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

Abstract

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.

Differential temporal release and lipoprotein loading in B. thetaiotaomicron bacterial extracellular vesicles

“…To minimise these effects, chemically defined media that is free of vesicle contaminants should be used. Alternatively, if rich media is necessary, vesicles should be depleted prior to use, as recently proposed (Le et al, 2023). By utilising a chemically defined media (BDMr6) this study demonstrates that B. thetaiotaomicron releases distinct sub-populations of vesicles.…”

Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains

“…The BHI broth used as a bacterial growth broth in this study contains components from beef heart and calf brains and may include vesicles and vesicle‐like structures (Le et al, 2023). We did not find any vesicle‐like particles in the negative control samples with TEM, but we cannot conclude that the broth is vesicle‐free.…”

“…Therefore, it is not possible to completely distinguish the different particle populations from each other (Giebel & Helmbrecht, 2017; Guerreiro et al, 2018). Le et al (2023) detected dark structures on TEM pictures of EV‐like particles (EVLPs) from BHI, but after using eukaryotic EV markers they concluded that the EVLPs in BHI probably are large protein aggregates and not true EVs. Finally, several other factors can affect the NTA results, among them the potential fusion of vesicles (Grava et al, 2022).…”

Campylobacter jejuni extracellular vesicles harboring cytolethal distending toxin bind host cell glycans and induce cell cycle arrest in host cells

“…C. jejuni EVs were isolated as described ( 33 ) with some modifications. In brief, C. jejuni strains were inoculated to an optical density (OD 600 ) of 0.1 in depleted Brain Heart Infusion (D-BHI; BD Bacto) broth containing Skirrow’s selective supplement and tetracycline (10 µg/mL) or kanamycin (20 µg/mL), as required.…”

“…EVs (100 µg/mL) were visualized by negative staining on the FEI Tecnai Spirit electron microscope (Fei Company), as described previously ( 33 ). Images were randomly selected across the grids at 42,000 x magnification.…”