Extended Data Fig. 2: Evaluation of phenotypic activity of MsbA inhibitors.
From: Structural basis for dual-mode inhibition of the ABC transporter MsbA
a, α-MsbA western blot of solubilized extracts from an E. coli CFT073 UPEC imp strain overexpressing MsbA from a pBAD24 vector in the presence of 2% arabinose (lane 1), an E. coli CFT073 imp strain (lane 2), an E. coli CFT073 imp strain with the arabinose-titratable msbA expression system in the presence of 2% arabinose (lane 3), an E. coli CFT073 imp strain with the arabinose-titratable msbA expression system in the presence of 0.01% arabinose (lane 4) and an E. coli CFT073 imp strain with the arabinose-titratable msbA expression system in the presence of 0.002% arabinose (lane 5). Bacteria were grown for 3 h at 37 °C before collection. The blot is representative of n = 2 independent experiments. For gel source images, see Supplementary Fig. 1. b, Dose–response curves of compounds on E. coli CFT073 imp strains expressing endogenous (WT) and overexpressed (HIGH, in the presence of 0.2% arabinose) levels of MsbA. Data are mean ± s.e.m. from two independent experiments except for G247 (n = 4). IC50 values (in parentheses) were determined by fitting the inhibition dose–response curve with a nonlinear four-parameter inhibition model (see Methods section ‘MsbA ATPase assay’). c, Representative (20 isolated cells from two independent experiments) thin section electron micrographs of CFT073 lptD(imp4213) and CFT073 wild-type strains. Treatment of the lptD(imp4213) strain with G592 (middle left) or G907 (middle right) and wild-type UPEC with G247 (bottom) leads to membrane stacking (red arrow) and enlargement of cells, similar to cells depleted of MsbA (top right). Scale bars, 0.1 μm. d, e, Nucleotide and amino acid sequence changes (indicated in red) in two G592-resistant MG1655 ΔtolC clones (mut1 and mut2, d) and a G247-resistant wild-type E. coli clone (mut3, e, data not shown). For both panels, positions are relative to the start of msbA ORF. The frequency of resistance for G247 on wild-type E. coli was 3.9 × 10−9 (data not shown).
