A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium
A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium
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DOI：
10.1074/jbc.m109.086603
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发表时间：
2010-02-26
影响因子：
4.8
通讯作者：
Finlay, B. Brett
中科院分区：
文献类型：
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作者：
Deng, Wanyin;de Hoog, Carmen L.;Finlay, B. Brett
肠致病性大肠埃希菌、肠出血性大肠埃希菌和啮齿柠檬酸杆菌属于附着性和消隐性（A/E）细菌病原体。它们与宿主肠上皮细胞紧密结合，引发肠道微绒毛的消失，引起腹泻。其发病机制的核心是III型分泌系统（T3SS），该系统由称为肠细胞消失位点（LEE）的致病性岛编码。T3SS用于将LEE和非LEE编码的效应蛋白注射到宿主细胞中，这些效应蛋白调节宿主信号通路和免疫反应。识别这些效应物并阐明它们的功能是了解这些病原体分子发病机制的核心。在这里，我们使用高灵敏度和定量的SILAC（细胞培养中氨基酸稳定同位素标记）质谱分析了C. rodentium的III型分泌组。该方法不仅证实了以往通过常规生化和蛋白质组学技术鉴定的几乎所有已知的分泌蛋白和效应蛋白，而且还鉴定了一些新的分泌蛋白。验证了这些新蛋白的t3ss依赖性分泌，其中5个蛋白被转运到培养细胞中，代表新的或额外的效应物。编码这些效应物的基因的缺失突变体在啮齿鼠中产生，并在小鼠感染模型中进行了测试。本研究全面表征了C. rodentium的III型分泌组，扩展了A/E病原体的III型分泌蛋白和效应物的库，并证明了使用基于silac的定量蛋白质组学作为鉴定蛋白质分泌系统底物的工具的简单性和敏感性。
Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.