A, shown are structures of compounds used and visualization of the proteins from the pulldown assay. Structures of the inhibitory compounds, ME0052 and ME0055, and derivatives, ME0055-Aff and ME0052-Bio, used for the identification of target proteins are shown. Supplemental Protocol S1 details the syntheses. ME0055-Aff was used in affinity chromatography experiments to enrich for putative target proteins; ME0055-Bio was used for Far Western experiments. B, visualization of the protein fractions from the affinity chromatography procedure is shown. Lysates from E. coli O157 were incubated with ME0055-Aff and washed, and then proteins were eluted using 20 and 200 μm unlabeled ME0055. The beads were stripped using 1% acetic acid. Equal volumes of samples from each fraction were separated by SDS-PAGE and then visualized using Colloidal Blue Stain. Proteins were excised, subjected to in-gel trypsin digestion, and analyzed by LC-electrospray ionization tandem MS. Each protein identified from the gel is numbered sequentially and is listed in supplemental Table S4.

Far Western probing to examine binding of ME0052-Bio to putative target proteins. A, putative target proteins were cloned into pET151 and expressed in E. coli BL21 (λDE3) cells before lysis, separation, and transfer to a nitrocellulose membrane. ME0052-Bio was used as a probe, and interactions were detected using HRP-conjugated streptavidin. Proteins probed were (or encoded by) Tpx (1), FolX (2), z2714 (3), z3974 (4), WrbA (5), SurA (6), and StcE (7). Lane 8 contains bacterial lysate with no overexpressed protein as a negative control. The same protocol was used to subsequently probe proteins from E. coli O157 (EC), Y. pseudotuberculosis (YP), S. typhimurium (ST), P. aeruginosa (PA), and S. flexneri (SF) or empty vector control (−ve). Panels show overexpression of target proteins: WrbA (B), Tpx (C), FolX and FolB (D). Mutation of Tpx Cys-61 (C61S) affected binding of ME0052-Bio (E). The addition of 200 μm unlabeled ME0052 (F–H) strongly affected the binding of the biotinylated probe.

NMR chemical shift perturbation of ypTpx by ME0052. A, ¹⁵N HSQC spectra of 100 μm ypTpx in the absence (black) and presence (red) of 200 μm ME0052. A selection of the most perturbed cross peaks is indicated with their assignments, and a perturbed glutamine side chain is indicated (Q). B, a histogram of the weighted amide chemical shift perturbation by residue reveals clusters of residues affected by ligand binding.

Mapping of residues involved in drug binding and ligand docking. A, shown is a schematic representation of the structure of ecTpx (PDB code 3HVV (43)) colored purple by the shifts observed when ME0052 is added to ypTpx. Subunit A is in dark gray, and subunit B is in lighter gray. B, shown is a surface representation of the ecTpx dimer, with the docked compound ME0052 (cyan) based on the residues with the highest shift (purple sticks) using Autodock Vina. The figures were created in PyMOL (56).

AUC analysis of ME0052 binding to ypTpx. A line graph shows the binding of ME0052 to purified ypTpx during AUC experiments. The lines represent different redox states for Tpx: oxidized (black diamonds), reduced (blue inverted triangles) and forced reduced mutant, C61S (green triangles). The amount of bound ME0052 was used to calculate the K_d values described in the text.

Comparison of gene expression between bacteria cultured with salicylidene acylhydrazides and defined mutants. Microarray data were filtered for transcripts that showed significant changes in gene expression (>1.5-fold, p < 0.05) when treated with salicylidene acylhydrazides (ME0052, ME0053, ME0054) and that also showed >5-fold changes in transcript abundance when compared with the defined mutants. A, shown is a graphic representation of gene expression. Red lines indicate genes associated with the T3SS, blue lines indicate transcripts associated with flagella and motility, and gray lines represent other genes. B, shown is a heatmap of gene expression. Gene names are indicated to the right of the heatmap, and experimental conditions (inhibitor or relevant gene deletion or mutation) are indicated below. Coloring of boxes indicates relative expression (green, increased; red, repressed) as indicated on the left. Genes have been grouped according to their function or genetic location in three major classes: motility, pO157, and T3SS.

Role of Tpx for appropriate expression of flagella and EspA filaments. Bacteria were cultured in LB medium (A–C) or DMEM (D–F) to promote expression of flagella or EspA filaments. Wild-type bacteria (A) express flagella when visualized using immunofluorescence microscopy. The Tpx mutant (B) is non-motile and aflagellate, a phenotype that could be partially restored by introduction of plasmid-borne tpx (C). Culture in DMEM results in 45% of wild-type bacteria expressing EspA filaments (D). Deletion of tpx increased this proportion to 79% of the population (E), and complementation by plasmid-borne tpx returned this to wild-type levels (F). Images are composites created from differential interference contrast and immunofluorescence overlays.