FIG 5.

Mer-A 2026B and piericidin A1 inhibit Yersinia type III secretion in vitro more robustly than several previously identified T3SS inhibitors. (A) WT Y. pseudotuberculosis was incubated for 2 h under type III secretion-inducing conditions in the presence of various concentrations of the piericidin derivative Mer-A 2026B or DMSO. The secretome was precipitated with trichloroacetic acid and analyzed by SDS-PAGE analysis. The intensity of the Coomassie blue-stained band consistent with the size of YopE was quantified relative to DMSO-treated Y. pseudotuberculosis. The identity of the indicated YopE band was confirmed by Western blotting (data not shown). (B) The experiment in panel A was repeated using piericidin A1 and the previously identified, commercially available T3SS inhibitors C15, C22, and C24 (16) and MBX1641 (17) at a final concentration of 71 μM (data not shown) (16, 17, 21). Aurodox was used at a final concentration of 12.5 μM (21). The average inhibition of YopE secretion by the T3SS inhibitors compared to DMSO [(inhibitor-treated YopE band intensity)/(DMSO-treated YopE band intensity)] and SEM from 3 or 4 independent experiments is shown. *, P < 0.05, and ** P < 0.02 (Student t test) relative to DMSO-treated WT Y. pseudotuberculosis.