CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon, Lyon, France.
Methods Mol Biol. 2024;2715:563-575. doi: 10.1007/978-1-0716-3445-5_35.
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 and/or kill the targeted cell. The translocated proteins, called effector proteins, can directly interfere with the normal processes of the targeted cell, preventing elimination of the pathogen and promoting its multiplication. The function of the 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, over 300 effector proteins substrate 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 represent an active field or research and are key to understanding the bacterial molecular weaponry. Translational fusion of the 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-resistance protein into a highly versatile reporter system to investigate protein transfer events associated with microbial infection of host cells. We here described a simple protocol to assay translocation of an effector protein by the Icm/Dot system of the human pathogen Legionella pneumophila. Taking advantage that the protonophore CCCP inhibits the secretion activity, this simple protocol can be derived into a time course analysis to follow the kinetic of effector translocation into target cells.
在细菌分泌系统中，III 型、IV 型和 VI 型分泌系统使细菌能够将蛋白质直接分泌到靶细胞中。这种特殊形式的分泌，称为“易位”，对于许多病原体改变和/或杀死靶细胞至关重要。被易位的蛋白质，称为效应蛋白，可以直接干扰靶细胞的正常过程，防止病原体的消除并促进其增殖。效应蛋白的功能因考虑的病原体和靶细胞而异。此外，通常没有灵丹妙药，效应蛋白的数量可以从少数到数百不等，例如，人类病原体嗜肺军团菌的 Icm/Dot 型 IV 分泌系统的底物有超过 300 种效应蛋白。鉴定、检测和监测每个效应蛋白的易位是一个活跃的研究领域，是理解细菌分子武器的关键。效应蛋白与已知活性的报告蛋白的翻译融合仍然是监测效应蛋白易位的最佳方法。TEM-1 内酰胺酶的荧光底物的开发将这种抗生素抗性蛋白转化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。我们在这里描述了一种简单的方案，用于测定人类病原体嗜肺军团菌的 Icm/Dot 系统的效应蛋白的易位。利用质子载体 CCCP 抑制分泌活性，这个简单的方案可以衍生为时间过程分析，以跟踪效应蛋白进入靶细胞的动力学。
