苏云金芽胞杆菌噬菌体受体结合蛋白决定其宿主特异性机制研究

Bacteriophages (phages) are virus that infect bacteria and lots of them exhibit high host specificity, which only infect the bacteria of one species or one genus, sometimes only one isolate. The receptor binding protein (RBP) encoded by phage is reported to determine the host specificity of phage. Bacillus thuringiensis (Bt) is a Gram-positive entomopathogenic bacterium and is widely used in producing insecticidal agents, while phage contamination can result in severe losses of Bt fermentation. Therefore, the study of Bt phage is of great important. However, there is rarely report on Bt phage, especially the interaction mechanism between Bt phage and Bt. Two Bt phages, BtCS33 and phiCM3, were isolated by our laboratory and they exhibited extremely different host range. The genomes of the two phage were sequenced and showed high genome similarity. In this study, by using the two phages and the corresponding host bacteria, we intend to resolve the crystal structures of the two phage RBPs, analyze the activitation mechanism of these RBPs, identify the phage binding receptor on Bt strain, and reveal the interaction mechanism between RBP and phage binding receptor during phage absorption. In addition, we also plan to screen phage-resistant Bt strains, and analyze the mutations of phage-resistant Bt strain that associated with RBP activation and RBP binding. This project is the first study on the activation of phage RBP during phage absorption and is also the first systematic study on the interaction mechanism between Bt phage RBP and Bt strain. The finish of this study will reveal the mechanism of Bt phage RBP in determining the phage host specificity and provide leads for screening phage-resistant Bt strain and for controlling phage contamination during Bt fermentation.

噬菌体是细菌病毒，常具有很高的宿主特异性，噬菌体编码的受体结合蛋白(RBP)是决定噬菌体宿主特异性的重要原因之一。苏云金芽胞杆菌(Bt)是革兰氏阳性昆虫病原菌，被广泛用以发酵生产生物杀虫剂，但噬菌体的污染可导致Bt发酵的重大损失，而目前对Bt噬菌体的研究较少，尚无Bt噬菌体受体结合蛋白和结合受体的研究。本项目拟采用两株基因组序列高度相似、却有着不同宿主特异性的Bt噬菌体为研究对象，采用结构生物学、生物化学等研究方法，解析其RBP的三维结构、鉴定噬菌体的结合受体、研究RBP和受体相互作用的分子机制，揭示RBP与宿主菌相互作用过程中的活化机制，筛选抗噬菌体Bt菌株。本项目为首次开展噬菌体RBP活化机制和Bt噬菌体RBP与Bt互作分子机制的研究。本项目的实施和完成可阐明Bt噬菌体受体结合蛋白决定其宿主特异性的机制，为筛选抗噬菌体高效杀虫Bt菌株和防治发酵过程中Bt噬菌体的污染提供理论指导。

噬菌体具有很高的宿主特异性性，而噬菌体的受体结合蛋白是决定噬菌体宿主特异性的重要原因之一。由于苏云金芽胞杆菌（Bt）多样的杀虫活性，其被广泛的用于生产生物杀虫剂，但在Bt发酵生产过程中，噬菌体污染可导致重大减产乃至发酵失败。故而，本项目围绕Bt噬菌体受体结合蛋白与宿主相互作用这一核心，主要开展的研究工作包括抗噬菌体Bt突变菌株筛选及重获侵染能力噬菌体突变株筛选、噬菌体在细菌上的吸附受体鉴定及噬菌体结合关键氨基酸位点鉴定、噬菌体受体结合蛋白鉴定及与细菌相互作用分子机制分析等研究工作。研究结果发现细菌鞭毛合成蛋白FlhA为噬菌体在细菌上的潜在吸附受体，该蛋白第一个跨膜域的缺失突变可导致噬菌体抗性的产生；噬菌体受体结合蛋白由三个噬菌体尾部蛋白构成，该三个蛋白的协同突变可导致噬菌体重获对噬菌体抗性菌株的侵染能力，发现了一种新的噬菌体-细菌协同共进化机制；筛选获得了抗噬菌体Bt突变株，该突变株对噬菌体表现出了较好的抗性，可用于抗噬菌体Bt工程菌的制备。本项目的实施和完成，系统的研究了噬菌体受体结合蛋白与宿主细菌的相互作用方式，阐明了噬菌体受体结合蛋白决定噬菌体宿主特异性的分子机制，研究成果对认识噬菌体与细菌的相互作用及协同共进化均有着重要的意义，可为筛选抗噬菌体高效杀虫Bt菌株和防治发酵过程中Bt噬菌体的污染提供理论指导，还可为构建噬菌体疗法噬菌体工程菌株提供理论指导。