# Effect of membrane vesicles produced under different pH conditions on the ability of Enterococcus faecalis to tolerate stressful environments and macrophages’ inflammatory response

## TL;DR

This study shows how membrane vesicles from Enterococcus faecalis help the bacteria survive harsh conditions and influence immune responses in macrophages.

## Contribution

The study reveals how pH-specific membrane vesicles enhance bacterial stress tolerance and modulate macrophage inflammation.

## Key findings

- MVs at pH 7.0 and 9.0 increase E. faecalis tolerance to alkaline, hyperosmotic, and chlorhexidine environments.
- MVs induce M1 macrophage polarization and ROS production, affecting inflammatory pathways.
- Transcriptomic analysis identified DEGs involved in cytokine and immune signaling pathways after MV exposure.

## Abstract

Enterococcus faecalis is frequently identified in persistent apical periodontitis lesions and possesses the ability to survive within the alkaline environment of the root canal system after treatment. Previously, we found that the secretion of pro-inflammatory cytokines by macrophages is stimulated by membrane vesicles (MVs) produced by E. faecalis at pH levels of 7.0 and 9.0. However, the influence of MVs on the survival of E. faecalis under stress conditions and the regulatory impact on macrophage-mediated inflammation require further elucidation. The present study demonstrated that the incorporation of pH 7.0 and 9.0 E. faecalis MVs enhances the bacterium’s tolerance to alkaline conditions, hyperosmolarity, oligotrophic environments, and chlorhexidine gluconate. Furthermore, the findings revealed that E. faecalis MVs at these pH levels induce M1 polarization of macrophages and promote intracellular ROS production. Transcriptomic analysis and quantitative reverse transcription polymerase chain reaction data indicated that, following treatment of macrophages with MVs under neutral and alkaline conditions, 208 and 302 differentially expressed genes (DEGs), respectively, were identified. These DEGs are predominantly involved in cytokine and cytokine receptor signaling pathways, chemokine signaling pathways, and NOD-like receptor signaling pathways. In summary, the results demonstrate that E. faecalis MVs produced under neutral and alkaline conditions contribute to the bacterium’s resistance to extreme environments and play a regulatory role in modulating macrophage inflammatory responses via multiple cytokine signaling pathways.

Persistent apical periodontitis is a prevalent oral disease that may result in tooth loss. Enterococcus faecalis is a common pathogen in the root canals of persistent apical periodontitis, exhibiting a remarkable capacity to endure extreme stress conditions. E. faecalis has been observed to secrete membrane vesicles, which are associated with bacterial pathogenicity, stress responses, and modulation of host immune regulation. The findings of this study suggest that the release of membrane vesicles by E. faecalis under varying pH conditions is correlated with the regulation of macrophage immune-inflammatory responses and the ability to withstand stressful environments. This research provides valuable insights into the pathogenic mechanisms underlying persistent E. faecalis infections in persistent apical periodontitis.