The Rab-binding Profiles of Bacterial Virulence Factors during Infection.
The Rab-binding Profiles of Bacterial Virulence Factors during Infection.
复制标题
DOI：
10.1074/jbc.m115.700930
复制
发表时间：
2016-03-11
期刊：
影响因子：
--
通讯作者：
Frankel G
中科院分区：
文献类型：
--
作者：
So EC;Schroeder GN;Carson D;Mattheis C;Mousnier A;Broncel M;Tate EW;Frankel G
嗜肺军团菌（Legionella pneumophila）是军团病的病原体，利用其IV型分泌系统将300多种效应蛋白转运到宿主细胞中。这些效应子破坏宿主细胞信号传导途径以确保细菌增殖。尽管其发病机制的重要性，大多数效应的作用还有待确定。理解效应子功能的关键是识别它们在感染过程中结合的宿主蛋白。我们以前开发了一种新的串联亲和纯化（TAP）的方法，使用六聚组氨酸和BirA特异性生物素化标签分离易位效应复合物从感染的细胞，其组成随后通过质谱破译。在这里，我们进一步推进了TAP方法的工作流程，并确定了效应物SidM和利达的感染依赖性相互作用组，这些相互作用组先前被报道在体外混杂地结合多种Rab GTP酶。在这项研究中，我们定义了一个严格的Rab GTP酶的子集，在感染过程中由SidM和利达靶向，包括Rab 1A，1B，6和10;此外，利达靶向Rab 14和18。总之，这项研究说明了这种方法的权力，以配置文件的细菌效应器在感染过程中的细胞内相互作用。
Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection.