DEVELOPMENT OF NEW ANTIMICROBIAL PEPTIDE AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS

本项目的目的是开发一种新的抗甲氧西林金黄色葡萄球菌的抗菌肽。我们克隆了金黄色葡萄球菌主要自溶素的编码基因，发现atl基因产物负责细胞簇的分离。我们还发现金黄色葡萄球菌裂解酶的酶谱图在生长过程中发生了变化，表明atl基因产物在分泌后进行了加工。免疫电镜观察表明，atl基因产物特异性定位于金黄色葡萄球菌的细胞表面。结合青霉素处理金黄色葡萄球菌的独特自溶数字数据，我们得出结论，atl基因产物是青霉素处理金黄色葡萄球菌自溶所必需的。在此基础上，我们合成了几个与推测保留信号区域对应的寡肽，并研究了它们对atl基因产物保留和青霉素诱导的自溶的影响。在低离子强度环境下，发现肽A10、A11、A14和A16降低金黄色葡萄球菌和cfu的浊度。通过测定浊度的降低，发现A10和A14抑制自溶，A11和A16诱导自溶。这些肽含有正电荷的赖氨酸，添加50 mM NaCl可以阻断这些寡肽的活性。透射电镜研究清楚地表明，A11或A16诱导金黄色葡萄球菌自溶的薄片显示出与atl基因产物定位位点对应的细胞壁缺陷。另一方面，A10处理后的细胞薄切片未见细胞裂解，但出现了细胞膜的紊乱。这些结果强烈表明，这些合成肽能够通过干扰atl基因产物的保留来显示抗菌作用。

The aim of this project was to develop a new antimicrobial peptide against methicillin resistant Staphylococcus aureus. We cloned the gene encoding major autolysin of S.aureus and found that atl gene products are responsible for cell separation of clusters. We also found that zymographic pattern of S.aureus lytic enzyme alters during growth and suggested that atl gene products undergo processing after it is secreted. Immunoelectronmicroscopical observation demonstrated that atl gene products are specifically localized on cell surface of S.aureus. Together with the data of unique autolytic figures of penicillin-treated S.aureus, we concluded that atl gene products are necessary for the autolysis of S.aureus treated with penicillin. Based on these observation, we synthsized several oligopeptides corresponding to the region of putative retention signal and investigated their effect on retention of atl gene products and penicillin induced autolysis. Under low ionic strength miliu, peptide A10, A11, A14 and A16 were found to decrease turbidity of S.aureus and cfu. By measuring decrease in turbidity, A10 and A14 were found to be inhibitory to autolysis and A11 and A16 were found to induce autolysis. These peptides contain positively chraged lysines, and addition of 50 mM NaCl was found to block the activity of these oligopeptide. TEM study clearly demonstrated that thin section of S.aureus which was induced autolysis by A11 or A16 shows the defect of cell wall corresponding to the site atl gene products localize. On theother hand, thin section of cells treated with A10 did not show any incidence of cell lysis, but revealed disturbance of cell membrane. These results strongly susggested that these synthtic peptides are able to show antimicrobial effect by interfering retention of atl gene products.