Visualizing TamA and TamB on Edwardsiella tarda

Detecting of TamA_Et on bacterial surface by fluorescence microscopy was performed as reported previously (Li et al., 2016 (link)). Briefly, E. tarda TX01 was cultured in LB medium to OD₆₀₀ of 0.8 and resuspended in PBS (pH 7) to 10⁸ CFU/ml. The bacterial suspension was dropped on a glass slide and incubated for 12 h at 28°C. The antibody against rTamA_Et, rTamB_Et, or rTrx was added to bacterial suspension. The cells were incubated at 37°C for 1 h and then washed three times with PBS (pH 7). Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Abcam, Cambridge, United Kingdom) was added to the bacteria, followed by incubation at 37°C for 1 h in the dark. After staining with 4, 6-diamino-2-phenyl indole (DAPI) (Invitrogen, Carlsbad, CA, United States), bacteria were visualized using a confocal microscope (Carl Zeiss, Oberkochen, Germany). To determine bacterial damage under acidic conditions, E. tarda TX01, TX01ΔtamA, TX01ΔtamB, TX01ΔtamA/tamA, and TX01ΔtamB/tamB were cultured as above and resuspended in PBS of pH 5 to 10⁸ CFU/ml. The cells were incubated at 28°C for 2 h. After incubation, bacterial cells were treated with propidium iodide (PI) (Majorbio Biotech, Shanghai, China) and DAPI for 15 min in the dark according to the manufacturer’s instructions. The cells were then subjected to microscopy as above.