FIG 7.

Influence of INP0341 and INP1750 on bacterial motility and YscN-ATPase activity. (A) Effect of INP0341 and INP1750 on flagellar motility. (Top) Swarming (left) and swimming (right) motility. Bacteria were grown from an OD₆₂₀ of 0.1 to an OD₆₂₀ of 0.8 with constant shaking in the presence of 0.8% DMSO (vehicle) or 80 μM INP0341 or INP1750; then, 3-μl portions of these cultures were placed in the center of agar LB plates and grown overnight at 37°C. The area covered by bacteria was evaluated using QuantityOne software (Bio-Rad). Swarming was evaluated using 0.5% agar for CHA (illustrated in the pictures) and CHAΔexsA strains that were precultivated with INPs in low-calcium medium (LB broth, 5 mM EGTA, 20 mM MgCl₂) and swimming using 0.3% agar for CHA precultivated with INPs in low-calcium medium or in RPMI 1640 or DMEM added by 10% serum (right). (Bottom) Concentration effect relationship for swimming, with bacteria grown with increasing concentrations of INP1750 in RPMI 1640 or DMEM added by 10% serum, as described above. Values are means ± the SEM of two independent experiments performed in duplicate. Statistical analyses were performed (#, P < 0.05; ##, P < 0.01; ###, P < 0.001). A Student t test was used to compare values measured in the presence of INP0341 or in the presence of INP1750. (B) Effect of INP0341 and INP1750 on purified YscN T3SS ATPase. His-YscN (25 μg/ml) was incubated with INP0341 or INP1750 for 1 h; ATP (4 mM) was then added to the samples for 30 min, and the ATPase activity was measured as the amount of free phosphate liberated. Experiments were performed in duplicates. (Top) Percentage of inhibition of the enzymatic activity in the presence of 80 μM concentrations of each INP. (Bottom) Residual activity as a function of INP concentrations. Statistical analyes were performed (#, P < 0.05; ##, P < 0.01; ###, P < 0.001). A Student t test was used to compare values measured in the presence of INP0341 or in the presence of INP1750.