苏云金芽胞杆菌噬菌体phiCM3双穿孔素宿主裂解系统作用机理研究

Bacteriophage is the most diverse organism in nature. It amplificated by invading and lysing host bacteria. In general, the phage host lysis system consists of a holin and an endolysin. Among these, holin could form holes in the bacterial cell membrane and provide conveniences for endolysin through the channel, and holin also could control the lysis time. However, only rare phage was found to possess two holins, and until now, there is no research on mechanism of dual-holin phage host lysis system. In this study, we intend to construct the mutant phages that the genes contained in the phage host lysis system was deleted and detect the lysis ability of mutant phages by using the method of biochemistry, microbiology and molecular biology, and carry out the research on the interaction model and dynamic state of two holins in vitro, in vivo and in the cell membrane, we aim to understand the hole model formed by holins, and to clarify the biology signification of “additional” holin protein in the phage host lysis system. The project is a first systematic research on the interaction mechanism of two holins in the phage host lysis system. The accomplishment of this work will clarify the novel lysis mechanism, and raise the understanding of routines of phage life activities. It provides a theoretical guidance for artificial construct new phage which has the effect of preventing and controlling the pathogenic bacteria of medicine.

噬菌体是自然界中最多样的生命体,其通过侵染宿主细菌、完成个体复制、并裂解宿主细菌来繁殖。噬菌体宿主裂解系统通常由一个穿孔素蛋白及一个裂解酶组成，其中穿孔素在细菌细胞膜上形成供裂解酶通过的孔道并控制细菌裂解时间，但有极少数噬菌体被发现具有两个穿孔素编码基因，而目前尚无对双穿孔素噬菌体宿主裂解系统作用机制的研究。本研究拟通过生物化学、微生物学及分子生物学等研究手段，构建宿主裂解系统突变噬菌体并分析其裂解能力、研究两个穿孔素蛋白在体外、细菌胞内及细胞膜上的互作模式及作用动态、解析双穿孔素蛋白的孔道形成模式、进而阐明“额外”的穿孔素在噬菌体宿主裂解系统中的生物学意义。本项目为首次系统研究具有两个穿孔素的噬菌体宿主裂解系统的作用机制及生物学意义；该工作的完成将阐明新型的噬菌体宿主裂解机制、可增加对噬菌体生命活动规律的认识、为人工构建具有防治医学病原菌效果的新型噬菌体提供理论指导。

噬菌体是自然界中最多样的生命体,其通过侵染宿主细菌、完成个体复制、并裂解宿主细菌来繁殖。噬菌体宿主裂解系统通常由一个穿孔素蛋白及一个裂解酶组成，其中穿孔素在细菌细胞膜上形成供裂解酶通过的孔道并控制细菌裂解时间，但有极少数噬菌体被发现具有两个穿孔素编码基因，而目前尚无对双穿孔素噬菌体宿主裂解系统作用机制的研究。本项目围绕苏云金芽胞杆菌噬菌体phiCM3所具备的双穿孔素宿主裂解系统，主要研究内容为明确双穿孔素对裂解宿主细菌的必要性、阐明该噬菌体宿主裂解系统中各关键组件的功能，增加对噬菌体宿主裂解系统多样性的认知。项目研究团队通过对双穿孔素噬菌体裂解系统中基因在大肠杆菌中的重组表达，构建了具备该噬菌体不同宿主裂解系统基因组片段的大肠杆菌工程菌株，验证了各基因在宿主裂解中的必要性；通过体外的裂解酶活性测定，明确了宿主裂解系统裂解酶的生物学功能及其酶学特征。此外，本项目还通过对传统的具有单一穿孔素的噬菌体裂解系统的研究，探讨了不同噬菌体宿主裂解系统的差异，增加了对噬菌体宿主裂解系统多样性的认知。最后，受噬菌体phiCM3裂解酶菌种特异性机制启发，构建了基于噬菌体裂解酶细胞壁结合结构域的苏云金芽胞杆菌分离系统。本项目的开展和完成丰富了对噬菌体宿主裂解系统多样性的认知，可为噬菌体疗法在防治病害微生物中的应用提供了理论指导。