Allombert Julie, Vianney Anne, Charpentier Xavier
CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon, 69100, Villeurbanne, France.
Methods Mol Biol. 2017;1615:489-499. doi: 10.1007/978-1-4939-7033-9_34.
Among the bacterial secretion systems, the Type III, IV, and VI secretion systems enable bacteria to secrete proteins directly into a target cell. This specific form of secretion, referred to as translocation, is essential for a number of pathogens to alter or kill targeted cells. The translocated proteins, called effector proteins, can directly interfere with the normal processes of the targeted cells, preventing elimination of pathogens and promoting their multiplication. The function of effector proteins varies greatly depending on the considered pathogen and the targeted cell. In addition, there is often no magic bullet, and the number of effector proteins can range from a handful to hundreds, with, for instance, a substrate of over 300 effector proteins of the Icm/Dot Type IV secretion system in the human pathogen Legionella pneumophila. Identifying, detecting, and monitoring the translocation of each of the effector proteins represents an active field of research and is key to understanding the bacterial molecular weaponry. Translational fusion of an effector with a reporter protein of known activity remains the best method to monitor effector translocation. The development of a fluorescent substrate for the TEM-1 beta-lactamase has turned this antibiotic-resistant protein into a highly versatile reporter system for investigating protein transfer events associated with microbial infection of host cells. Here we describe a simple protocol to assay the translocation of an effector protein by the Icm/Dot system of the human pathogen Legionella pneumophila.
在细菌分泌系统中，III型、IV型和VI型分泌系统能使细菌将蛋白质直接分泌到靶细胞中。这种特殊的分泌形式，即转位，对于许多病原体改变或杀死靶细胞至关重要。被称为效应蛋白的转位蛋白可直接干扰靶细胞的正常进程，阻止病原体被清除并促进其增殖。效应蛋白的功能因所涉及的病原体和靶细胞的不同而有很大差异。此外，通常没有万灵药，效应蛋白的数量从几个到数百个不等，例如，人类病原体嗜肺军团菌中Icm/Dot IV型分泌系统有超过300种效应蛋白的底物。识别、检测和监测每种效应蛋白的转位是一个活跃的研究领域，也是理解细菌分子武器的关键。将效应蛋白与具有已知活性的报告蛋白进行翻译融合仍然是监测效应蛋白转位的最佳方法。TEM-1β-内酰胺酶荧光底物的开发已将这种抗生素抗性蛋白转变为一种高度通用的报告系统，用于研究与宿主细胞微生物感染相关的蛋白质转移事件。在此，我们描述了一种简单的实验方案，用于检测人类病原体嗜肺军团菌的Icm/Dot系统介导的效应蛋白转位。
