FIG 2.

The N-terminal 20 to 23 amino acid residues of translocators (EspA, EspB, and EspD), secreted T3SS components (EscF and EscI), and both LEE-encoded (Tir and EspZ) and non-LEE-encoded (NleA and NleD) effectors all confer type III-specific secretion and translocation when fused to TEM-1. (A) Type III secretion assay (using the same conditions as described for Fig. 1) of TEM-1 fusions to the N-terminal secretion signals of EspA, EspB, EspD, EscF, EscI, Tir, EspZ, NleA, and NleD in EPEC WT, ΔescN, and ΔsepD strains. Whole-cell lysates (WCL) and secreted proteins (SP) were resolved by SDS-PAGE and analyzed by Western blotting using mouse antibodies against TEM-1. (B) Type III translocation assay of the same set of strains as described for Fig. 2A. EPEC WT and ΔescN strains carrying different TEM-1 fusion constructs were used to infect cultured HeLa cells. The infected cells were loaded with CCF2/AM and measured for fluorescence using a microplate reader. Fluorescence quantification data are presented as the emission ratio between blue fluorescence at 460 nm and green fluorescence at 530 nm. Positive type III translocation is indicated by higher 460/530 (nm/nm) ratios by WT EPEC but not the ΔescN mutant. The results shown are mean values with standard deviations from triplicates in one representative experiment out of 3 experiments.