FIG 4.

Additional Fn during cultivation of host cells and especially during infection results in a significant increase in bacterial invasion of A549 cells but not of pHOBs. All cells were cultured in growth medium supplemented with Fn-depleted bovine serum. A concentration of 50 μg/ml exogenous Fn was added during cultivation [Fn(C)] (cells were washed before infection) or during infection [Fn(I)]. (A and B) A549 cells (A) or pHOBs (B) were infected with S. carnosus TM300(pFNBA4) for 1 h. Afterwards, extracellular staphylococci were removed by lysostaphin treatment. Subsequently, the host cells were detached and the number of cells was determined. Host cells were lysed, and the number of viable intracellular bacteria was assessed by plate counting. Data are means ± SD from three independent experiments. *, P < 0.05, one-way ANOVA followed by Dunnett’s multiple-comparison test. (C and D) Imaging flow-cytometric analysis (bacterium-positive host cells [C], spot count [D]) of A549, infected with S. aureus 6850-GFP for 1 h, followed by lysostaphin step and fixation. Data represent the means ± SD from three independent experiments. ***, P ≤ 0.001, one-way ANOVA followed by Dunnett’s multiple-comparison test and two-way ANOVA followed by Bonferroni posttests (spot count).