FIG. 4.
Persistence and heterogeneity of gene expression of intracellular P. aeruginosa. A. Transepithelial cell resistance (Rt) of MTEC incubated with PAO1-GFP for 4 h, treated with fresh medium containing gentamicin daily for up to 72 h. The dashed line represents the level of Rt associated with maintenance of epithelial cell tight junctions (46). Values are the means ± standard deviations of replicate samples from three independent preparations. B. Gentamicin survival assay of recovered intracellular bacteria (CFU/well) from MTEC incubated with PAO1-GFP as in panel A. Values are the means ± standard deviations of triplicate samples from three independent experiments. C. Laser scanning confocal photomicrograph (x, y) and z-axis reconstruction of MTEC stained with rhodamine-labeled phalloidin (red), obtained after incubation with PAO1-GFP for 4 h followed by gentamicin for 24 h. Bar = 10 μm. D. Immunofluorescence photomicrograph of P. aeruginosa strain CS1 cultured using planktonic conditions, applied to a glass slide, and then immunostained for FlgA (red) and/or OprF (green). Accompanying DAPI (4′,6′-diamidino-2-phenylindole)-stained images are below (background was inverted to white for contrast). Bar = 1 μm. E. Confocal photomicrograph (x, y) and z-axis reconstruction of MTEC obtained after incubation with CS1 immunostained for FlgA (red) and OprF (green). White lines indicate Transwell membrane and apical cell border. Bar = 10 μm.