Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Collins, Shannon M.; Nice, Justin B.; Chang, En Hyung; Brown, Angela C.

doi:10.3791/62429-v

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“…Density gradient centrifugation (DGC) and size exclusion chromatography (SEC) have increasingly been introduced into EV purification workflows, with the former more commonly used to purify BEVs, whereas the latter is most frequently used for eukaryotic EVs (Akbar et al., 2022; Collins et al., 2021). These methods each have advantages and disadvantages (Hong et al., 2019; Konoshenko et al., 2018); a major strength of both methods being their ability to sub‐fractionate heterogenous populations of BEVs by density and size which is important for the characterisation of distinct BEV subpopulations (Dauros Singorenko et al., 2017; Hong et al., 2019).…”

Section: Introductionmentioning

confidence: 99%

A new isolation method for bacterial extracellular vesicles providing greater purity and improved proteomic detection of vesicle proteins

Steele

Ying

et al. 2023

J of Extracellular Bio

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Contaminants within cell culture media often co-isolate with eukaryotic extracellular vesicles (EVs) thus affecting their biological properties. It has yet to be investigated if this is also true for bacterial EVs (BEVs), especially for organisms grown in complex culture media containing animal-derived products. To address this question, we isolated BEVs from the fastidious bacterium Helicobacter pylori grown in either standard Brain Heart Infusion (BHI) medium or BHI depleted of animal-derived products (D-BHI). We show that BEVs prepared from bacteria grown in D-BHI medium have similar morphologies, size ranges and yields to those prepared from standard medium. Similarly, no differences were found in the ability of H. pylori BEVs to induce IL-8 responses in epithelial cells. However, H. pylori BEVs prepared from D-BHI medium were of higher purity than those prepared from standard medium. Importantly, proteomic analyses detected 3.4-fold more H. pylori proteins and 10-fold fewer bovine-derived proteins in BEV samples prepared from D-BHI rather than the standard method. Fifty-seven H. pylori proteins were uniquely detected in BEV samples prepared from D-BHI. In conclusion, we have described an improved method for BEV isolation. Furthermore, we demonstrate how animal-derived products in bacteriological culture media may adversely affect proteomic analyses of BEVs. K E Y WO R D S bacterial extracellular vesicles, Helicobacter pylori, outer membrane vesicles, proteomics  INTRODUCTIONCells belonging to all three domains of life release spherical membrane-bound nanostructures, known as extracellular vesicles (EVs) (Gill et al., 2019). The EVs produced by bacteria (BEVs) are 20-400 nm in size and contain a range of products, such as proteins, nucleic acids, lipopolysaccharide and peptidoglycan, which may contribute to infection and to horizontal gene transfer (Gao & van der Veen, 2020;Kaparakis-Liaskos & Ferrero, 2015) BEVs are generally isolated by either ultracentrifugation or ultrafiltration, though both techniques result in relatively crude preparations (Klimentová & Stulík, 2015). Ultracentrifugation has been reported to promote vesicle and extra-vesicle proteinThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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