PCR are used to find the downstream target genes regulated by Lon and RarA, and to verify their universality among clinical strains, respectively. Sequential peptide affinity purification and bacterial two-hybrid assays are used to screen and validate the interaction of possible proteins interacting with Lon, Lon with RamRA and RarA. Finally, the molecular regulatory pathways that mediate tigecycline resistance with Lon protease and RamRA as the core regulon will be delineated to provide an important support in preventing and controlling dissemination of tigecycline-resistant bacteria and delaying resistance development trends.