嗜肺军团菌RpoS调控II型分泌系统效应蛋白磷脂酶A分子机理的研究

Legionella pneumophila is a Gram-negative, facultative intracellular pathogen leading to lung destruction and consequently to a severe form of pneumonia, Legionnaires’ disease. The key question about Legionnaires’ disease is how the bacteria manage to survive intracellularly and cause pneumonia. One common virulence factor was employed by many pathogenic microorganisms via protein secretion systems. In the different infection stages, effector proteins are transferred from the bacterium to the phagosomal space or to specific places in the host cell allowing both intracellular establishment and subsequent escape. RpoS was shown to be necessary for intracellular infection and replication of the bacterium in host cell and expression of virulence traits present in the transmissive growth phase. The regulation protein RpoS play a role in the control of type II-dependently secreted effector proteins, which are yet identified including two phospholipase A (PlaA and PlaC) and one protease ProA. However, the three dimentional struture and regulating mechanism of RpoS involved in type II secretion system is still unknow. In this proposed project, our research objects is regulating mechanism of RpoS involved in type II secretion system effector proteins. Structural analysis in combination with biochemical analysis can provide the detailed relationship between RpoS and effector proteins.These studies can not only strengthen our understanding the structrure and biological function of RpoS and regulating effector proteins, but also pave the road for clarifying about pathogenic mechanism of the Legionella pneumophila intracellular infection and replication. ..

嗜肺军团菌是一类能引起军团菌肺炎的兼性革兰氏阴性胞内病原细菌。关键致病机制是嗜肺军团菌在感染过程中利用一套独特的分泌系统向宿主细胞内注射大量的毒力因子，即效应蛋白，这些毒力因子操控宿主细胞的各种胞内活动途径，从而在宿主细胞内生存和大量增殖，最终逃逸并侵染其他细胞。不同于其他细菌RpoS功能，嗜肺军团菌RpoS调控II型分泌系统效应蛋白PlaA与PlaC的磷脂酶A活性及ProA金属蛋白酶活性，从而增加病菌的细胞毒性和感染细胞的能力，对于在宿主细胞中胞内生存及复制是至关重要的，但是由于缺乏结构信息，使得我们对RpoS的调控机理并不清楚。本项目拟以嗜肺军团菌调控蛋白RpoS及其所调控II型分泌系统效应蛋白为研究对象，对其进行生物化学及结构生物学研究，剖析RpoS及调控的效应蛋白之间的作用机制，获悉RpoS和其调控的效应蛋白结构与生物学功能关系，进一步阐明嗜肺军团菌在宿主胞内侵染和复制的致病机理。

嗜肺军团菌能够入侵宿主肺泡内巨噬细胞和释放毒力因子并实现胞内增殖，最终导致肺部感染。嗜肺军团菌II型与IVB型分泌系统是产生毒力因子的两种重要系统。II型分泌系统分泌至少25个毒力因子中有18个具有蛋白酶，磷酸酶，磷脂酶和几丁质酶等酶活性，促进嗜肺军团菌在巨噬细胞和肺上皮细胞的增殖，增加细胞毒性和感染细胞的能力。RpoS 蛋白（RNA polymerase sigma factor）是一个主要的调控因子，主要参与II型分泌系统的调控，RpoS不仅对于在宿主细胞中胞内生存及复制时必需的，同时还参与调控嗜肺军团菌II型分泌系统中具有效应蛋白的转录与表达。本项目对RpoS和RpoS调控的II型分泌系统毒力因子进行生化分析和结构生物学研究，理解其结构与功能关系，进一步阐述毒力因子发挥功能的分子机制。目前已取得多个研究成果，对RpoS及其调控的II型分泌系统中毒力因子（PlaA、PlaC、ProA、LapB、VirK、MapA等）进行较为系统的结构和功能的研究，已发表5篇SCI论文、5篇硕士毕业生论文，已在蛋白质数据库注册了5个蛋白质晶体结构（5GNE、5XTA、5ESW、5ESX、5H6X）和已解析代注册的2个结构。