抗菌肽P7抑制大肠杆菌的非膜作用机制

Non-membrane mechanisms of antimicrobial peptide P7 against Escherichia coli

摘要:

[目的] 研究抗菌肽P7抑制大肠杆菌的非膜作用机制。[方法] P7与溴化乙锭竞争结合大肠杆菌基因组DNA的荧光光谱，分析P7与DNA的结合方式；流式细胞术分析P7与大肠杆菌基因组DNA结合对细菌细胞周期的影响；采用磁珠富集和PCR扩增相结合的方法分析P7特异结合的DNA序列；通过实时荧光定量PCR分析P7对大肠杆菌DNA复制和SOS损伤修复基因表达的影响；核酸染料的荧光分析研究P7对大肠杆菌DNA和RNA合成的影响。[结果] P7以嵌插的方式作用于大肠杆菌基因组DNA碱基对并形成肽-DNA复合物，使溴化乙锭-DNA复合体系的荧光强度减弱。P7可以显著增加大肠杆菌细胞周期中S期细胞数目，抑制大肠杆菌DNA复制。P7特异性结合rnhA使该基因表达水平显著下调2.24倍。同时，在肽的影响下参与大肠杆菌DNA复制相关的ssb、dnaG、ligB和rnhA基因的表达水平显著下调（P<0.05），DNA损伤修复的recA和recN基因显著上调（P<0.05）。P7可降低大肠杆菌DNA和RNA的合成。[结论] P7特异性地结合rnhA序列引起大肠杆菌DNA的损伤并抑制大肠杆菌的DNA复制。在P7的影响下，参与大肠杆菌DNA复制相关的基因的表达水平下调，DNA损伤修复基因显著上调，同时抑制大肠杆菌DNA和RNA的合成。

关键词:抗菌肽;抑菌机制;细胞周期;DNA结合

Abstract:

[Objective] The molecular mechanism of antimicrobial peptide P7 against Escherichia coli was studied.[Methods] The binding mode between P7 and DNA was analyzed through fluorescence spectroscopy of P7 binding with E. coli genome DNA. The effects of P7 on E. coli cell cycle were determined through flow cytometry. Magnetic beads coupled with peptide were ussed to enrich peptide DNA-binding fragments, and PCR methods were used to analyze specific DNA to which P7 bound with. The influence of P7 on the gene expression levels of DNA replication and SOS damage and repair was analyzed through quantitative real-time PCR. The effects of P7 on E. coli DNA and RNA synthesis were analyzed according to the fluorescence spectra of nuclear stains.[Results] P7 intercalated into the base pairs of E. coli genomic DNA and then formed peptide-DNA complexes. As a result, the fluorescence intensity of the EB-DNA complex decreased. P7 could significantly increase the number of E. coli cells in phase S. The effect of P7 on normal E. coli cell cycle could significantly inhibit the DNA replication of E. coli. The binding of P7 with rnhA down-regulated the gene expression level by 2.24 times. The gene expression levels of ssb, dnaG, ligB, and rnhA that participated in E. coli DNA replication significantly decreased, and the gene expression levels of recA and recN in DNA damage and repair were significantly up-regulated under the effect of P7. P7 reduced E. coli DNA and RNA synthesis.[Conclusion] P7 also bound with rnhA. This binding resulted in DNA damage and inhibition of DNA replication of E. coli. P7 down-regulated the gene expression level of DNA replication, and the gene expression levels of DNA damage and repair were significantly up regulated. P7 reduced DNA and RNA synthesis of E. coli.

Key words:antimicrobial peptide;antibacterial mechanisms;cell cycle;DNA-binding