ExoU-induced host cell rounding and plasma membrane blebbing prior to cell death.

A) Cytoskeletal changes and membrane perturbation are induced before cell death. Fibroblasts were infected with PA103ΔUT + exoU-S142A or + exoU. Top panels: Microtubules (MT) and P. aeruginosa were immunolabeled with an anti-tubulin antibody (green) and an anti-LPS antibody (red), respectively. Bottom panels: The actin cytoskeleton was stained with phalloidin (red) and labeled PA103 is shown in green. ExoU-mediated cytoskeletal disruption is indicated with arrows. Scale bar: 25 µm. Data shown are represenative of 6 experiments. B) ExoU-induced morphological changes in host cells during infection analyzed by time-lapse microscopy. HeLa cells were infected with a P. aeruginosa strain expressing both GFP and ExoU at an MOI of 2.5. The CellMask plasma membrane stain (red) and permeant Hoechst 33342 (blue) were added 20 min before the start of imaging. Cell rounding is marked with an arrow (+ 3 min) and membrane blebbing is marked with an arrowhead (+ 9 min). The acquired images were cropped (scale bar: 20 µm). Data shown are represenative of 6 experiments. C) The timing of membrane blebbing compared to the onset of plasma membrane damage. Cells were infected with an ExoU-expressing strain at an MOI of 5, incubated in the presence of the dyes as used for Fig 2B. The timing of plasma membrane damage was determined as SYTOX green influx, which is shown in bottom panels as corresponding to the multichroic images (top panels). Cell rounding and membrane blebbing are indicated with an arrow and arrowheads, respectively (in the +5 min, multichroic panel). The influx of SYTOX green stained from the edge of the nucleus (an arrow in the +30 min panel) progressing towards the center of the nucleus over time (an arrowhead in the +40 min panel). Scale bar: 10 µm. Data shown are represenative of 21 experiments.