Crawford J Adam, Kaper James B
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Mol Microbiol. 2002 Nov;46(3):855-68. doi: 10.1046/j.1365-2958.2002.03214.x.
Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to translocate into host cells several effector molecules that are required for virulence. One of these, the translocated intimin receptor, Tir, inserts into the host cell cytoplasmic membrane, where it functions as the receptor for intimin, an outer membrane adhesin expressed by EPEC. A chaperone for Tir, called CesT, is required for stability of Tir in the EPEC cytoplasm. In this study, the cyaA gene reporter system was used to identify domains in Tir that mediate secretion into the culture supernatant and translocation into host cells. A Tir-CyaA fusion containing the first 15 N-terminal residues of Tir was secreted and translocated into HeLa cells by a deltatirdeltacesT mutant; however, maximal secretion and translocation was observed with the first 26 N-terminal residues of Tir. Fusions containing progressively larger N-terminal sequences of Tir were also efficiently secreted and translocated into HeLa cells by the deltatirdeltacesT strain. However, in a deltatir mutant that expresses CesT, Tir26-CyaA and an additional fusion containing the first 69 N-terminal residues of Tir were not secreted or translocated, but fusions containing larger N-terminal Tir sequences were secreted and translocated by the deltatir mutant. Wild-type EPEC secreted and translocated the Tir15-CyaA fusion, whereas longer fusions, such as Tir26-CyaA and Tir69-CyaA, were translocated to higher levels, similar to what was observed with the deltatirdeltacesT mutant. A Tir-CyaA fusion containing the CesT binding domain was translocated into HeLa cells more rapidly in the presence of CesT compared with translocation in the absence of CesT. Collectively, these results suggest that an N-terminal domain of 26 amino acids functions as a CesT-independent signal that is capable of delivering Tir into both the culture supernatant and the cytosol of host cells. Furthermore, in addition to its role in the stability of Tir, CesT may function in translocation by mediating rapid delivery of Tir into host cells.
肠致病性大肠杆菌（EPEC）利用III型分泌系统将几种毒力所需的效应分子转运到宿主细胞中。其中之一是易位紧密素受体（Tir），它插入宿主细胞质膜，在那里作为紧密素的受体发挥作用，紧密素是EPEC表达的一种外膜黏附素。Tir的一种伴侣蛋白CesT是Tir在EPEC细胞质中稳定所必需的。在本研究中，使用cyaA基因报告系统来鉴定Tir中介导分泌到培养上清液和转运到宿主细胞中的结构域。含有Tir前15个N端残基的Tir-CyaA融合蛋白被一个缺失tir和cesT的突变体分泌并转运到HeLa细胞中；然而，当含有Tir前26个N端残基时，观察到最大分泌和转运。含有逐渐增大的Tir N端序列的融合蛋白也被缺失tir和cesT的菌株有效分泌并转运到HeLa细胞中。然而，在表达CesT的缺失tir突变体中，Tir26-CyaA和另一个含有Tir前69个N端残基的融合蛋白没有被分泌或转运，但含有更大Tir N端序列的融合蛋白被缺失tir突变体分泌并转运。野生型EPEC分泌并转运Tir15-CyaA融合蛋白，而更长的融合蛋白，如Tir26-CyaA和Tir69-CyaA，转运水平更高，这与缺失tir和cesT突变体的情况类似。与不存在CesT时的转运相比，在存在CesT的情况下，含有CesT结合结构域的Tir-CyaA融合蛋白更快地转运到HeLa细胞中。总体而言，这些结果表明，一个26个氨基酸的N端结构域作为一个不依赖CesT的信号，能够将Tir递送到培养上清液和宿主细胞的细胞质中。此外，除了其在Tir稳定性中的作用外，CesT可能通过介导Tir快速递送到宿主细胞中而在转运中发挥作用。
