少棘蜈蚣抗菌肽Scolopin 2-NH2的抗菌作用机制研究

Mechanism of Antimicrobial Peptide Scolopin 2-NH2 Isolated from Scolopendra subspinipes mutilans

旨在探究抗菌肽Scolopin-2(S-2)及其酰胺化修饰产物Scolopin-2-NH2(S-2-N)的二级结构、抗菌活性及其理化性质,并从细胞水平和分子水平揭示其抗菌作用机制。按最小抑菌浓度测定法检测抗菌肽S-2/S-2-N对4种菌株的抗菌活性;用不同条件处理S-2/S-2-N,测定其理化性质;用圆二色谱仪检测抗菌肽S-2/S-2-N的二级结构;以S-2-N/S-2处理大肠杆菌,用流式细胞仪检测二者穿透细胞膜的效率及菌体细胞膜完整性;通过流式细胞术和RT-PCR技术分析S-2-N/S-2对细菌细胞周期及细胞内DNA复制和修复相关基因的影响。除了具有和母体肽S-2相似的耐热性、耐酸碱性及抵抗离子强度和消化酶的能力,S-2-N还具有更强的抗菌活性;流式细胞仪检测结果显示,短时间内(30 min)二者均能引起一定程度的细胞膜损伤,但抗菌肽S-2-N穿透细胞膜的效率更高;流式细胞术及RT-PCR技术分析结果显示,S-2-N/S-2抑制了大肠杆菌细胞周期的进行,下调了基因dnaA、dnaB、dnaG和SSB的表达,同时促进了RecA和RecN的表达。抗菌肽S-2经酰胺化修饰后,具有比母体肽更强的抗菌活性。二者的抗菌作用机制为:破坏细菌细胞膜,结合细菌DNA和RNA、影响DNA的二级结构,阻滞细菌细胞周期的进行、影响细菌细胞内与DNA复制和修复相关基因的表达。

This work aims to elucidate the secondary structure,antibacterial activity and physicochemical properties of antibacterialpeptide Scolopin-2(S-2)and S-2-amidated Scolopin-2-NH2(S-2-N),and to reveal their antibacterial mechanism at the cell and molecularlevel. The method of minimum inhibitory concentration was used to detect the antimicrobial activity of S-2/S-2-N against 4 strains,differenttreatments were applied to measure their physicochemical properties,and circular dichroism spectroscopy was to determine the secondarystructure S-2/S-2-N. After Escherichia coli was treated with S-2-N and S-2 respectively,flow cytometry was used to detect the efficiency ofpeptides penetrating cell membrane and cell membrane integrity. Flow cytometry and RT-PCR technology were together to analyze the effectsof peptides on cell cycle and the genes related to intracellular DNA replication and repair. In addition to the heat resistance,acid and alkalineresistance,the resistance to ionic strength and the ability of digestive enzymes,the S-2-N also had stronger antibacterial activity than thematernal peptide S-2. The results of flow cytometry showed that in a short period of time(30 min)both peptides caused cell membranedamaged,while the efficiency of S-2-N penetrating membrane was higher. The analysis of flow cytometry and RT-PCR demonstrated that thebacterial cell cycle was blocked by S-2-N and S-2,which lowered the expressions of gene dnaA,dnaB,dnaG and SSB,but promoted theexpressions of RecA and RecN. In conclusion,the antibacterial peptide S-2-N amidated from S-2 has stronger antibacterial activity than maternalpeptide,and both function by the same antibacterial mechanism,i.e.,destroying the bacterial cell membrane,combining bacterial DNA andRNA,affecting the secondary structure of DNA,blocking the cell cycle of bacteria,and affecting the expressions of genes associated with DNA replication and repair in cells.