Characterization of TccP-mediated N-WASP activation during enterohaemorrhagic Escherichia coli infection
Characterization of TccP-mediated N-WASP activation during enterohaemorrhagic Escherichia coli infection
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DOI：
10.1111/j.1462-5822.2006.00723.x
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发表时间：
2006-09-01
影响因子：
3.4
通讯作者：
Frankel, Gad
中科院分区：
文献类型：
--
作者：
Garmendia, Junkal;Carlier, Marie-France;Frankel, Gad
宿主细胞骨架的破坏是肠出血性大肠杆菌（EHEC）感染的标志。肠出血性大肠杆菌移位跨膜受体蛋白Tir（移位的内膜受体），其将细胞外细菌连接到真核细胞肌动蛋白细胞骨架，触发在粘附细菌下方形成富含肌动蛋白的细胞。Tir介导的EHEC的肌动蛋白增加需要TccP（Tir细胞骨架偶联蛋白），TccP是最近发现的III型分泌系统效应蛋白，其在易位后结合并激活Wiskott-Aldrich综合征蛋白（N-WASP），其进而激活肌动蛋白相关蛋白2/3复合物，导致肌动蛋白的局部聚合。在这项研究中，截短的N-WASP和TccP衍生物的产生和测试在体外肌动蛋白聚合和上皮细胞感染试验。N-WASP的GT3结合结构域（GBD）的C-末端氨基酸253-276被鉴定为对于TccP：N-WASP蛋白质：蛋白质相互作用、TccP介导的N-WASP活化和肌动蛋白聚合的诱导是必需的，尽管不是足够的。来自EHEC O 157：H7菌株EDL 933的TccP由独特的N端结构域和六个富含脯氨酸的重复序列组成。TccP的N-末端内的进行性缺失揭示了残基1-21对于其易位是必要的和足够的，而氨基酸1-181，包括N-末端易位信号和两个富含脯氨酸的重复序列，足以在EHEC感染的上皮细胞和体外肌动蛋白聚合测定中触发肌动蛋白聚合。本研究定义了TccP的模块化结构域结构和TccP介导的N-WASP激活和EHEC诱导的宿主肌动蛋白细胞骨架重塑的分子基础。
Subversion of the host cell cytoskeleton is the hallmark of enterohaemorrhagic Escherichia coli (EHEC) infection. EHEC translocates the trans-membrane receptor protein Tir (translocated intimin receptor), which links the extracellular bacterium to the eukaryotic cell actin cytoskeleton, triggering formation of actin-rich pedestals beneath adherent bacteria. Tir-mediated actin accretion by EHEC requires TccP (Tir cytoskeleton coupling protein), a recently discovered type III secretion system effector protein which, following translocation, binds and activates Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the actin-related protein 2/3 complex leading to localized polymerization of actin. In this study, truncated N-WASP and TccP derivatives were generated and tested in in vitro actin polymerization and epithelial cell infection assays. The C-terminal amino acids 253-276 of the GTPase binding domain (GBD) of N-WASP were identified as essential, although not sufficient, for TccP:N-WASP protein:protein interaction, TccP-mediated N-WASP activation and induction of actin polymerization. TccP from EHEC O157:H7 strain EDL933 consists of a unique N-terminal domain and six proline-rich repeats. Progressive deletions within the N-terminus of TccP revealed that residues 1-21 are necessary and sufficient for its translocation, while amino acids 1-181, encompassing the N-terminal translocation signal and two proline-rich repeats, are sufficient for triggering actin polymerization in EHEC-infected epithelial cells and in in vitro actin polymerization assays. This study defines the modular domain structure of TccP and the molecular basis of TccP-mediated N-WASP activation and EHEC-induced remodelling of the host actin cytoskeleton.