Characterization of Legionella effector proteins

为了确定未鉴定的嗜肺乳杆菌效应蛋白的功能，必须建立其重组生产和纯化的方案。为了生产蛋白质，将选定的嗜肺乳杆菌效应蛋白的开放阅读框引入大肠杆菌菌株，大肠杆菌是实验室中常用的用于生产重组蛋白的表达宿主。随后，通过将嗜肺乳杆菌的标签与亲和树脂结合，从大肠杆菌裂解液中分离出嗜肺乳杆菌效应蛋白。通过凝胶基质分离（SDS - PAGE）测定收获的效应蛋白的产率和稳定性，并将蛋白冷冻保存以供进一步分析。

In order to determine the function of uncharacterized L. pneumophila effector proteins a protocol for their recombinant production and purification had to be established. For protein production, the open reading frames of selected L. pneumophila effector proteins were introduced into the bacterial strain Escherichia coli, an expression host commonly used in laboratories for the production of recombinant proteins. The L. pneumophila effector proteins were subsequently isolated from E. coli lysate through binding of their tag to an affinity resin. Yield and stability of the harvested effector proteins was determined by gel matrix separation (SDS PAGE) and the proteins were stored in a frozen state for further analyses. The L. pneumophila effector LidA has previously been shown to interact with various host cell proteins of the Rab family of GTPases. These Rab proteins are molecular switches that can increase or reduce the amount of vesicles transported between membrane-bound compartments within eukaryotic cells. Many of these pathways are critical to cellular function and viability, and their misregulation has been identified as cause for various genetic disorders in humans. L. pneumophila has been found to benefit from host cell vesicle transport processes. The pathogen hijacks transport vesicles from selected trafficking routes in order to transform its surrounding vacuole into a compartment that mimics host cell organelles. To understand the molecular details of vesicle hijacking we studied the effect of LidA on Rab GTPases in vitro and determined the importance of Rab function for L. pneumophila virulence using tissue culture infection studies combined with fluorescence microscopy. The results from our ongoing studies will increase our understanding of the complexity of bacterial infections and will help to learn more about the role of certain host-pathogen interaction for L. pneumophila virulence.