Analysis of the Escherichia coli extracellular vesicle proteome identifies markers of purity and culture conditions

Abstract

Bacteria release nano-sized extracellular vesicles (EVs) into the extracellular milieu. Bacterial EVs contain molecular cargo originating from the parent bacterium and have important roles in bacterial survival and pathogenesis. Using 8-plex iTRAQ approaches, we profiled the EV proteome of two Escherichia coli strains, uropathogenic (UPEC) 536 and probiotic Nissle 1917. For these strains, we compared the proteome of crude input EVs prepared by ultracentrifugation alone with EVs purified by either density gradient centrifugation (DGC) or size exclusion chromatography (SEC). We further compared the proteome of EVs from bacterial cultures that were grown in iron-restricted (R) and iron-supplemented (RF) conditions. Overall, outer membrane components were highly enriched, and bacterial inner membrane components were significantly depleted in both UPEC and Nissle EVs, in keeping with an outer membrane origin. In addition, we found enrichment of ribosome-related Gene Ontology terms in UPEC EVs and proteins involved in glycolytic processes and ligase activity in Nissle EVs. We have identified that three proteins (RbsB of UPEC in R; YoeA of UPEC in RF; BamA of Nissle in R) were consistently enriched in the DGC- and SEC-purified EV samples in comparison to their crude input EV, whereas conversely the 60 kDa chaperonin GroEL was enriched in the crude input EVs for both UPEC and Nissle in R condition. Such proteins may have utility as technical markers for assessing the purity of E. coli EV preparations. Several proteins were changed in their abundance depending on the iron availability in the media. Data are available via ProteomeXchange with identifier PXD011345. In summary, we have undertaken a comprehensive characterization of the protein content of E. coli EVs and found evidence of specific EV cargos for physiological activity and conserved protein cargo that may find utility as markers in the future. Abbreviation: DGC: density gradient centrifugation; DTT: 1,4-dithiothreitol; EV: extracellular vesicles; FDR: false discovery rate; GO: Gene Ontology; R: iron-restricted; RF: iron-supplemented; iTRAQ: isobaric tags for relative and absolute quantitation; OMV: outer membrane vesicle; SWATH-MS: sequential window acquisition of all theoretical mass spectra; SEC: size exclusion chromatography.

Horizontal Gene Transfer: From Evolutionary Flexibility to Disease Progression

“…In nature, transformation, transduction, and conjugation are the principal mechanisms of HGT. Other mechanisms involve gene transfer agents (GTAs), nanotubes, and membrane vesicles (MVs) [which are also called extracellular vesicles (EVs) or exosomes] (Figure 1; Dauros Singorenko et al, 2017;Hong et al, 2019). Accordingly, the production of recombinant DNA can be achieved by directly transforming DNA or RNA from a donor, and subsequent integrating of foreign genetic material into the recipient cell's genome.…”

Lactobacillus casei extracellular vesicles stimulate EGFR pathway likely due to the presence of proteins P40 and P75 bound to their surface

“…Also, EVs from probiotic bacteria have comparable effects to whole bacteria, increasing the number of T reg cells, in case of L. rhamnosus JB-1 9 , and promoting the survival of Caenorhabditis elegans exposed to vancomycin-resistant E. faecium by L. plantarum WCFS1 10 . Analysis of EVs from probiotic lactobacilli, like Lactobacillus rhamnosus , L. plantarum , L. reuteri and L. casei , showed that they contained glycolytic enzymes, chaperonins 9 , 11 – 14 and ligase functions 15 . In addition to proteins and membrane phospholipids, EVs from Gram-positive bacteria also contain lipoteichoic acid (LTA).…”

Effect of the Extracellular Vesicle RNA Cargo From Uropathogenic Escherichia coli on Bladder Cells

“…Moreover, while the quantity of EVs produced by extraintestinal pathogenic E. coli is not affected by iron starvation, changes in the EV proteome reflect a stressed cellular state (Chan et al, 2017). Finally, we have seen that iron-replete conditions cause UPEC EVs to be enriched in outer membrane components, while low iron conditions caused no enrichment, suggesting evidence of two different EV biogenesis mechanisms or differential packaging dependent on iron availability (Hong et al, 2019).…”

“…Our previous investigations into proteome of EVs showed that UPEC 536 EVs are enriched in rRNA-binding proteins in contrast to Nissle EVs, which are enriched in proteins involved in glycolytic process and ligase activity, leading us to believe that differential packaging of RNA may be causing some of the differences seen between UPEC and Nissle EV-RNA (Hong et al, 2019). A key investigation of this study was to delineate the effects of E. coli EV-RNA on host cell responses.…”

“…Importantly, we identified that UPEC-derived EVs carried proteins enriched in ribosome-related processes, themselves carriers of RNA (Hong et al, 2019). RNA is considered an important and complex communication signal within eukaryotes, prokaryotes, or trans-kingdom (Zeng et al, 2019).…”