A rapid method for isolation of bacterial extracellular vesicles from culture media using epsilon-poly-L–lysine that enables immunological function research

Abstract

Both Gram-negative and Gram-positive bacteria can release vesicle-like structures referred to as bacterial extracellular vesicles (BEVs), which contain various bioactive compounds. BEVs play important roles in the microbial community interactions and host-microbe interactions. Markedly, BEVs can be delivered to host cells, thus modulating the development and function of the innate immune system. To clarify the compositions and biological functions of BEVs, we need to collect these vesicles with high purity and bioactivity. Here we propose an isolation strategy based on a broad-spectrum antimicrobial epsilon-poly-L-lysine (ϵ-PL) to precipitate BEVs at a relatively low centrifugal speed (10,000 × g). Compared to the standard ultracentrifugation strategy, our method can enrich BEVs from large volumes of media inexpensively and rapidly. The precipitated BEVs can be recovered by adjusting the pH and ionic strength of the media, followed by an ultrafiltration step to remove ϵ-PL and achieve buffer exchange. The morphology, size, and protein composition of the ϵ-PL-precipitated BEVs are comparable to those purified by ultracentrifugation. Moreover, ϵ-PL-precipitated BEVs retained the biological activity as observed by confocal microscopy studies. And THP-1 cells stimulated with these BEVs undergo marked reprogramming of their transcriptome. KEGG analysis of the differentially expressed genes showed that the signal pathways of cellular inflammatory response were significantly activated. Taken together, we provide a new method to rapidly enrich BEVs with high purity and bioactivity, which has the potential to be applied to BEVs-related immune response studies.

Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9

“…Following confirmation of MPER expression on the surface of EcN-MPER clones, we analysed if MPER could be detected in outer membrane vesicle (OMV) – rich fractions isolated from EcN-MPER culture supernatant. Slightly modifying the method previously described by Wei et al [ 59 ], a single colony of EcN-MPER from an overnight culture on LB agar was inoculated into 10 mL of LB broth. This was followed by overnight incubation at 37 °C with shaking at 220 rpm.…”

The Experimental Study of Periodontal Ligament Stem Cells Derived Exosomes with Hydrogel Accelerating Bone Regeneration on Alveolar Bone Defect

“…Subsequently, these samples were filtered through a 0.22 μm filter and centrifuged at 100,000× g for 70 min. After the supernatant was removed, pellets were resuspended in PBS and centrifuged again at 100,000× g for 70 min [ 28 ]. Exosome pellets were resuspended in PBS and frozen at −80 °C for subsequent use.…”

Bacterial extracellular vesicles: biotechnological perspective for enhanced productivity

“…Meanwhile, for the quantification of BEVs through lipids, the FM4-64 assay is widely reported (Klimentová and Stulík 2015 ). However, it is not always possible to correlate the protein and lipid content with the BEV quantity since the composition and size of the vesicles may vary depending on their production conditions, bacterial strain, and isolation method, among others (Hirayama and Nakao 2020 ; Bitto et al 2021 ; Wei et al 2022 ). Dry weight has been reported as a relative measure of vesiculation (Deatherage et al 2009 ; Klimentová and Stulík 2015 ; McMahon et al 2012 ).…”