Centre for Molecular Microbiology and Infection, Imperial College London, UK.
Cell Microbiol. 2010 Dec;12(12):1718-31. doi: 10.1111/j.1462-5822.2010.01503.x.
Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C-terminal class I PSD-95/Disc Large/ZO-1 (PDZ)-binding motif. Using a PDZ array screen we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co-immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA-tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C-terminal PDZ-binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map-induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti-apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.
肠致病性大肠杆菌（EPEC）菌株是引起腹泻的病原体，它利用 III 型分泌系统将效应蛋白转运到宿主细胞中，从而在人类肠道中定植和繁殖。Map、EspI 和 NleH1 是保守的 EPEC 效应蛋白，它们都具有 C 末端的 I 类 PDZ 结合基序。通过 PDZ 结构域阵列筛选，我们鉴定了 Na(+)/H(+) 交换体调节因子 2（NHERF2），这是一种参与极化上皮中离子通道锚定和回收的支架蛋白，它包含两个 PDZ 结构域，是 Map、EspI 和 NleH1 的共同靶标。我们使用重组蛋白和共免疫沉淀实验证实了 NHERF2 与每种效应蛋白结合。我们构建了稳定表达 HA 标记的 NHERF2 的 HeLa 细胞系，发现 Map、EspI 和 NleH1 通过其 C 末端 PDZ 结合基序共定位并与细胞内 NHERF2 相互作用。NHERF2 的过表达增强了 Map 诱导的丝状伪足的形成和持久性，加速了 EspI 向高尔基体的运输，并减弱了 NleH1 的抗凋亡活性。多个 T3SS 效应子与单个支架蛋白的结合是独特的。我们的数据表明，NHERF2 可能作为质膜分选位点，为控制细胞内时空效应蛋白活性提供了一种新的调节机制。
