# Oxidative Stress Reshapes Porphyromonas gingivalis Outer Membrane Vesicles and Impairs OMV-Mediated Invasion and Persistence in Trophoblast Cells

## TL;DR

Oxidative stress changes the size and protein content of Porphyromonas gingivalis outer membrane vesicles, reducing their ability to help the bacteria invade and persist in trophoblast cells.

## Contribution

This study reveals how oxidative stress alters OMV properties and impairs their role in bacterial invasion of trophoblast cells.

## Key findings

- Oxidative stress reduces OMV size and surface charge while increasing membrane fluorescence.
- OMVs from stressed bacteria fail to enhance P. gingivalis invasion and persistence in trophoblast cells.
- Proteomic analysis shows selective enrichment of specific proteins in OMVs under oxidative stress.

## Abstract

Background: Porphyromonas gingivalis outer membrane vesicles (OMVs) are key mediators of host–pathogen interactions and have been implicated in both periodontal disease and systemic conditions, including pregnancy complications. Although OMV production and cargo are known to be influenced by environmental stress, how oxidative stress reshapes P. gingivalis OMVs and their functional impact on trophoblast cells remains poorly understood. Here, we investigated how exposure to hydrogen peroxide (H2O2) affects OMV biogenesis, composition, and their ability to modulate bacterial invasion in trophoblast cells. Methods: P. gingivalis was cultured anaerobically and exposed to 30 mM H2O2 during the final 24 h of growth. OMVs were isolated by differential ultracentrifugation and characterized by nanoparticle tracking analysis and transmission electron microscopy and OMV protein cargo was analyzed by proteomics. Functional effects were assessed using invasion and persistence assays in HTR-8/SVneo trophoblast cells pretreated with OMVs. Results: Oxidative stress did not significantly alter total OMV yield but resulted in smaller vesicles (control OMV 168.2 ± 8.7 nm vs. OMV from H2O2-treated cultures 130.0 ± 13.8 nm) with reduced negative surface charge and increased membrane-associated FM4-64 fluorescence. Proteomic analysis revealed a remodeling of the OMV protein cargo under oxidative stress, including the selective enrichment of a von Willebrand factor type A domain-containing protein. Functionally, OMVs from control cultures led to a 2.5-fold increase in P. gingivalis invasion and a 4-fold increase in intracellular persistence in trophoblast cells, whereas OMVs produced under oxidative stress failed to promote these processes. Conclusions: Together, these findings highlight oxidative stress as a key determinant of OMV-mediated host–pathogen interactions at the maternal–fetal interface.