FIG. 2.

Evaluation of inhibition of type III and type II secretion in P. aeruginosa. P. aeruginosa ExoS-secreting strain PAKΔTY was grown under T3SS-inducing conditions (LB plus 5 mM EGTA) for 3 h in the presence of the indicated concentrations of compounds, and culture medium (1 ml) was concentrated in SDS-PAGE sample buffer, separated by SDS-12.5% PAGE, and stained with Coomassie blue. The positive control, DMSO plus EGTA, was treated with 5 mM EGTA but not inhibitors, and the negative control, DMSO without EGTA, was treated with neither EGTA nor inhibitors. The identity and molecular weights of protein markers are as follows: porcine myosin (200K), E. coli β-galactosidase (116K), rabbit muscle phosphorylase B (97K), bovine albumin (66K), ovalbumin (45K), and bovine carbonic anydrase (29K). (A) Secreted proteins from cells treated with EGTA and the five validated T3SS inhibitors (Table 3). The band corresponding to 49K ExoS is marked. (B) Secreted proteins from cells treated with EGTA and serial dilutions of T3SS inhibitor compound 1. (C) Effects of T3SS inhibitors on type II secretion of elastase. P. aeruginosa PA14 cells were grown in LB medium for 16 h in the presence of 50 μM of the indicated compounds. As controls, PA14 and PA14 xcpQ::Tn cells were grown in LB in the presence of the equivalent concentration of DMSO, and PA14 was grown in the presence of a type II secretion inhibitor (compound 7941790; Chembridge, Inc.). Culture medium corresponding to equivalent numbers of cells was harvested by centrifugation and incubated with shaking for 6 h with Congo Red-elastin. Digested soluble Congo Red was measured by the A₄₉₅ in two independent assays and plotted (gray and black bars).