Characterization of Legionella effector proteins

为了确定未表征的嗜肺军团菌效应蛋白的功能，必须建立其重组生产和纯化方案。为了生产蛋白质，将选定的嗜肺军团菌效应蛋白的开放阅读框引入细菌菌株大肠杆菌中，大肠杆菌是实验室中常用的表达宿主，用于生产重组蛋白。 随后通过将其标签与亲和树脂结合，从大肠杆菌裂解物中分离出嗜肺军团菌效应蛋白。通过凝胶基质分离（SDS PAGE）测定收获的效应蛋白的产量和稳定性，并将蛋白质储存在冷冻状态以供进一步分析。 嗜肺军团菌效应子 LidA 先前已被证明与 GTPases Rab 家族的各种宿主细胞蛋白相互作用。这些 Rab 蛋白是分子开关，可以增加或减少真核细胞内膜结合区室之间运输的囊泡数量。其中许多途径对细胞功能和活力至关重要，它们的失调已被确定为人类各种遗传性疾病的原因。已发现嗜肺军团菌受益于宿主细胞囊泡运输过程。病原体劫持来自选定运输路线的运输囊泡，以便将其周围的液泡转变为模仿宿主细胞细胞器的隔室。为了了解囊泡劫持的分子细节，我们在体外研究了 LidA 对 Rab GTPases 的影响，并利用组织培养感染研究结合荧光显微镜确定了 Rab 功能对嗜肺军团菌毒力的重要性。我们正在进行的研究结果将增加我们对细菌感染复杂性的理解，并将有助于更多地了解某些宿主-病原体相互作用对嗜肺军团菌毒力的作用。

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.