Characterization of Legionella pneumophila genes with impact on bacterial uptake into host cells and on bacterial virulence

嗜肺军团菌是一种在细胞内复制的细菌，是军团病的病原体。寄主细胞的成功寄生需要细菌的有效黏附和进入。一些军团菌的黏附和吸收促进因子已经被描述，但是具体的潜在机制还没有得到广泛的评估。在我们的前期工作中，我们在阿米巴感染模型中筛选了11,300个转座子诱变的嗜肺乳杆菌克隆，并鉴定了98个非致病原性克隆。进一步对选定的突变体在巨噬细胞中的黏附/摄取特性进行了测试，发现有7个克隆明显缺陷。在其中两个克隆中，转座子击中了众所周知的军团菌黏附/摄取基因，但这方面的五个基因是新的。这五个基因中的三个还没有对嗜肺乳杆菌进行功能评估。因此，这项研究的目的是检查各自的蛋白质是否直接或间接地促进细菌的黏附/摄取过程。此外，一个选定的蛋白质将被提纯，它对摄取过程的影响将被检测，并将确定它的真核相互作用伙伴。此外，还将确定细菌在实验室培养基和真核细胞中生长过程中的蛋白质定位，同时将监测由于蛋白质而引起的宿主细胞细胞骨架的变化。

Legionella pneumophila is an intracellularly replicating bacterium and the causative agent of Legionnaires' disease. Successful parasitism of host cells involves efficient adherence and entry of the bacteria. Some of the Legionella adherence- and uptake-promoting factors have been already described, however specific underlying mechanisms have not been extensively evaluated. In our prework, we screened 11,300 transposon-mutagenized L. pneumophila clones in an amoebae-infection model and we identified 98 apathogenous clones. Selected mutants were furthermore tested for adherence/uptake properties in macrophages and seven clones were found dramatically deficient. In two of the clones, the transposon hit well known Legionella adherence/uptake genes, but five genes in that respect were novel. Three of those five genes have not yet been functionally evaluated for L. pneumophila. The aim of the presented study is therefore to examine whether the respective proteins directly or indirectly contribute to bacterial adherence/uptake processes. Furthermore, one selected protein will be purified, its effect on uptake processes examined, and its eukaryotic interaction partner will be identified. Additionally, the protein localization during bacterial growth in laboratory medium and in eukaryotic cells will be determined and in parallel the changes in host cell cytoskeleton due to the protein will be monitored.