Characterization of Legionella effector proteins

为了确定非特征化L的函数。必须建立其重组生产和纯化的方案。对于蛋白质生产，选择L.将嗜肺菌效应蛋白引入细菌菌株大肠杆菌（Escherichia coli）中，大肠杆菌是实验室中通常用于生产重组蛋白的表达宿主。 洛杉矶嗜肺菌效应蛋白随后从E.大肠杆菌裂解物通过其标签结合到亲和树脂上。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，并将蛋白以冷冻状态储存用于进一步分析。 洛杉矶嗜肺菌效应子利达先前已显示与GTP酶的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转运的囊泡的量。这些途径中的许多对细胞功能和活力至关重要，并且它们的失调已被确定为人类各种遗传疾病的原因。L.已发现嗜肺菌受益于宿主细胞囊泡转运过程。病原体从选定的运输路线劫持运输囊泡，以便将其周围的液泡转化为模仿宿主细胞器的隔室。为了了解囊泡劫持的分子细节，我们研究了利达对体外Rab GTP酶的作用，并确定了Rab功能对L。使用组织培养感染研究结合荧光显微镜观察嗜肺菌毒力。我们正在进行的研究的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L。嗜肺菌毒力

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.