Transcriptional analysis of THP-1 cells stimulated by bacterial extracellular vesicles enriched by ultracentrifugation and an ε-poly- L-lysine-based method
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ABSTRACT: We propose an isolation strategy based on a broad-spectrum antimicrobial epsilon-poly-L–lysine (ε-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 ε-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 ε-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 ε-PL-based method also retained the in vitro biological activity as the commonly used ultracentrifugation.
Project description:Both Gram-negative and Gram-positive bacteria can release vesicle-like structures referred 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 cell, thus modulating the development and function of the innate immune system. To clarify the compositions and biological functions of BEVs, these vesicles need to be collected 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 pH and ionic strength of the media, followed by ultrafiltration step to remove ε-PL and achieve buffer exchange. Protein compositions of the BEVs enriched by ultracentrifugation and ε-PL-based method are measured using LC-MS/MS. The resultant data indicate that protein compositions of the ε-PL-precipitated BEVs are comparable to those purified by ultracentrifugation.
Project description:Transcriptional analysis of THP-1 cells stimulated by bacterial extracellular vesicles enriched by ultracentrifugation and an ε-poly- L-lysine-based method
Project description:ε-poly-L-lysine (ε-PL) is a high value, widely used natural antimicrobial peptide additive for foods and cosmetic products that is mainly produced by S. albulus. In previous work, we developed the high-yield industrial strain S. albulus WG-608 through successive rounds of engineering. Here, we use integrated physiological, transcriptomic, and proteomics association analysis to resolve the complex mechanisms underlying high ε-PL production by comparing WG-608 with the progenitor strain M-Z18. Our results show that key genes in the glycolysis, glyoxylate, and L-lysine biosynthesis pathways are differentially upregulated in WG-608, while genes in the biosynthetic pathways for fatty acids, various branched amino acids, and secondary metabolite by-products are downregulated. This regulatory pattern results in the introduction of more carbon atoms into L-lysine biosynthesis and ε-PL production. Furthermore, transcriptional and translational upregulation of genes involved in the tricarboxylic acid cycle, oxidative phosphorylation, and pentose phosphate pathway also increase the pools of available NADH, ATP, and NADPH. In addition, significant changes in the regulation of DNA replication, transcription, and translation, two component systems, and quorum sensing may facilitate the adaptability to environmental pressure, thus further regulating the ε-PL biosynthesis. This study enables comprehensive understanding of the biosynthetic mechanisms of ε-PL in S. albulus WG-608, while providing a theoretical foundation development of advanced Streptomycetaceae microbial cell factories.
Project description:Total RNA secreted or excreted by C. elegans incubated for 5h or 24h in M9 culture media and isolated. Extracellular vesicles were isolated by differential ultracentrifugation, and subjected to miRNA sequencing.
Project description:IntroductionBacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. MethodsTo gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment.ResultsParticle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions.ConclusionsOur study showed that the choice of medium and the duration of culturing significantly influence both E.coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E.coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.
Project description:This study examined microRNA expression of cells maintained in media prepared with replete serum (EVR) or with serum depleted of extracellular vesicles (EVs) by either of two methods: standard overnight ultracentrifugation (UC-EVD) or a proprietary method used by Thermo Fisher (TF-EVD).
Project description:The helminth Acanthocheilonema viteae serves as model organism for research on parasitic filarial nematodes. Total RNA secreted or excreted by 1500 adult female and male A. viteae over 3 weeks was isolated from culture media previously processed by differential ultracentrifugation, and subjected to miRNA sequencing.
Project description:Thy1 + cells isolated from male F344 rats with Dgalactosamine-induced liver injury were cultured for one week. Bone marrow mesenchymal cells isolated from from the femurs and tibias of a 5-week old male F344 rat. Small hepatocytes (SH) and mature hepatocytes (MH) were isolated from normal livers of 7-week old male F344 rat, Extracellular vesicles (EVs) were separedt from culture media by ultracentrifugation.
Project description:THP-1 cells were cultured and EVCs were isolated after treatment with dexamethasone, LPS and a mixture of dexamethasone/LPS or non treated (control) by ultracentrifugation. Proteins were separated by 1D SDS-PAGE, tryptic digested and analyzed by nanoLC-MSMS
Project description:After isolation, islets were cultured in a serum-exosomes-free culture media for one week. Collected culture media were centrifuged first at 300g for 20 min to pellets cells and then at 10000g for 20 min to discard dead cells and cell debris. Exosomes were then isolated from the supernatant by ultracentrifugation at 110000g for 70 min. Exosomes were collected in a minimal volume of PBS,and added three times the volume of Trizol LS to extract exosomes RNA.