Transcriptional analysis of THP-1 cells stimulated by bacterial extracellular vesicles enriched by ultracentrifugation and an e-poly- L-lysine-based method

We propose an isolation strategy based on a broad-spectrum antimicrobial epsilon-poly-L–lysine (e-PL) to precipitate bacterial extracellular vesicles (BEVs) at a relatively low centrifugal speed (10,000 × g). Compared to the standard ultracentrifugation (UC) strategy, our method can enrich BEVs from large volumes of media inexpensively and rapidly. The precipitated BEVs can be recovered by adjusting pH and ionic strength of the media, followed by ultrafiltration step to remove e-PL and achieve buffer exchange. To address whether BEVs isolated by these two methods lead to different host responses, we isolated Escherichia coli (E. coli) and Staphylococcus aureus BEVs from bacterial culture media using ultracentrifugation and our e-poly- L-lysine-based method, respectively. Then we stimulated THP-1 cells with the isolated BEVs and the global transcript profiles were evaluated by the RNA-seq technique. Compared with the unstimulated THP-1 cells, BEV isolated by both methods could trigger a striking alteration in the gene expression pattern. Among the functions significantly enriched by these DEGs were immune response and leucocyte chemotaxis. The results indicated that the BEVs isolated by the e-PL-based method also retained the in vitro biological activity as the commonly used ultracentrifugation. Overall design: Totally 15 samples were analyzed, including unstimulated THP-1 cells as controls, THP-1 cells treated with E. coli BEVs isolated by ultracentrifugation/e-PL-based method and S. sureus BEVs isolated by ultracentrifugation/e-PL-based method.