a β-lactamase based reporter system for esx dependent protein translocation in mycobacteria基于β-lactamase记者esx系统依赖的蛋白质在分枝杆菌易位.pdfVIP

A b-Lactamase Based Reporter System for ESX Dependent Protein Translocation in Mycobacteria 1 ¨ 1 1,2 Tobias Rosenberger , Juliane K. Brulle , Peter Sander * ¨ 1 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland, 2 Nationales Zentrum fur Mykobakterien, Zurich, Switzerland Abstract Protein secretion is essential for all bacteria in order to interact with their environment. Mycobacterium tuberculosis depends on protein secretion to subvert host immune response mechanisms. Both the general secretion system (Sec) and the twin- arginine translocation system (Tat) are functional in mycobacteria. Furthermore, a novel type of protein translocation system named ESX has been identified. In the genome of M. tuberculosis five paralogous ESX regions (ESX-1 to ESX-5) have been found. Several components of the ESX translocation apparatus have been identified over the last ten years. The ESX regions are composed of a basic set of genes for the translocation machinery and the main substrate - a heterodimer. The best studied of these heterodimers is EsxA (ESAT-6)/EsxB (CFP-10), which has been shown to be exported by ESX-1. EsxA/B is heavily involved in virulence of M. tuberculosis. EsxG/H is exported by ESX-3 and seems to be involved in an essential iron- uptake mechanism in M. tuberculosis. These findings make ESX-3 components high profile drug targets. Until now, reporter systems for determination of ESX protein translocation have not been developed. In order to create such a reporter system, a truncated b-lactamase (‘bla TEM-1) was fused to the N-terminus of EsxB, EsxG and EsxU, respectively. These constructs have then been tested in a b-lactamase (B