Figure 3.

Characterization and validation of the hit compounds. (A) Structure, effect on PLC activity, pyoverdine production and growth in LB in the presence of TAT-1, -2, -3, -4, -5. (B) Structure, effect on PLC activity, elastase and growth in LB in the presence of TSS-1. PLC activity was measured in a P. aeruginosa PAO1 culture grown in MMST with added NPPC. 80 μl of the culture was aliquoted in a 384-well microtiter plate in the presence of different concentrations of the inhibitors and grown at 37°C for 7 h, and A₄₀₅ was measured. Pyoverdine production was measured in a P. aeruginosa PAO1 culture grown in CAA in a 384-well microtiter plate in the presence of different concentrations of the inhibitors. Bacteria were incubated at 37°C for 7 h, and fluorescence was measured at emission/excitation 460/405 nm. Elastase activity was measured in 100 μl of cell-free supernatants of P. aeruginosa PAO1 cultures grown at 37°C for 10 h in LB added with different concentrations of the tested compounds. Growth was measured in a P. aeruginosa PAO1 culture grown in LB at 37°C for 7 h in a 384-well microtiter plate in the presence of different concentrations of the inhibitors. Activities have been normalized by the cell density and subtracted of background values. Bars represent standard deviation from the mean, n = 3.