Tn1721/Tn4401嵌合体结构中blaKPC基因的表达调控机制研究

KPC type Carbapenemase which can hydrolyze nearly all of the β-lactam antibiotics and is detected in 58% CRE strains in China. Previous study reviewed great difference of the Carbapenem resistance in KPC strains, however, the mechanism has not yet been elucidated. In China, the blaKPC gene is mainly located in the Tn1721/Tn4401 chimera. Our pilot study discovered that in low Carbapenem resistant strain, the blaKPC gene carries the controversial promoter P1 and a putative promoter PX in upstream, and forms into a tandem promoter P1X, while in high resistant isolate, it contains only the P1. Especially, the untranslated region (UTR) of blaKPC mRNA generated by P1X promoter may form a strong hairpin structure while it may not by P1. Compared with strains containing promoter P1X, KPC mRNA and Carbapenem resistance of bacteria with only P1 were 2- or 4-fold higher, but isolates with PX were basically the same. Hence, we speculated that P1 is more active but PX is the dominant promoter, and the UTR may regulates KPC translation. This study plan to shed light on the regulation mechanism of blaKPC gene expression from going into the core promoter of blaKPC and its transcription traits, and the role of UTR in KPC translation. This work may contribute to precision anti-infection therapy and will provide new sight for antibiotic developing.

KPC酶能够水解几乎所有β-内酰胺类抗生素，在我国CRE菌株中的检出率高达58%。前期发现产KPC酶菌株的碳青霉烯耐药性具有高度差异，但其机制尚未明确。国内KPC基因(blaKPC)主要位于Tn1721/Tn4401嵌合体结构。初步研究发现，低耐药菌嵌合体中的blaKPC基因携带争议启动子P1并在上游插入潜在启动子PX，构成串联启动子P1X，且转录的mRNA非编码区(UTR)可形成复杂的发夹结构；高耐药菌则仅有P1而UTR无发夹。分析启动子重组菌株的KPCmRNA表达量和碳青霉烯耐药性，发现P1比P1X分别高2倍和4倍，而PX则与P1X基本一致。我们推测P1活性高但PX是显性启动子，且UTR可调控KPC翻译。本项目拟深入研究嵌合体中blaKPC基因的核心启动子、转录特征以及UTR的调控作用，进而明确KPC菌株碳青霉烯耐药差异机制，可为精准抗感染治疗提供实验依据并为抗生素研发探索新思路。

KPC酶能够水解几乎所有β-内酰胺类抗生素，在我国CRE菌株中的检出率高达58%。前期发现产KPC酶菌株的碳青霉烯耐药性具有高度差异，但其机制尚未明确。本项目比较分析了国外Tn4401和国内Tn3-Tn4401嵌合体(CTB)结构中blaKPC基因的表达差异，发现CTB的碳青霉烯类耐药性介于Tn4401a和Tn4401b之间。发现并验证了blaKPC基因的新型启动子PX、PY，其中PY是嵌合体中blaKPC基因的核心启动子，并且缺失PX和P1反而分别增强CTA和CTC等嵌合体亚型中blaKPC基因表达和菌株的碳青霉烯耐药性。同时，发现5’ UTR中PY和P1启动子之间的干预序列(IVS)长度具有重要的调控作用，缩短CTB或延伸CTA或进一步缩短CTA的IVS长度均可显著抑制blaKPC基因表达，其中CTA为最优化结构。以上成果先后发表于(Antimicrob Agents Chemother，2019；Antimicrob Agents Chemother，2020)。此外，分析了本地区CRKP菌株的分子流行病学特征，发现了一起ST15型CRKP菌株引起的院内暴发流行。建立了一种细菌耐药性快速检测方法，用于判断肺炎克雷伯菌对亚胺培南的耐药性。本项目研究发表学术论文5篇，申报发明专利1项，为新型抗菌药物研发提供新靶点，并为碳青霉烯耐药性快速检测提供了新思路。