Quantitative Mass Spectrometry Catalogues Salmonella Pathogenicity Island-2 Effectors and Identifies Their Cognate Host Binding Partners
Quantitative Mass Spectrometry Catalogues Salmonella Pathogenicity Island-2 Effectors and Identifies Their Cognate Host Binding Partners
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DOI：
10.1074/jbc.m111.224600
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发表时间：
2011-07-08
影响因子：
4.8
通讯作者：
Finlay, B. Brett
中科院分区：
文献类型：
--
作者：
Auweter, Sigrid D.;Bhavsar, Amit P.;Finlay, B. Brett
革兰氏阴性细菌病原体已经开发出专门的分泌系统，将细菌蛋白质直接转移到宿主细胞中。这些细菌效应物是毒力的核心，并重新编程宿主细胞过程，以有利于细菌的生存、定植和增殖。了解由特定病原体编码的全套效应器是了解细菌疾病的关键。此外，一旦这些效应器进入宿主细胞，识别这些效应器参与的分子组件对于确定每个效应器的作用机制至关重要。在这项工作中，我们使用细胞培养中氨基酸的稳定同位素标记(SILAC)这一强大的定量蛋白质组学技术来鉴定沙门氏菌致病岛-2三型分泌系统(SPI-2 T3SS)分泌的蛋白质，并表征SPI-2效应器的宿主相互作用伙伴。我们确认了许多已知的SPI-2效应器，并能够确定该分泌系统的几个新的候选底物。我们验证了先前发表的宿主蛋白-效应器结合对，获得了11个新的相互作用，其中三个被进一步研究并通过相互免疫共沉淀证实。这里确定的宿主细胞相互作用伙伴表明，沙门氏菌SPI-2效应器以协调的方式针对细胞过程，如细胞附着和细胞周期控制，而这些过程在感染背景下被低估。这项研究中概述的技术是特异和敏感的，是识别效应器和它们的宿主目标的强大工具，很容易适用于其他细菌病原体的研究。
Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.