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

## TL;DR

E-cigarette liquids affect the production and properties of bacterial vesicles linked to periodontitis and other diseases.

## Contribution

Shows that e-liquids alter outer membrane vesicle biogenesis and composition in Aggregatibacter actinomycetemcomitans.

## Key findings

- Cinnamon and menthol e-liquids inhibit A. actinomycetemcomitans growth, with cinnamon being more toxic.
- E-liquid exposure reduces OMV production and shifts vesicle size toward larger particles.
- Cinnamon e-liquids increase OMV protein and DNA content, altering their composition.

## Abstract

Aggregatibacter actinomycetemcomitans 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 A. actinomycetemcomitans virulence by altering OMV production. To test this, we examined the impact of cinnamon and menthol flavored E-liquid exposure on A. actinomycetemcomitans growth as well as the production and composition of the resulting OMVs. Both cinnamon and menthol E-liquids inhibited A. actinomycetemcomitans 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 A. actinomycetemcomitans, potentially influencing its virulence and pathogenicity.