常见致病军团菌O抗原基因簇分子进化机制的研究

O-antigen, a bacterial pathogenic factor of the highest degree of biological diversity, is one of the most suitable materials for the study of molecular evolution at the DNA level. Legionnaires' disease is caused by Legionella spp., which are Gram-negative bacteria, capsule-free and facultative of intracellular parasitic. The bacteria do not grow in normal media but grow and reproduce in still water, water pipe or sewer, and are opportunistic human pathogens. The common pathogenic spieces include L. pneumophila, L. micdadei, L. bozemanii and L. longbeachae. The genetic mechanism underlining the molecular evolution of Legionella pneumophila O-antigen remain elusive, as thus far only one of the O-antigen gene clusters, L. pneumophila O1, was determined..Building up on the extensive foundation works of the applicant's laboratory, the project will determine the DNA sequences of 15 O-antigen gene clusters of 10 pathogenic Legionella spp., predict the functions of each of the genes using bioinformatics, compare comprehensively the sequences of the 15 O-antigen gene clusters, analyze systematically the genetic features of the molecular evolution, and elucidate the genesis and dissemination mechanisms of their diversity, as a first attempt in the world to fill up the missing knowledge of the field. In addition, specific DNA molecular markers based on the O-antigens will be identified, molecularly typed and their rapid detection methods established, to lay a foundation for rapid detection of the clinical pathogenic bacteria.

O抗原是细菌致病因子。其具有生物界最丰富的多样性，故而是在DNA水平研究分子进化最好的材料之一。军团菌属兼性胞内寄生革兰氏阴性杆菌,无荚膜，在静止的水源或人工管道中或下水道生长、繁殖并导致人军团病。军团菌为条件致病菌，常见致病种包括：嗜肺军团菌、米克戴得军团菌、波兹曼军团菌、长滩军团菌等。迄今为止国内外仅破译了一种嗜肺军团菌的O抗原基因簇，致病军团菌O抗原的遗传基础和分子进化方面的研究则仍属空白。本项目在申请人前期工作基础上，将对10种致病军团菌共15个血清型的O抗原基因簇进行破译，并利用生物信息学方法预测各个基因的功能，综合比较O抗原基因簇序列，系统分析O抗原的遗传基础的分子进化特征，进而阐明军团菌O抗原多样性的产生和扩散机制，以弥补了世界上军团菌O抗原分子进化方面研究的空白；本项目还将筛选基于O抗原的特异分子标识，建立分子分型和快速检测方法，为致病菌的快速检测奠定基础。

军团菌属兼性胞内寄生革兰氏阴性杆菌,无荚膜，在静止的水源或人工管道中或下水道生长、繁殖并导致人军团病。军团菌为条件致病菌，常见致病种包括：嗜肺军团菌、米克戴得军团菌、波兹曼军团菌、长滩军团菌等。致病军团菌O抗原的遗传基础和分子进化方面的研究仍属空白。本课题顺利完成任务，获得成果如下：.(1) 已收集到军团菌49 株，其中标准菌株 39株，包括嗜肺军团菌15种血清型的标准株和非嗜肺军团菌的标准株，临床分离株10 株，奠定了O抗原基因簇破译及芯片研发的基础。.(2) 对4种致病军团菌，包括嗜肺军团菌3个亚种（肺炎亚种、弗雷泽亚种、牧场亚种和米氏军团菌的O抗原基因簇序列破译。.(3) 对上述军团菌O抗原基因簇中的所有基因进行功能预测，从而获得第一手致病军团菌O抗原的遗传信息。.(4) 通过生物信息学分析，从16S-23S rRNA基因间区（16S-23S rRNA internal transcribed spacer，简称ITS），筛选并确定了嗜肺军团菌和米克戴德军团菌不同种间的特异分子标识；进一步依据O抗原基因簇的特异基因，筛选并确定了嗜肺军团菌3个亚种（肺炎亚种、弗雷泽亚种、牧场亚种）的特异分子标识。.(5) 申请国内发明专利2项，发表SCI论文1篇。.(6) 培养硕士生2名。