# Investigation on physical and physiological properties of extracellular vesicles derived from Enterococcus faecalis

## TL;DR

This study explores the physical and biological properties of extracellular vesicles from the bacteria Enterococcus faecalis, revealing how different preparation methods affect their characteristics and immune responses.

## Contribution

The study provides new insights into the biological characteristics of EVs from E. faecalis, which were previously poorly understood.

## Key findings

- EVs prepared using different filtration methods showed distinct size distributions.
- Each EV preparation had unique protein profiles and induced different cytokine responses in macrophages.
- EVs increased macrophage viability and size, indicating potential immune-modulating effects.

## Abstract

Extracellular membrane vesicles (EVs) are nanosized particles that contain various molecules originating from their parental cells and are produced by all three domains of life, including bacteria. Bacterial EVs are known to contribute to bacterial infections and immune responses in various human diseases. Enterococcus faecalis is an opportunistic pathogen. In this study, we examined the physical and physiological properties of EVs generated by E. faecalis, including particle size, protein composition, and cytokine-inducing profiles. To this end, we isolated EVs from bacteria under different preparation processes, and also a certain condition with the addition of EGCG. First, the bacterial culture supernatants were directly ultracentrifuged (named “Rough”), or filtered through 0.45- or 0.22 µm pore-sized membrane filters (named as “0.45 µm” or “0.22 µm,” respectively). EVs from EGCG-treated bacteria were prepared using a 0.45 µm pore-sized membrane filter and named “EGCG + 0.45 µm.” Each EV sample was subjected to DLS, SDS-PAGE, and cytokine array analyses. DLS results showed that the differently prepared EVs had distinct size distributions depending on the filtration process. SDS-PAGE results revealed unique protein profiles that differentiated EVs under each condition. Treatment of macrophages with each EV sample markedly increased cell viability and size. The cytokine profiles produced by macrophages in response to each EV preparation revealed both common and distinguishable factors. This study has significance in revealing aspects of the biological characteristics of EVs produced by E. faecalis, which have previously been largely unknown.