Characterization of Legionella virulence mechanisms

为了确定未鉴定的嗜肺乳杆菌效应蛋白的功能，建立了重组蛋白的生产和纯化方法。在蛋白生产方面，将选定的嗜肺乳杆菌效应蛋白的开放阅读框引入到细菌菌株EscherichiaColi中，该菌株是实验室中通常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)对收获的效应蛋白的得率和稳定性进行了测定，并将其冷冻保存以供进一步分析。 我们的最新研究表明，嗜肺乳杆菌SIDD蛋白是宿主蛋白Rab1的一种新的调节剂。Rab1是一种分子开关，可以增加或减少真核细胞内膜结合的隔室之间的囊泡运输量。Rab1对细胞功能和活性至关重要，它的错误调节可能会导致人类功能紊乱。嗜肺性乳杆菌已被发现受益于宿主细胞的囊泡运输过程。病原体从选定的运输路线劫持运输小泡，以便将其周围的液泡转变为模拟宿主细胞细胞器的隔室。为了了解囊泡劫持的分子细节，我们研究了军团菌蛋白对Rab1活性的影响。我们发现，效应蛋白SIDD具有一种新的酶活性，使军团菌能够在感染过程中精确控制Rab1的开发时间。我们的研究结果不仅增加了我们对细菌感染的复杂性的了解，还将有助于更多地了解蛋白质如何在我们自己的细胞内受到调控，以及它们的错误调控如何导致疾病。

In order to determine the function of uncharacterized L. pneumophila effector proteins a protocol for their recombinant production and purification was 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. Our latest studies revealed that the protein SidD from L. pneumophila is a novel modulator of the host protein Rab1. Rab1 is a molecular switch that can increase or reduce the amount of vesicles transported between membrane-bound compartments within eukaryotic cells. Rab1 is critical to cellular function and viability, and its misregulation can cause 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 Legionella proteins on the activity of Rab1. We discovered that the effector protein SidD has a novel enzymatic activity that allows Legionella to precisely control the timing of Rab1 exploitation during infection. The results from our studies not only increased our understanding of the complexity of bacterial infections but will also help to learn more about the mechanism how proteins may be regulated within our own cells, and how their misregulation causes disease.