Monitoring Effector Translocation with the TEM-1 Beta-Lactamase Reporter System: From Endpoint to Time Course Analysis

Abstract

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.