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.
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