邻菲罗啉抗大肠杆菌作用机制的初步研究

Study on the antibacterial mechanism of 1,10-Phenanthroline monohydrate against E. coli

关键词:  邻菲罗啉  大肠杆菌  耐药性  抗菌活性  作用机制

Key words:  1,10-Phenanthroline monohydrate  E. coli  antimicrobial resistance  antibacterual activity  action mechanism

摘要:

为分析邻菲罗啉对大肠杆菌的抗菌作用机制，本研究测定了邻菲罗啉对大肠杆菌的最小抑菌浓度及最小生物被膜抑制浓度，并通过扫描电镜观察邻菲罗啉对大肠杆菌微观形态的影响，随后利用RNA-seq技术和qPCR对邻菲罗啉处理大肠杆菌差异表达基因进行分析。结果表明：1）终浓度100 μmol/L的邻菲罗啉可完全抑制大肠杆菌生长，50 μmol/L可抑制其生物被膜形成。2）电镜结果显示邻菲罗啉可使大肠杆菌菌体皱缩，细胞膜及细胞壁受到破坏。3）使用邻菲罗啉处理大肠杆菌后，786个基因出现显著上调，850个基因出现显著下调，差异表达基因主要参与细菌代谢过程、双组分系统、群体感应、抗生素合成等通路，如下调基因ompW在革兰氏阴性菌生物被膜形成、黏附、应激耐受方面发挥重要作用，PepN蛋白是宿主抵抗细菌感染的靶点，上调基因citT编码蛋白可影响细菌的能量供应及药物外排系统。4）选择9个与生物被膜形成及耐药性相关基因进行qPCR验证，与转录组测序结果一致。综上，邻菲罗啉对大肠杆菌具有显著的抑制作用，且可调控大肠杆菌代谢及耐药基因表达水平。本研究初步分析了邻菲罗啉对大肠杆菌的抗菌作用机制，为寻找抗生素替代药物提供新思路。

Abstract:

In order to analyze the antibacterial mechanism of 1，10-PHEN against E. coli， the minimum inhibitory concentration and the minimum biofilm inhibition concentration of 1，10-PHEN on E. coli was determined， and the effect of 1，10-PHEN on the micro-morphology of E. coli by scanning electron microscope was observed. Then， the differentially expressed genes in E. coli treated with 1，10-PHEN by RNA-seq technology and qPCR were analyzed. The results showed that： 1） The final concentration of 100 μmol/L 1，10-PHEN could completely inhibit the growth of E. coli， and 50 μmol/L 1，10-PHEN could inhibit the formation of its biofilm. 2） The results of electron microscope showed that 1，10-PHEN could shrink E. coli and destroy its cell membrane and cell wall. 3）1，10-PHEN could shrink E. coli and destroy its cell membrane and cell wall. After treating E. coli with 1，10-PHEN， 786 genes were significantly up-regulated and 850 genes were significantly down-regulated. The differentially expressed genes mainly participated in bacterial metabolism， two-component system， quorum sensing， antibiotic synthesis， and other pathways. The down-regulated gene ompW played an important role in biofilm formation， adhesion and stress tolerance of gram-negative bacteria， and PepN protein was the target of host to resist bacterial infection. Up-regulating the protein encoded by citT gene can affect the energy supply and drug excretion system of bacteria. 4） Nine genes related to biofilm formation and drug resistance were selected for qPCR verification， which was consistent with the results of transcriptome sequencing. In conclusion， 1，10-PHEN exhibits a significant inhibitory effect on the antibacterial activity of E. coli， and exerts regulatory control over the metabolism and expression levels of drug resistance genes in this bacterium. This study provides preliminary insights into the antibacterial mechanism of 1，10-PHEN against E. coli， and offers novel perspectives for identifying alternative antibiotics.