EXTRACELLULAR VESICLES FROM BACILLUS SUBTILIS 168 AND THEIR EFFECT ON HUMAN HCT-116 CELLS

BARNECH CAROLINA; GAONA BRUNO; MALAMUD FLORENCIA; COLUCCIO, LESKOW FEDERICO; CIMOLAI, M. CECILIA

Conferencia; saic; 2023

Bacillus subtilis is a Gram-positive bacterium that has demonstrated probiotic properties and can be isolated from various environments with many applications. Bacterial extracellular vesicles (EVs) are nanoscale structures composed of lipids, proteins, and nucleic acids that are released into the extracellular medium by membrane blebbing. Our hypothesis is that secreted EVs may serve as communication particles. Our aim was to characterize Bacillus subtilis EV in detail and to evaluate their possible role in inter-kingdom-related responses.EV isolation was performed by several steps of centrifugation, filtration and ultracentrifugation of culture supernatants obtained from Bacillus subtilis 168. The resulting EV samples were extensively characterized by TEM, DLS and macromolecules content. The EV protein load was qualitatively analyzed by SDS-PAGE and proteomics (Orbitrap nanoHPLC). Gene ontology analysis revealed the presence of proteins related to metabolic processes, biological regulation, localization and response to stimulus. Furthermore, EV generated under oxidative stress (H2O2 58 μM, non-lethal dose) were enriched in stress response related proteins . Therefore, the ability of these bacterial EV to modify the eukaryotic cellular response to stress was analyzed by treating human colon cancer cells (HCT-116). EV pre-treatment (0.5 μg/well; 2 h) showed protection when cells were loaded with DCFDA (2',7'-dichlorofluorescin diacetate) and challenged with H2O2 to detect intracellular reactive oxygen species (ROS). In summary, we could demonstrate that EV are secreted into the extracellular medium and that their cargo is modified by environmental conditions. Considering that bacterial EV could protect cells from external insults, it is plausible that EV represents a new cell-cell communication medium that needs to be further investigated.