Fig. 4

C-di-AMP enhances migration and invasion abilities of both non-AIEC K12 and AIEC LF82 strains. A Representative images of the bacterial growth zone (swimming diameters) of the non-AIEC K12 and AIEC LF82 strains in the presence of 5.0 μM c-di-AMP. Each bacterial strain (OD₅₇₀ = 1.0) was added to the center of a plate containing Luria Bertani (LB) medium containing 0.3% agar and 5.0 μM c-di-AMP, and then the plate was incubated at 37 °C for 16 h. B Bacterial swimming diameters on LB agar plates were measured using the ImageJ software. Data are presented as mean ± SD. *P < 0.05, **P < 0.01; P-values were determined using the student’s t-test. C Each bacterial strain was precultured in LB broth containing 5.0 μM c-di-AMP for 16 h, and then added to Caco2 cells at a multiplicity of infection (MOI) of 1:100 (1 × 10⁵ bacteria/well). After 3 h incubation, each bacterial strain was added to Caco-2 cells (MOI of 1:100 (1 × 10.⁵ bacteria/well) for 3 h and incubated for 3 h in DMEM containing 400 μg/mL kanamycin to kill extracellular bacteria. The Caco2 cells were lysed with PBS containing 1% Triton X-100, and the lysates were plated on LB agar. Colony-forming units (CFU) were counted after 24 h of incubation. Data are presented as the mean ± SD. **P < 0.01; P-values were determined using the student’s t-test. (n = 3)