FIG 1 .

Single-cell imaging of Tir translocation by EPEC. HeLa cells preloaded with CCF2 were infected with EPEC expressing Tir-BlaM from the native promoter and mCherry as a bacterial marker. Infection was carried out under the microscope, and images were captured using phase-contrast, green channel (intact CCF2), blue channel (hydrolyzed CCF2), and red channel (bacteria) microscopy for 3 h at 90-s intervals. (A) Selected frames taken from Movie S2 in the supplemental material. The respective time points postinfection are indicated above each frame. A pioneering microcolony is indicated by the yellow arrow, and the white arrow indicates a recruited microcolony. (B) Translocation dynamics of 11 HeLa cells. The raw data are represented by the shift from green (substrate) to blue (product). Yellow dots indicate 50% of maximal product accumulation level, red dots indicate attachment time of the pioneering microcolony, and red asterisks indicate attachment time of a second or recruited microcolony (where it existed). (C) Plot of the dynamics of translocation into the 11 cells shown in panel B. The data were normalized using the attachment time of the pioneering microcolony as t₀. Translocation levels are presented using a scale of 0.0 to 1.0. (D) Selected frames taken from Movie S3. The respective time points postinfection are indicated above each frame. Yellow arrows indicate blebbing buds generated by a cell resistant to translocation.