Figure 3. The ndk null-mutant enhances P. aeruginosa virulence in A549 cells through T3SS activation.

Prior to cell infection, the indicated bacterial strains were cultured in LB broth for 12 h. The bacterial pellets and culture supernatants were collected after centrifugation. A549 cells were infected with the indicated strains (MOI = 50). In addition, A549 cells were treated with bacterial culture media 5-fold diluted in DMEM. At 2 h post-infection, the cell viability was detected. (a) CCK-8 assay. The percentage of cytotoxicity was calculated according to the following formula: cytotoxicity % = 1 − (OD_{450 infected cells}/OD_{450 sham-infected control}) × 100%. (b) Calcein-AM staining. Scale bar: 50 μm. Representative images from triplicate experiments are shown. (c) Fluorescence intensity per cell from (b) was analyzed by Image pro-Plus software (IPP, edition 6.0) (n_cell = 90). Data are expressed as the mean ± SD from three independent experiments. *Indicates P < 0.05 compared to PAO1 strain. ^#Indicates P < 0.05 compared to Δndk strain.