金黄色葡萄球菌噬菌体穿孔素及其与裂解酶CHAP片段重组蛋白的构建与作用机制

The lytic system of phage infecting bacterium consists of holin and lysin which destroy the cell membrane and cell wall respectively. The bactericidal mechanisms of holin and lysin are different from that of antibiotics. Bacteria that are resistant to antibiotics also exhibit sensitive to holin and lysin. Hence, holin and lysin belong to novel bactericidal agents. In the previous studies, we obtained a lysin named LysGH15 (encoded by phage GH15) which shows broad host-range and strong lytic activity against Staphylococcus aureus in vitro and in vivo, especially multi-drug resistant S. aureus. The three-dimensional structure and molecular function mechanism of LysGH15 have been further revealed. The CHAP domain of LysGH15 is responsible for the strong lytic activity. Recently, HolGH15, the holin protein of phage GH15, also has been expressed and shows efficient bactericidal activity against S. aureus, Listeria monocytogenes, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Thus, HolGH15 shows a great potential in the treatment of infections caused by drug-resistant bacteria. In the following work, the engineering strains will be screened and the expression system will be optimized for the sake of obtaining an enormous amount of HolGH15. The bactericidal mechanism of HolGH15 will be researched afterwards. In order to obtain the fusion protein which possesses much stronger and broader bactericidal activity, the recombinant protein CHAP-HolGH15 (or HolGH15-CHAP) will be constructed. Furthermore, the synergistic bactericidal mechanism of HolGH15 and CHAP will be studied comprehensively. Through these researches, the efficacious drugs which possess great potential in treating infectious caused by drug-resistant bacteria, especially by mult-drug resistant S. aureus, will be obtained.

穿孔素（holin）和裂解酶（lysin）共同构成噬菌体的二元裂解系统，分别可以特异性地破坏细菌的细胞膜和细胞壁，具有与抗生素完全不同的作用机制，是新概念的抗菌物质。前期本课题组获得了对金黄色葡萄球菌具有广谱、高效裂解活性的裂解酶LysGH15，揭示了其三维结构和分子作用机制，该裂解酶的CHAP片段发挥了主要的裂解活性。近期课题组成功表达了穿孔素HolGH15，它可以有效裂解金黄色葡萄球菌、单核增生李斯特杆菌、枯草芽孢杆菌、大肠杆菌和铜绿假单胞菌，对治疗耐药菌感染具有很大的应用潜力。本项目首先通过工程菌株的筛选和工艺的优化以解决HolGH15难以大量获得的问题，并进一步揭示其作用机制；重点将HolGH15与LysGH15的CHAP片段进行融合表达，深入研究二者的协同作用机制，以获得裂解活性更强、裂解谱更宽的重组蛋白。这将为耐药细菌、尤其是耐药性金黄色葡萄球菌感染的治疗提供有效的新型药物。

噬菌体的穿孔素（holin）和裂解酶（lysin）共同构成了噬菌体的二元裂解系统。在噬菌体感染细菌的过程中，holin可以在细菌的细胞膜上形成孔道，而lysin则可以通过holin形成的孔道到达并水解细菌的细胞壁。噬菌体的holin和lysin具有与抗生素完全不同的作用机制，是新概念的抗菌物质。尤其是在当前细菌耐药性问题日趋严重的背景下，深入研究噬菌体的holin和lysin更具有重要的意义。本项目对噬菌体GH15的穿孔素蛋白进行了全面的研究：首先确定了GH15编码穿孔素的基因，并且对穿孔素HolGH15进行了表达，进一步的研究表明HolGH15在细菌外部同样可以发挥抗菌的活性，抗菌谱宽于裂解酶LysGH15，且对金葡菌以外的单核增生李斯特菌、枯草芽孢杆菌、大肠杆菌和铜绿假单胞菌等其它细菌同样具有显著的抗菌作用，值得一提的是，HolGH15在低温下仍然可以杀灭单增李斯特菌，在食品的防腐方面显示出应用潜力；通过一系列研究表明HolGH15是通过破坏细菌的细胞膜以增加细菌的通透性，进而使细菌内容物大量、快速地泄漏，从而导致细菌快速死亡；成功构建了GFP-HolGH15融合蛋白，提高了HolGH15的获得率；CHAP片段是LysGH15发挥抗菌活性的关键部分，因此本研究构建了CHAP-HolGH15融合蛋白，初步研究表明该融合蛋白表现出优于HolGH15和CHAP的抗菌活性，比二者表现出更强的裂解活性和更宽的裂解谱，在防治金黄色葡萄球菌感染、消除其生物被膜、控制李斯特菌方面表现出应用潜力。