Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity

Abstract

Prokaryotes release membrane vesicles (MVs) with direct roles in disease pathogenesis. MVs are heterogeneous when isolated from bacterial cultures so Density Gradient Centrifugation (DGC) is valuable for separation of MV subgroups from contaminating material. Here we report the technical variability and natural biological heterogeneity seen between DGC preparations of MVs for Mycobacterium smegmatis and Escherichia coli and compare these DGC data with size exclusion chromatography (SEC) columns. Crude preparations of MVs, isolated from cultures by ultrafiltration and ultracentrifugation were separated by DGC with fractions manually collected as guided by visible bands. Yields of protein, RNA and endotoxin, protein banding and particle counts were analysed in these. DGC and SEC methods enabled separation of molecularly distinct MV populations from crude MVs. DGC banding profiles were unique for each of the two species of bacteria tested and further altered by changing culture conditions, for example with iron supplementation. SEC is time efficient, reproducible and cost effective method that may also allow partial LPS removal from Gram-negative bacterial MVs. In summary, both DGC and SEC are suitable for the separation of mixed populations of MVs and we advise trials of both, coupled with complete molecular and single vesicle characterisation prior to downstream experimentation.

Extracellular Vesicles with Possible Roles in Gut Intestinal Tract Homeostasis and IBD

“…Microvesicles that separated at approximately 10 to 14,000 g are heterogeneous. In contrast, microvesicles separated at 100,000 g are homogeneous [17,[23][24][25]. Apoptotic bodies with large populations originate from membrane blebbing and cellular disassembly from cell fragmentation when the cytoskeleton breaks at the beginning of apoptosis.…”

Mycobacterial extracellular vesicles and host pathogen interactions

“… 2014a ; Dauros Singorenko et al. 2017 ). However, given the propensity of lipids to organize into vesicles-like structures, it is important to establish that recovered nanoparticles are not byproducts of cell lysis.…”

Electroporation of outer membrane vesicles derived from Pseudomonas aeruginosa with gold nanoparticles

“…In their native form, OMVs contain high amounts of lipopolysaccharides (LPS) that can elicit immune responses; thus, several studies have been done to modify bacterial strains into detoxified LPS to reduce the endotoxicity of OMVs [30]. In addition, the findings of Dauros Singorenko et al [31] have shown that samples purified using size exclusion chromatography contained lower amounts of LPS when compared to the crude sample. For further verification, cytotoxicity studies of the system developed here are planned for future work.…”