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. 2019 Mar 18;5(6):1001–1012. doi: 10.1021/acsinfecdis.9b00048

Figure 3.

Figure 3

Evidence of inhibitor probe binding to purified MmpL3tb-GFP and flow-cytometry-based competition binding assay using purified MmpL3tb-GFP. (A) Comparative analysis of the binding of indolecarboxamide probes to MmpL3tb-GFP bound to polystyrene particles. MmpL3tb-GFP-coated beads were incubated for 15 min at room temperature with different concentrations of North 100 and North 114. After three washes in PBS pH 7.0–5% glycerol, the TAMRA mean fluorescence intensity (MFI) of the beads was analyzed by flow cytometry. The histograms show the absence of binding of the TAMRA-mannose probe to MmpL3tb-GFP-coated beads, while North 100 and North 114 bind to particles coated with different concentrations of MmpL3tb-GFP. The graph shows the concentration-dependent binding of North 100 and North 114 to particles coated with MmpL3tb-GFP; no binding is observed on particles coated with GFP alone. The MFI reported are mean values ± SD of technical triplicates and are representative of at least two independent experiments. (B) Flow-cytometry-based competition binding assay performed on purified MmpL3-GFP. Cells were cotreated with 2 μM North 114 and increasing concentrations of the inhibitors as described in the Methods. The concentrations of inhibitors are indicated under the x-axis. Shown on the y-axis are the MFI of the polystyrene particles from each treatment group expressed relative to that of particles not treated with any inhibitor (relative fluorescence value [RFI] arbitrarily set to 1). MFIs were determined by analyzing 10 000 particles under each condition. The data reported are mean values ± SD of technical duplicates and are representative of at least three independent experiments. Asterisks denote statistically significant decreases in fluorescence intensity between no inhibitor control (blue bars) and beads cotreated with North 114 and various concentrations of the inhibitors pursuant to the Student’s t-test (P < 0.05). No significant displacement was seen with the negative control drugs isoniazid (INH) and rifampicin (RIF), whose mechanisms of action are independent of MmpL3tb (with the exception of INH at 2 μM but not 4 μM).