菲氏军团菌依赖DotA蛋白在宿主细胞内增殖及致病作用

Legionella feeleii is a Gram-negative intracellular bacterium, which caused Legionnaires disease. However, the mechanisms of the bacterial intracellular growth and pathogenesis are still not clear. In our previous researches, we found that L. feeleii could proliferate in host cells, induced cytokines secretion and caused the cells death. It has been demonstrated that DotA protein could mediate the intracellular proliferation of Legionella strains and cause the inflammatory reaction of host cells. Based on the current researches and the homology of Legionellae, we inferred: 1. In order to proliferate in the host cells, L. feeleii could utilize DotA protein to recruit the protein of the host cells. 2. L. feeleii could translocate the flagellin via DotA protein into the cytoplasm, active caspase-1 and induce cytokines secretion and pyroptosis. In this study, we will make L. feeleii dotA mutant, and try to investigate the mechanism of bacterial proliferation via infection of the mammalian cells. In combination of infection experiments that are performed in vitro and in vivo, the process of bacterial flagellin that was translocated into the cytoplasma and induction of inflammation will be analyzed. This research will elucidate the mechanisms of L. feeleii infection and the pathogenesis of L. feeleii caused respiratory diseases. This project will provide reliable theoretical basis for analyzing human respiratory system inflammation that caused by Legionella and other intracellular bacteria.

菲氏军团菌是革兰阴性胞内菌，可引起军团病，但其胞内增殖及致病机制尚未阐明。我们前期研究发现，菲氏军团菌可在宿主细胞内繁殖并诱导细胞因子释放及细胞死亡。已有研究证实，DotA蛋白能够调节军团菌的胞内增殖及引起宿主细胞炎症反应。基于当前研究现状及军团菌属同源性，我们推断: 1.菲氏军团菌利用DotA蛋白募集宿主蛋白在细胞内增殖。2.菲氏军团菌依赖DotA蛋白将鞭毛蛋白转运至胞质，调节活化caspase-1，诱导炎性细胞因子释放和焦亡，引起炎症反应。本研究拟以野生型与dotA基因缺陷型菲氏军团菌感染宿主细胞，探究其胞内增殖机制，结合野生型及caspase-1基因缺陷型小鼠巨噬细胞及小鼠感染实验，分析DotA蛋白转运鞭毛蛋白进入宿主细胞后，诱发宿主炎症性反应的过程，揭示菲氏军团菌引起呼吸系统疾病的本质，对解析军团菌及其它胞内菌引起人类呼吸系统炎症反应的致病机理提供可靠理论基础。

菲氏军团菌属革兰阴性杆菌，是军团菌属家族中重要病原体之一，能够引起人体呼吸系统炎症性疾病并导致死亡。人体吸入被菲氏军团菌污染的水滴或气溶胶后，可出现高热、咳嗽、寒战、头痛、肌痛等症状。菲氏军团菌可在哺乳动物类宿主细胞内生长繁殖，且能够诱导宿主细胞炎症反应，但其详细致病机制尚未阐明。本项目以解析菲氏军团菌在宿主细胞内的增殖及其毒力因子导致宿主疾病的致病作用为主要研究内容，分别对菲氏军团菌在人体细胞系内的繁殖，该菌利用毒力因子诱导宿主细胞炎症反应和体内感染对小鼠的致病作用，其鞭毛蛋白的表达及鞭毛结构的电子显微镜下观察，以及该菌对氯化钠的敏感程度、对宿主细胞膜的打孔能力及分泌蛋白分解酶等方面做出探究与证实。我们发现，菲氏军团菌可依赖DotA蛋白在人体U937及A549细胞系内的增殖。其血清2群细菌可表达FlaA蛋白，电子显微镜下能观察到完整的鞭毛结构，同时该菌感染人体细胞系后，可诱导宿主细胞释放炎性细胞因子，但菲氏血清1群军团菌不具有鞭毛结构，且不可诱导宿主细胞释放炎性细胞因子。此外，由于菲氏血清1群军团菌致病力较弱，其不能在ddY小鼠肺部增殖且不可导致小鼠疾病。指数生长期菲氏血清1群军团菌比指数生长后期的细菌对氯化钠敏感，而指数生长后期菲氏血清2群军团菌比指数生长期的该菌对氯化钠敏感，但二者均不具备对宿主细胞膜打孔的能力，且不能分泌蛋白分解酶。本研究的开展与实施或可为解析菲氏军团菌的致病机制提供可靠的实验数据和依据，同时为我们后续深入研究该菌毒力因子引起宿主炎症反应性疾病奠定坚实基础。