Figure 6. FadA increases the permeability of the endothelial barrier.

A–C. Confluent HUVEC monolayers in transwells were incubated with 50 μg/ml FadAc (■), 0.5 UI/mL Thrombin (◇), or untreated (×) (A); or with increasing concentrations of FadAc (B); or with 50 μg/ml FadAc in combination with varying concentrations of GST or GST-VE-cadherin_415–534, or untreated (C). Cell permeability was assessed by adding Texas Red-conjugated dextran to the upper chamber (final concentration = 1 mg/ml). At indicated times (A), or after 30 min (B and C), aliquots from the lower chamber were taken and the fluorescence was measured. D. Cells were incubated with F. nucleatum 12230 (■), fadA-deletion mutant US1 (▲), fadA-complementing strain USF81(×), or E coli DH5α (□), each at an MOI=100, followed by the addition of fluorescent dextran. The permeability assay was performed as described above. E. E. coli was added to the transwell inserts with or without HUVEC, or in combination with F. nucleatum 12230, US1, or USF81. At the indicated times, aliquots were taken from the lower chamber and plated on LB agar plates followed by incubation in air. The rate of E. coli penetration was calculated as percent of bacteria recovered from the lower chamber over the total bacteria added to the upper chamber. Data are the means ± SD from at least three independent experiments performed in duplicates or triplicates. * p<0.05 and *** p<0.001 based on t test (A to D) and ANOVA test followed by Bonferroni’s post test (E).