Figure 3.

Pre-incubation of cells with sphingomyelinase (rHlb) prevented rHla-mediated formation of microscopically visible gaps in airway epithelial cell layers. Cellular changes, like loss of contacts to neighboring cells, changes in cell shape, and formation of paracellular gaps (white arrows) induced by S. aureus alpha-toxin (rHla, added at 0 h) in initially confluent cell layers of human airway epithelial model cells (16HBE14o- or S9 cells) were inhibited by pre-incubation of cells with S. aureus beta-toxin (rHlb), a sphingomyelinase. Treatments of cells with rHlb alone showed no cellular changes (data not shown). Phase contrast images were taken at different time points after adding PBS (vehicle control, PBS), 2000 ng/mL rHla, or rHla in the continued presence of 5000 ng/mL rHlb, respectively. Images were taken at the indicated times from time-lapse movies (Biostation IM, Nikon) monitoring the cells over 24 h.