研究发现绝大多数军团菌都会引起人类肺部感染，其中嗜肺军团菌感染性很强，且分布较广，是引起重症肺炎的主要病原体之一。目前，研究者们已对嗜肺军团菌的分布范围、传播途径、感染机制、生长环境、毒力因子和宿主免疫应答等方面进行了深入探索。嗜肺军团菌主要存在于水体环境中，通常以气溶胶的形式经呼吸道传播引起人类肺部感染和类似流感的症状，严重可致死。一经感染，嗜肺军团菌通过吞噬作用侵入肺泡巨噬细胞。嗜肺军团菌通过 Icm/Dot Ⅳ型分泌系统分泌效应蛋白以躲避宿主细胞的识别和消化机制，部分效应蛋白帮助其在宿主体内的存活和繁殖，增加其致病性。如今，嗜肺军团菌的诊断方法主要有细菌培养、抗原检测、PCR 检测等。抗生素一般用于治疗由嗜肺军团菌感染引起的疾病，但耐药性问题一直还未突破，应加强预防感染的措施。因此，精准的靶向治疗显得极为紧迫，对嗜肺军团菌效应蛋白的结构分析为靶向军团菌药物的开发提供了重要的基础。

嗜肺军团菌效应蛋白 RavL 和 lpg1959 自发现以来二者各自的功能还不明确。本论文第一部分采用蛋白质-脂质覆盖实验和微量热泳动实验探索与 RavL 相互作用的底物，并采用酶联免疫吸附实验验证其水解产物，最后证明 RavL 是一种磷脂酰肌醇-5-磷酸酶。之后用 AlphaFold 预测了 RavL 蛋白的全长结构，并与本课题组之前解析的部分结果进行比对，联用分子动力学模拟技术和分子对接方法寻找 RavL 关键的活性氨基酸位点，有助于设计实验探究酶的活性中心。最后应用计算机辅助技术筛选 RavL 截短体蛋白的抑制剂，为军团菌病的治疗和新药物的开发提供了思路。

第二部分首先采用定点突变的研究方法找到了 RavL 与镍结合的关键氨基酸位点。其次为了获得具有规则形状和高衍射率的蛋白质晶体，设计了 RavL 蛋白 31-261 aa、24-158 aa+ 177-258 aa 的氨基酸序列的截短体。然后对 lpg1959 蛋白的纯化条件进行了探索，但lpg1959 截短体蛋白的纯化条件还需要进一步尝试。最后我们通过体外 pull-down 和体内细胞实验验证了 RavL 与 lpg1959 的相互作用，并借助 AlphaFold 的预测结果设计了验证二者相互作用位点的实验，为更好地了解效应蛋白在感染过程中发挥的作用奠定基础。

Studies have found that most Legionella bacteria can cause human lung infection, among which Legionella pneumophila is highly infectious and widely distributed, and is one of the pathogens causing severe pneumonia. At present, researchers have carried out in-depth exploration of Legionella pneumophila's distribution range, transmission route, infection mechanism, growth environment, virulence factors and host immune response. Legionella pneumophila is mainly found in the water environment and is usually transmitted through the respiratory tract in the form of aerosols to cause lung infections and flu-like symptoms in humans, which can be fatal in severe cases. Once infected, Legionella pneumophila invades alveolar macrophages through phagocytosis. Legionella pneumophila secretes effector proteins through the Icm/Dot Ⅳ secretory system to avoid the recognition and digestion mechanism of host cells. Some effector proteins help the survival and replication of Legionella pneumophila in the host, increasing its pathogenicity. Nowadays, the main diagnostic methods of Legionella pneumophila include bacterial culture, antigen detection, PCR detection and so on. Antibiotics are commonly used to treat diseases caused by Legionella pneumophila infection, but the problem of drug resistance has not been broken through and measures to prevent infection should be strengthened. Therefore, precise targeted therapy is extremely urgent. Structural analysis of Legionella pneumophila effector protein provides an important basis for the development of targeted Legionella drugs.

Since the discovery of Legionella pneumophila effectors RavL and lpg1959, their respective functions have been unclear. In the first part of this paper, protein-lipid covering experiment and microthermokinetic experiment were used to explore the substrate interacting with RavL, and enzyme linked immunosorbent experiment was used to verify its hydrolytic products. Finally, RavL was proved to be a phosphatidylinositol-5-phosphatase. Then, AlphaFold was used to predict the full-length structure of RavL protein, which was compared with the partial results previously analyzed by our research group, and the key active amino acid sites of RavL were searched by molecular dynamics simulation technology and molecular docking method, which  was helpful to design experiments to explore the active center of enzyme. Finally, the computer aided technology was used to screen the inhibitors of RavL truncated body protein, which provided ideas for the treatment of legionella disease and the development of new drugs.

In the second part, site-specific mutation was used to identify the key amino acid sites of RavL binding to nickel. Secondly, in order to obtain protein crystals with regular shape and high diffraction rate, truncated bodies of amino acid sequences of RavL proteins 31-261 aa, 24-158 aa+177-258 aa were designed. Then the purification conditions of lpg1959 protein were explored, but the purification conditions of lpg1959 truncated body protein still need further attempts.

Finally, we verified the interaction between RavL and lpg1959 through pull-down in vitro and cell experiments in vivo, and designed experiments to verify the interaction sites of RAVL and lpg1959 based on the predicted results of AlphaFold, which laid a foundation for a better understanding of the role of effector proteins in the process of infection.