Sub-Inhibitory Concentrations of Metronidazole Enhance Production, Virulence Factor Loading, and Endothelial Cytotoxicity of Porphyromonas gingivalis Extracellular Vesicles

<i>Porphyromonas gingivalis</i> (<i>P. gingivalis</i>), a key periodontal pathogen, has been linked to atherosclerosis development. The clinical failure of antibiotics to improve cardiovascular outcomes necessitates alternative explanations. This study examines how sub-inhibitory concentrations of metronidazole affect the biogenesis and pathogenic potential of <i>P. gingivalis</i> extracellular vesicles (EVs) on human umbilical vein endothelial cells (HUVECs). EVs were isolated from both untreated bacteria (N-EVs) and those treated with sub-inhibitory concentrations of metronidazole (M-EVs) through ultracentrifugation. Characterization included transmission electron microscopy (TEM), nanoparticle tracking analysis, and Western blotting for virulence factors. HUVECs were evaluated using viability, migration, cell death assays, ROS detection, NF-κB activation imaging, and cytokine measurement. Sub-inhibitory concentrations of metronidazole increased EV production by 2.3-fold and enriched M-EVs with virulence factors (lipid A LPS, Kgp, RgpA). M-EVs demonstrated significantly stronger cytotoxicity, causing greater impairment of HUVEC viability and migration, alongside increased cell death. Mechanistically, M-EVs induced elevated mitochondrial and cellular ROS, promoting NF-κB activation and enhancing secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). Sub-inhibitory concentrations of metronidazole exacerbate endothelial injury by amplifying EV production and virulence factor loading in <i>P. gingivalis</i>, offering a mechanistic explanation for the limited cardiovascular benefits of antibiotic therapy in periodontitis patients.