Bacillus subtilis subtilis 168 EXTRACELLULAR VESICLES IN STRESS-RELATED RESPONSE.

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

Congreso; SAMIGE; 2023

Bacillus subtilis is a Gram-positive spore-forming bacterium that can be isolated from diverse environments and has various biotechnological applications. Bacterial extracellular vesicles (EV) are nanoscale structures liberated to the extracellular medium, composed of lipids, proteins and nucleic acids. EV production by Gram-positive bacteria was long overlooked due to the belief that their thick peptidoglycan layer would prevent EV release, in consequence, Gram-positive EV functions and biological roles are yet not completely understood. We hypothesize that secreted EV could serve as communication particles in which signals (proteins, nucleic acids) are protected from extracellular proteases and nucleases. Thus, our aim was to extensively characterize B. subtilis derived EV and evaluate their possible role in stress related responses. EV isolation was performed by several steps of centrifugation, filtration and ultracentrifugation of Bacillus subtilis subtilis 168 culture supernatants, the resulting EV samples were exhaustively characterized. Ultrastructural features were analyzed by electron microscopy, images showed the characteristic EV cup-shape surrounded by lipid bilayer with electrodense center. The particle population had a mean diameter (Zave) of 128 ± 19 nm in a monomodal distribution determined by dynamic light scattering. EV RNA content (19.4 ± 3.5 ng/μl) was characterized by capillary electrophoresis revealing a predominant pattern of sRNA of under 200 nucleotides. EV protein cargo (0.046 ± 0.003 mg/ml) analyzed by SDS-PAGE showed a differential protein pattern compared to the whole cell proteome. Both EV RNA and protein contents were explored by means of next generation sequencing and proteomics (Qiaseq miRNA library - Illumina Novaseq and Orbitrap- nanoHPLC; n=1). Only a specific subset of RNAs and proteins were identified in EVs suggesting selective packaging into these extracellular particles. Moreover, modification of environmental conditions (oxidant stress: H2O2 58 μM, non-letal dose) showed changes in EV cargo, with enrichment in stress-response related proteins and modification of RNA patterns. In consequence, EV capacity to modify cell response to stress was analyzed. B. subtilis cells were treated with EV, washed and loaded with DCFDA (2’,7’-dichlorofluorescin diacetate; 50μM). This probe was used to detect intracellular reactive oxygen species and the response was followed fluorometrically. EV (0.25 μg) treated cells resulted in lower dye oxidation in the presence of H2O2 0.05 % (control: 2.27 ± 0.33; H2O2: 5.06 ±0.35; H2O2 + EV: 3.31±0.15 AU), reflecting a protective effect. In summary we could show that B. subtilis EV are secreted into the extracellular medium and that their cargo is modified by environmental conditions. Considering that EV were able to protect the cells from external insults, it is plausible that they represent a new means of cell-cell communication that needs to be further investigated.