Figure 4.

Loss of endothelial cell surface PECAM-1 by W83 whole bacteria and outer membrane vesicles (OMVs) is mediated by gingipains. Following a 1.5-h exposure to W83 or ΔK/R-ab whole bacteria or OMVs, human microvascular endothelial cells (HMEC-1) were removed from tissue culture plates and subjected to flow cytometric analysis for PECAM-1 cell surface abundance. Cells were gated using (A) side-scatter (SSC) and forward-scatter (FSC) voltages, then for (B) cell viability using TO-PRO-3 live/dead staining. Untreated cells were used as controls. Representative histograms and bar chart of 10,000 gated cells showing that PECAM-1 cell surface abundance is significantly decreased upon treatment with (C, D) W83 whole bacteria and (E, F) W83-derived OMVs compared to ΔK/R-ab-treated equivalents and untreated controls. PECAM-1 cell surface abundance was similar on HMEC-1 treated with ΔK/R-ab whole bacteria or ΔK/R-ab-derived OMVs to those observed on untreated controls. Data in D and F are presented as mean ± SD normalized median fluorescence index (nMFI) from 5 independent experiments with statistical significance determined by a 1-way analysis of variance with Tukey’s post hoc multiple comparisons test. **P < 0.01. ***P < 0.001.