Characterization of Legionella effector proteins

军团菌效应蛋白的表征

• 批准号: 8149395
• 负责人: Matthias Machner
• 金额: $ 51.53万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149395
• 关键词: Affinity; Air Conditioning; Binding; Biological; Disease; Environment; Escherichia coli; Eukaryotic Cell; Family; Freezing; Fresh Water; Gel; Goals; Guanosine Triphosphate Phosphohydrolases; HIV; Harvest; Hereditary Disease; Human; In Vitro; Infection; Laboratories; Learning; Legionella; Molecular; Process; Proteins; Recombinants; Sequence Homology; Transport Process

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.

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。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。 嗜肺乳杆菌效应器LIDA此前已被证明与Rab家族GTP酶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的隔室之间运输的囊泡的数量。其中许多途径对细胞功能和生存能力至关重要，它们的错误调控已被确定为人类各种遗传疾病的原因。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，我们在体外研究了Lida对Rab GTP酶的影响，并通过组织培养感染结合荧光显微镜研究确定了Rab功能在嗜肺乳杆菌毒力中的重要性。我们正在进行的研究结果将增加我们对细菌感染的复杂性的理解，并将有助于更多地了解某些宿主-病原体相互作用在嗜肺乳杆菌毒力中的作用。