Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 β-lactamase as a new fluorescence-based reporter
Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 β-lactamase as a new fluorescence-based reporter
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DOI：
10.1128/jb.186.16.5486-5495.2004
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发表时间：
2004-08-01
影响因子：
3.2
通讯作者：
Oswald, E
中科院分区：
文献类型：
--
作者：
Charpentier, X;Oswald, E
肠致病性大肠杆菌和肠出血性大肠杆菌（EPEC和EHEC）菌株是人和动物的病原体，它们通过III型分泌系统（TTSS）将效应蛋白注入宿主细胞。Cif是一种效应蛋白，它可诱导宿主细胞周期停滞以及肌动蛋白细胞骨架的重组。Cif由存在于大多数EPEC和EHEC菌株中的λ样原噬菌体编码。在这项研究中，我们通过使用一种基于效应蛋白与成熟TEM - 1β - 内酰胺酶的翻译融合的新型报告系统，分析了将Cif靶向TTSS的结构域。通过使用荧光性β - 内酰胺酶底物CCF2/AM，可在活的宿主细胞中直接检测到易位。我们表明，Cif的前16个氨基酸（aa）对于介导其易位到宿主细胞是必要且充分的。同样，效应蛋白Map、EspF和Tir（与TTSS在同一区域编码）的前20个氨基酸以III型依赖但不依赖伴侣蛋白的方式介导分泌和易位。缺失前20个氨基酸的截短形式的Cif不再被分泌和易位，但与Tir、Map或EspF的前20个氨基酸融合可恢复分泌和易位。此外，嵌合蛋白完全能够触发宿主细胞周期停滞和应力纤维形成。总之，我们的结果表明，Cif由一个C末端效应结构域和一个可交换的N末端易位信号组成，并且TEM - 1报告系统是研究毒素或效应蛋白向宿主细胞易位的便捷工具。
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase. Translocation was detected directly in living host cells by using the fluorescent P-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.