EspT-mediated Il-8 production is regulated via RAC1.
A. Alignment of IpgB1, Map EspM2 and EspT effector proteins; the point mutations at position W63 and Q117 are indicated in red.
B. U937 cells were infected with E2348/69 or E2348/69 expressing EspTWT, EspTW/A or EspTQ/Y. EspTW/A and EspTQ/Y mutants drastically reduced the level of EspT-mediated Il-8 secretion.
C and D. U937 cells were transfected with non-targeting (NT) siRNA or siRNA targeted against Rac1 (SiRac1) or Cdc42 (SiCdc42) for 48 h. Successful knock-down was confirmed by Western blotting (C). Transfected U937 cells were either uninfected (U/I) or infected with E2348/69 expressing EspT (EspT). siRac1 inhibits the level of EspT-mediated Il-8 secretion while siCdc42 showed no effect on the level of Il-8 secretion.
E. Pre-treatment with NSC23766 (NSC) for 1 h reduced the level of EspT-mediated Il-8 secretion. Data are means + SD and are representative of at least three independent experiments; P value determined by Student’s T-test (*P < 0.05) comparing EspT strain with all other conditions as indicated.