Presence and activity of wild-type and ΔK/R-ab Porphyromonas gingivalis gingipains on whole bacteria and outer membrane vesicles (OMVs). (A) Immunopositive bands of 45 kDa were observed in the W83 whole-cell and OMV samples but not in the gingipain-null ΔK/R-ab equivalents when protein extracts were analyzed by immunoblotting using the Rb7 antigingipain antiserum. (B) Cryo–electron microscopy (EM) micrographs showing mAb 1B5 immunogold-labeled W83 bacteria and OMV. The gingipain expression is mainly located to the cell wall in both W83 whole cells and OMVs (black arrows) but is absent in ΔK/R-ab equivalents (scale bar: whole bacteria = 100 nm; OMV = 50 nm). (C–F) Gingipain fluorometric enzyme activity assays showing the higher levels of activity of arginine-specific (Arg, C, E) and lysine-specific (Lys, D, F) protease in W83 whole cells and OMVs compared to ΔK/R-ab mutant equivalents. In C–F, data are mean ± SD of 5 independent experiments with each individual experiment performed in triplicate. Statistical significance was determined by 1-way analysis of variance, ***P < 0.001.

Increased endothelium permeability in vitro following treatment with Porphyromonas gingivalis whole cells and outer membrane vesicles (OMVs) is gingipain dependent. (A) Movement of fluorescently labeled 70 kDa dextran from the upper well to the lower well in a Transwell assay increased in a time-dependent manner in the absence of human microvascular endothelial cells (HMEC-1; insert only), whereas this movement was almost abolished when a confluent endothelium was cultured on the insert surface (monolayer). Endothelial monolayers were treated with (B) whole bacteria or (C) OMVs from either W83 or ΔK/R-ab for 1.5 h, and then dextran permeability across the endothelium was measured for up to 5 h; phosphate-buffered saline (PBS)–treated endothelium was used as controls. Increased endothelial permeability was significantly increased in a time-dependent manner following exposure to W83 when compared to ΔK/R-ab equivalents and untreated controls for both whole bacteria and OMVs. No significant differences were observed between ΔK/R-ab-treated and uninfected controls. Data are presented as mean ± SD of 3 independent experiments and were analyzed by 1-way analysis of variance followed by Tukey’s post hoc multiple comparisons test. *P < 0.05. **P < 0.01.

W83 outer membrane vesicles (OMVs) induce systemic disease in zebrafish larvae in a gingipain-dependent manner. (A) Kaplan-Meier survival plots of zebrafish larvae infected 30-h postfertilization (hpf) with phosphate-buffered saline (PBS) control, Porphyromonas gingivalis (Pg) W83 whole cells (WCs), Pg W83 OMVs, or ΔK/R-ab OMVs. Comparison of survival curves using the log-rank test shows significant differences between W83 whole cell–injected and W83 OMV-injected zebrafish compared to PBS controls. Survival curves of zebrafish larvae injected with ΔK/Ra-b OMVs were not statistically different from the PBS control (ns = no significant difference, ***P < 0.001). (B–D) Percentage live, edematous, and dead zebrafish larvae at (B) 24, (C) 48, and (D) 72 hpi showing that the percentage of diseased (dead + edematous) zebrafish was significantly increased following systemic infection with W83 OMVs compared to ΔK/R-ab OMVs at all time points (*P < 0.05, **P < 0.01 by 1-way analysis of variance with Tukey’s post hoc multiple comparisons test). (E) Representative micrographs showing the morphology of zebrafish larvae infected with PBS control, W83 whole cells (WCs), W83 OMVs, or ΔK/R-ab OMVs. W83 whole-cell and OMV-infected zebrafish showed marked edema around yolk sac and heart (black arrows). Scale bars = 500 µm. Data in A–D are mean ± SD pooled from 3 independent experiments with at least 39 zebrafish total per group.

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.

Inhibition of gingipain activity prevents ablation of PECAM-1 expression following W83 outer membrane vesicle (OMV) infection. W83 OMVs were treated with 2 µM KYT gingipain inhibitors for 1 h prior to human microvascular endothelial cell (HMEC-1) infection. HMEC-1 treated with W83 OMVs or untreated cells were used as controls. Flow cytometric analysis showed that the gingipain-specific inhibitor, KYT, prevented the loss of PECAM-1 cell surface abundance that was mediated by W83 OMVs. Data are mean ± SD normalized median fluorescence index (nMFI) from 4 independent experiments with statistical significance determined by a 1-way analysis of variance with Tukey’s post hoc multiple comparisons test. *P < 0.05. ***P < 0.001.