CAI-1 enhances T3SS activity of EPEC in a dose-dependent manner. (A) Chemical structure of CAI-1. (B) Synthetic CAI-1 was added (at concentrations of 0.2–100 μM) to WT EPEC grown under T3SS-inducing conditions (DMEM) for 6 h. The secreted proteins were concentrated from the supernatants of the bacterial cultures and analyzed by 12% SDS-PAGE and Coomassie blue staining. The ΔescN mutant strain, which is unable to secrete through the T3SS, was used as a negative control. The locations of the type III translocators EspA, EspB, and EspD are indicated to the right of the gel. Also indicated is the location of EspC, which an autotransporter that is also secreted during EPEC infection. We observed that addition of CAI-1 at a concentration of 5 μM or higher enhanced EPEC T3SS activity. (C) and (D) WT EPEC was grown in 1:1 (v/v) DMEM:plain LB mixture for 6 h in the presence or the absence of synthetic CAI-1 (2, 5, 10 and 25 μM). The secreted proteins were concentrated as described above and analyzed by SDS-PAGE and western blot analysis using anti-EspB and anti-EspA antibodies (C) while the bacterial pellets were analyzed by SDS-PAGE and western blot analysis using anti-Tir, anti-EscJ and anti-DnaK antibodies (D).