Fig. 6

Infection of Caco2 cells by S. aureus in the presence or absence of cell–cell junctions. Caco2 cells were seeded in 24-well plates at a density of 200,000 cells per well and cultured for 84 h prior infection. A Confocal fluorescence microscopy images of a Caco2 monolayer stained with antibodies specific for ZO-1 (red) and DAPI (blue). Scale bar: 50 µm. B Disruption of Caco2 cell–cell junctions by treatment for 45 min with EGTA in calcium-free media. C Start of tight junction restoration after EGTA treatment and 1 h incubation in a calcium-free medium and (D) complete restoration of tight junctions after 3 h in a calcium-free medium. E, F, G Infection of the Caco2 monolayer or the EGTA-disrupted Caco2 monolayer upon 3 h infection with GFP-expressing S. aureus. The percentage of living cells (compared to the uninfected control) that resulted in GFP + or GFP − cells after infection was measured by flow cytometry. GFP + Caco2 indicates the proportion of the cell population with GFP-expressing S. aureus and GFP − indicates the population that remained uninfected. F Bacterial adherence was measured immediately upon infection in the absence of lysostaphin and, accordingly, GFP + cells represent both adherent and intracellular bacteria. E, G Upon removal of extracellular bacteria by a 30-min incubation with lysostaphin only intracellular bacteria remain detectable. H, I Confocal fluorescence microscopy images of Caco2 cells in a closed monolayer (H) and upon EGTA treatment (I). Cells were stained with an antibody specific for ZO-1 (red) and DAPI (blue). GFP-expressing S. aureus are represented in green. Scale bar: 50 µm