两种合成抗菌肽的结构及抗菌作用机理

Solution structure and antibacterial mechanism of two synthetic antimicrobial peptides

摘要:

为深入了解两种新型人工抗菌肽mytilin-derived-peptide-1 (MDP-1) 和mytilin-derived-peptide-2 (MDP-2) 的溶液结构和抗菌机理并探讨两种抗菌肽之间活性差异的结构基础，采用二维核磁共振技术 (2-D NMR) 研究MDP分子的溶液结构；采用透射电镜技术 (Transmitted electron microscopy，TEM) 研究MDP分子对于大肠杆菌和藤黄叠球菌的作用机理。研究结果表明，MDP-1和MDP-2均采取了典型的β-发夹结构，其分子表面具有明显的疏水斑片，其分子中碱性氨基酸突出于分子表面；经MDP分子处理后的大肠杆菌以及藤黄叠球菌均出现细胞壁或细胞膜结构被破坏，并出现膜壁分离以及细胞质内缩现象。我们认为，MDP-1和MDP-2分子中的碱性氨基酸有助于MDP结合细菌表面的带负电荷的基团，同时其分子表面的疏水斑片有助于其插入到细菌细胞膜内；其疏水斑片面积以及碱性氨基酸在分子表面的拓扑结构差异是MDP-1和MDP-2活性差异的主要原因；电镜实验结果表明MDP-1和MDP-2的主要靶标是细菌细胞壁以及细胞膜；上述研究为深入了解MDP分子的结构与功能的关系以及将来基于MDP分子的药物研发奠定了基础。

Abstract:

Mytilin-derived-peptide-1 (MDP-1) and mytilin-derived-peptide-2 (MDP-2) are two truncated decapeptides with reversed sequence synthesized corresponding to the residues 20-29 of mytilin-1 (GenBank Accession No. FJ973154) from M. coruscus. The objective of this study is to characterize the structural basis of these two peptides for their antimicrobial activities and functional differences, and to investigate the inhibitory mechanism of MDPs on Escherichia coli and Sarcina lutea. The structures of MDP-1 and MDP-2 in solution were determined by 1H 2D NMR methods; the antibactericidal effects of MDPs on E. coli and S. lutea were observed by transmitted electron microscopy (TEM). Both MDP-1 and MDP-2 have a well-defined loop structure stabilized by two additional disulfide bridges, which resemble the-hairpin structure of mytilin-1 model. The surface profile of MDPs￠ structures was characterized by protruding charged residues surrounded by hydrophobic residues. TEM analysis showed that MDPs destroyed cytoplasmic membrane and cell wall of bacteria and the interface between the cell wall and membrane was blurred. Furthermore, some holes were observed in treated bacteria, which resulted in cell death. Structural comparison between MDP-1 and MDP-2 shows that the distribution of positively charged amino acids on the loop of MDPs is topologically different significantly, which might be the reason why MDP-2 has higher activity than MDP-1. Furthermore, TEM results suggested that the bactericidal mechanisms of MDPs against E. coli and S. lutea were similar. Both MDP-1 and MDP-2 could attach to the negatively charged bacterial wall by positively charged amino acid residues and destroy the bacteria membrane in a pore-forming manner, thus cause the contents of the cells to release and eventually cell death.