FIG 6 .

Functional fleQ is required for P. aeruginosa internalization in HeLa cells and opposes anti-internalization effects of ExoS. (A) Restoration of fleQ function in the PA103ΔexoUT mutant was accomplished with a V240G point mutation in mutant PA103ΔexoUT. HeLa cells were infected with the indicated strains, including transformation with pExoS. Extracellular bacteria were killed with 200 µg/ml amikacin at 3 h and imaged hourly from 4 h to 14 h postinfection; images are from the 10-h time point. (B) The percentage of cells showing bacterial internalization was determined, and the data in the indicated columns were compared using an unpaired t test. (C) The coding region of fleQ was deleted in strain PAO1 and mutant PAO1ΔexoSTY. HeLa cells were infected with the indicated strains transformed with T3SS-GFP reporter pJNE05 and imaged as described for panel A. Bar, 50 µm. (D) The percentage of cells showing bacterial internalization was determined as described for panel B, and data in the indicated columns were compared by unpaired t test. (E and F) Comparison of total levels of P. aeruginosa internalization in HeLa cells after infection with mutant PAO1ΔexoSTY (E) or mutant PA103ΔexoUT (F) with and without fleQ restoration and pExoS expression. Extracellular bacteria were eliminated with 200 µg/ml gentamicin at 3 h. At 4 h, total levels of internalized bacteria were enumerated by viable count. Data in the indicated columns were compared by unpaired t test.