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

Porphyromonas gingivalis (P. gingivalis), 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 P. gingivalis 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 P. gingivalis, offering a mechanistic explanation for the limited cardiovascular benefits of antibiotic therapy in periodontitis patients.