FIG 1.

Small molecule library screen using a FRET-based reporter assay. (A) Schematic depiction of the reporter translocation assay. Compounds that interfere with T4SS-mediated reporter delivery into infected cells will prevent β-Lac-mediated cleavage of the mammalian cell-permeable CCF4/AM substrate, retaining its green fluorescence, whereas reporter delivery into host cells will result in a shift in emission light from green to blue due to cleavage of the FRET pair. (B) RAW264.7 macrophages were challenged at an MOI of 20 with either Lp02 (functional T4SS) or Lp03 (defective T4SS) harboring plasmids encoding either βLac (control) or βLac-LidA. At the indicated time points (hours postinfection [hpi]), CCF4/AM was added to the cells, and fluorescence emission light was detected by epifluorescence microscopy. (C) Schematic overview of the different high-throughput screen stages. (D) Waterfall plot depicting the dose response to all compounds. Compounds that result in reduced reporter translocation (assessed by FRET) are shown in red, compounds without effect are shown in green, activators or false activators/fluorescent compounds are shown in blue. (E) Inhibition profile for representative hits from the FRET confirmation assay using an 11-point dose range. Data were globally fit using a Hill equation.