致病杆菌中SrfABC三元复合毒素的生物活性及作用机理研究

The entomopathogenic nematode-bacterium complex, one of the most promising new biological pesticides, is considered to be the potential alternative for Bacillus thuringiensis. When the nematode invades an insect host the symbiotic bacteria are released into the insect haemocoel, and the bacteria secrete a range of toxins to escape the host immune response, to destroy the insect midgut epithelial cells, to cause septicemia, which ultimately kills the infected insect. The tripartite toxin complex, SrfABC, which possesses both cytotoxicity and injectable insecticidal activity has been identified from Xenorhabdus stockiae HN_xs01 strain. Actually, srfABC operon is widely distributed among the pathogenic microorganisms and can be horizontally transferred, suggesting its important roles in escaping host immune response, exhibiting virulence and colonizing host. However, more experimental data are needed to unravel the biological activity and mechanism of action of SrfABC, which were formerly speculated by homology comparison. In order to elucidate the precise role of SrfABC in the process of invasion, the oral and injectable insecticidal activity and cytotoxicity of SrfABC will be measured firstly. The site of action and target cell of SrfABC will then be detected. The assembly model, enzymatic component, cellular target sites, and mode of action will then be studied to clarify the cytotoxic mechanism of SrfABC. This project will definitely provide theoretical basis for employing Xenorhabdus in pest control. Meanwhile, the project will also lay the foundation for biological function research of SrfABC in other pathogenic microorganism and provide new ideas for developing SrfABC tripartite toxin as tools for basic research, disease therapy, and biological prevention.

昆虫病原线虫-共生菌复合体被认为是继苏云金杆菌后、最具应用前景的新型生物杀虫剂。共生菌通过分泌多种毒素以抵御昆虫免疫系统、破坏昆虫肠道细胞、导致昆虫产生败血症而死亡。课题组从致病杆菌中鉴定到一类同时具细胞毒性和注射杀虫活性的SrfABC三元毒素。SrfABC在病原微生物中分布广泛、可通过基因水平转移而传递，暗示其在抵抗宿主防御、毒力发挥、宿主定殖中扮演着重要的角色。当前有关SrfABC生物活性和作用机理的报道是通过同源比对而进行的推测，缺乏实验数据支撑。为了阐明SrfABC在侵袭昆虫中的重要作用，本项目拟测定SrfABC的杀虫活性及细胞毒性，明确毒素的作用位点及靶细胞；研究SrfABC的酶活性组分、作用方式、细胞内靶位点、组装模式，阐明其细胞毒性的精确作用机制，为更好的利用致病杆菌进行害虫防治提供理论依据。本项目也将为其他病原微生物中SrfABC毒素的生物功能研究奠基础。

昆虫病原线虫共生菌被认为是继苏云金杆菌后、最具应用前景的新型生物杀虫剂。共生菌通过分泌多种毒素以抵御昆虫免疫系统、破坏昆虫肠道细胞、导致昆虫产生败血症而死亡。本研究对致病杆菌中的新型三元毒素SrfABC进行了研究。.SrfABC三组分在E.coli中共表达时，对棉铃虫和甜菜夜蛾幼虫表现出很高的注射杀虫活性；对昆虫细胞CF203、肿瘤细胞HeLa、 HEK293T及B16均表现出细胞毒性，导致数目减少、变圆、伪足消失、贴壁性降低、空泡化。Confocal观察显示：SrfABC毒素作用后，CF203细胞核凝聚、细胞骨架解聚、线粒体消失。Native-PAGE结合Western blot分析表明：当SrfABC三组分在E.coli中共表达能形成复合物。利用免疫共沉淀，进一步证实了SrfABC组分间存在相互作用。.SrfA、SrfB、SrfC蛋白单独作用时，对HeLa和CF203细胞均表现出明显细胞毒性，三者混合作用时毒性最大。Hoechst/PI染色和流式细胞仪检测，表明：三种组分均能造成细胞凋亡，并将细胞周期阻滞在G2/M期。Western blot证实：三种组分均能较高效率的、主动进入HeLa和CF203细胞。利用免疫荧光实验对各组分进行细胞定位，发现：在CF203细胞中，SrfA、SrfB、SrfC蛋白集中分布在细胞质；在HeLa细胞中，SrfA、SrfC分布在细胞质，SrfB分布在细胞核。.通过脂质体转染，在HeLa细胞和HEK293T细胞中表达了SrfA，SrfB，SrfC蛋白，SrfA蛋白分布于细胞质，并导致线粒体消失，细胞骨架解聚；SrfB，SrfC蛋白分布于细胞质和细胞核内，并导致线粒体消失。三组分的表达使两种细胞都出现不同程度的死亡，其中SrfA表达毒细胞死亡率最为显著。三组分的表达均能造成PARP蛋白剪切，引起细胞凋亡。Western Blot检测显示：SrfA蛋白在HeLa细胞和HEK293T细胞中发生了翻译后修饰，通过定点突变和糖苷酶处理，初步排除其修饰为糖基化。利用免疫沉淀及质谱技术筛选出SrfA蛋白的细胞靶标主要为骨架蛋白。.本项目研究了SrfABC毒素的杀虫活性及细胞毒性，明确了SrfABC的酶活性组分、作用方式、细胞内靶位点，阐明了其在抵抗宿主防御、毒力发挥中的作用，为更好的利用致病杆菌进行害虫防治提供理论依据。