E-cigarette liquids alter the biogenesis and properties of Aggregatibacter actinomycetemcomitans outer membrane vesicles

<i>Aggregatibacter actinomycetemcomitans</i> is an oral pathogen associated with aggressive forms of periodontitis as well as systemic diseases such as endocarditis. Through the secretion of outer membrane vesicles (OMVs), this bacterium establishes complex host-pathogen interactions, transmitting virulence determinants such as leukotoxin A (LtxA) that influence host cell behavior. Smoking of both traditional cigarettes as well as electronic cigarettes (ECIG) has been correlated with increased rates of periodontitis, yet the mechanisms regulating this have not been fully elucidated. We hypothesized that ECIG liquids (E-liquids), which contain numerous organic molecules, including nicotine and flavoring agents, might affect <i>A. actinomycetemcomitans</i> virulence by altering OMV production. To test this, we examined the impact of cinnamon and menthol flavored E-liquid exposure on <i>A. actinomycetemcomitans</i> growth as well as the production and composition of the resulting OMVs. Both cinnamon and menthol E-liquids inhibited <i>A. actinomycetemcomitans</i> growth at relatively low concentrations, with cinnamon E-liquid exhibiting greater toxicity than menthol. Bacteria treated with subinhibitory concentrations of the E-liquids produced fewer OMVs than untreated cells. While the average OMV size remained similar between treated and untreated cells, cinnamon E-liquid exposure shifted OMV production toward larger vesicles. Cinnamon E-liquid exposure also increased the protein and surface-associated DNA concentrations of the OMVs. SDS-PAGE showed significant differences in the protein composition between OMVs produced by untreated or treated bacteria, but no changes in membrane fluidity were detected among the OMV groups. Together, these results demonstrate that E-liquids modulate OMV biogenesis and composition in <i>A. actinomycetemcomitans</i>, potentially influencing its virulence and pathogenicity.