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

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

Uropathogenic <i>Escherichia coli</i> Releases Extracellular Vesicles That Are Associated with RNA

<div><p>Background</p><p>Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection.</p><p>Methods and Results</p><p>Using the uropathogenic <i>Escherichia coli</i> (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of <i>csrC</i>. It was estimated that 1% of MV RNA cargo is delivered into cultured cells.</p><p>Conclusions</p><p>These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process.</p></div

Bacteria release membrane vesicles that associate with protein and RNA.

<p>A. Contrast electron microscopy of budding UPEC with a white arrow pointing to the released MV. B. Contrast electron microscopy of isolated vesicle preparation by ultracentrifugation. C. Coomassie stained protein gel of MVs isolated from UPEC D. Agilent Tapestation gel for RNA from three replicate MVs isolated from UPEC plus one donor cell RNA. Intact ribosomal bands are labelled 23S and 16S as are the small RNA fragments. The green line marks the internal loading marker.</p

UPEC MV and their RNA cargo are delivered into human bladder cells <i>in vitro</i>.

<p>A. Confocal image of 5637 cells (red) stained with DAPI blue nuclear stain after treatment for 15 hr with 50μg/mL PKH26 green vesicles. B. Confocal image of 5637 cells (red) after treatment for 15 hr with 50μg/mL 5EU-labelled RNA vesicles (green with an arrow) C. Droplet digital RT-PCR validation of UPEC <i>csrC</i> rRNA into cells treated with 100μg/mL MVs across a 48 hr timeframe. Each treatment timepoint repeated in at least duplicate as represented by a closed spot. Red dotted lines mark the copies of <i>csrC</i> per μL from a standard curve of MV protein equivalents shown on the left.</p

Populations of RNAs found in vesicles from three size separated libraries.

<p>RFAM and RefSeq annotated sequencing reads classified by RNA-type after alignment to UPEC 536 genome.</p