碳青霉烯类耐药产气肠杆菌中外膜孔蛋白OmpF表达的sRNA调控机制研究

Carbapenem resistant Enterbactor aerogenes infections with high fatality rate tended to spread in the hospital. The deceased outer membrane porin expression with the unclear regulatory system was considered one of the primary carbapenem resistance mechanisms. We found a clinical carbapenem resistant isolate with declined OmpF expression without β-lactamase production or efflux pump in the previous studies. This project was planned to study on the regulatory mechanisms of decreased expression of outer membrane porin OmpF. This study intended to detect the differential sRNA expression levels between carbapenem sensitive and resistant isolates with application of High Throughput Sequencing and Bioinformatic Analysis, so as to screen the sRNA regulating the outer membrane porin OmpF expression. The expression level changes of the sRNA, ompF mRNA and OmpF protein during the carbapenem resistance induction would be detected. Building the sRNA gene knockout mutant and restore strains, we were going to study the regulatory mechanism of sRNA played in the carbapenem resistance mechanism. This study would help to understand the molecular mechanisms of carbapenem resistance in Enterobacter aerogenes and provide theoretical support for the development of promising drugs with probable new target.

碳青霉烯类耐药产气肠杆菌感染治疗困难，易在院内传播，外膜孔蛋白表达降低是其主要耐药机制之一，但外膜孔蛋白表达的具体调控机制不明。前期研究中我们发现一株碳青霉烯类耐药产气肠杆菌，不产β内酰胺酶也无外排机制参与，而外膜孔蛋白OmpF表达下降是其耐药机制。本项目旨在对外膜孔蛋白OmpF表达下降的调控机制进行研究，用高通量测序技术比较耐药与敏感临床株差异表达sRNA，通过生物信息学分析筛选调控外膜孔蛋白OmpF表达的sRNA；通过碳青霉烯类敏感株体外诱导为耐药株，构建sRNA基因敲除突变和回复突变株，并检测上述各菌株中sRNA，ompF mRNA和OmpF蛋白的表达变化，确认调控外膜孔蛋白OmpF的sRNA，从而明确碳青霉烯类耐药产气肠杆菌中外膜孔蛋白OmpF表达的sRNA调控新机制。本研究从sRNA水平深入探讨产气肠杆菌对碳青霉烯类耐药的分子机制，可为研发新的抗菌药物靶点提供理论依据。

近年来，碳青霉烯类耐药的肠杆菌科细菌检出率逐年升高，已逐渐成为一个严重的公共卫生问题。除了携带碳青霉烯酶，外膜孔蛋白的缺失或表达下降也是碳青霉烯类耐药的一个潜在重要机制。本研究旨在评价主要的外膜孔蛋白在对碳青霉烯类耐药的产气肠杆菌（carbapenem-resistant Enterobacter aerogenes, CREA）中的作用，以及明确小RNA相关的外膜孔蛋白表达下降和缺失机制。筛选2013-2015年华山医院分离的CREA通过SDS-PAGE和RT-qPCR方法明确外膜孔蛋白缺失或表达下降的菌株。进一步以全基因组测序，美罗培南诱导耐药，sRNA过表达，外膜孔蛋白回补和RT-qPCR试验方法探讨其外膜孔蛋白作用和分子机制。结果表明：共有4株CREA以外膜孔蛋白Omp35和/或Omp36缺失或表达下降为碳青霉烯类耐药的主要机制。美罗培南诱导耐药试验进一步表明在抗生素压力下外膜孔蛋白的缺失或表达下降是碳青霉烯类耐药中的重要一环。编码序列的突变和sRNA调控是外膜孔蛋白缺失的机制。其中sRNA micF和micC可以分别调控Omp35和Omp36蛋白的表达水平。