Dataset Information

The surface proteome of extracellular vesicles from Fasciola hepatica

ABSTRACT: Helminth parasites secrete extracellular vesicles (EVs) as a means of exporting effector molecules into the host microenvironment. Once released, the parasite-derived EVs are internalised by host immune cells and trigger a range of biological effects including modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that may effect ligand-receptor interactions with the target host cell surface that initiate vesicle docking and subsequent internalisation. Here we used a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of adult F. hepatica EVs and mass spectrometry to identified the surface proteins.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Fasciola Hepatica (liver Fluke)

TISSUE(S): Protein Secretion, Secretory Cell

DISEASE(S): Fascioliasis

SUBMITTER: Mark Robinson

LAB HEAD: Mark Robinson

PROVIDER: PXD007782 | Pride | 2019-11-12

REPOSITORIES: Pride

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Publications

Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells.

de la Torre-Escudero Eduardo E Gerlach Jared Q JQ Bennett Adam P S APS Cwiklinski Krystyna K Jewhurst Heather L HL Huson Kathryn M KM Joshi Lokesh L Kilcoyne Michelle M O'Neill Sandra S Dalton John P JP Robinson Mark W MW

PLoS neglected tropical diseases 20190118 1

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membr ...[more]

PMID: 30657764

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Project description:Fascioliasis is a major neglected tropical disease that infects humans and ruminant species worldwide. Recent discoveries have identified that liver fluke extracellular vesicles are released into the host’s internal environment. To understand extracellular vesicle biology further, isolation of the extracellular vesicle is necessary for downstream applications. The most widely used purification method involves differential centrifugation however; there is no ‘gold standard’ to date of extracellular vesicle isolation. Recently, size exclusion chromatography has been used to successfully purify EVs. In this study, liver flukes were collected to compare size exclusion chromatography EV purification, to the widely accepted method of differential centrifugation. Transmission electron microscopy and atomic force microscopy showed that size exclusion chromatography extracellular vesicles were smaller in size and diversity than differential centrifugation purified extracellular vesicles. Protein concentration and western blotting suggested that size exclusion chromatography purification had a high EV purity to protein yield ratio in comparison to differential centrifugation. Mass spectrometry analysis discovered a greater amount of protein identifications and unique peptide hits in differential centrifugation purified EVs with less diverse functionality, than size exclusion chromatography purified EVs. However, gene enrichment analysis against the Fasciola hepatica genome showed comparable results between purification methods. Furthermore, western blotting revealed that glutathione s-transferase sigma could be a potential marker for liver fluke EVs. These results suggest that size exclusion chromatography purification produces a higher EV purity to protein yield ratio and maintains extracellular vesicle composition, so therefore is a better suited method for further downstream experimentation with liver fluke extracellular vesicles, compared to differential centrifugation.

Project description:Malignant germ-cell-tumours (GCTs) are characterised by microRNA (miRNA/miR-) dysregulation, with universal over-expression of miR-371~373 and miR-302/367 clusters regardless of patient age, tumour site, or subtype (seminoma/yolk-sac-tumour/embryonal carcinoma). These miRNAs are released into the bloodstream, presumed within extracellular-vesicles (EVs) and represent promising biomarkers. Here, we comprehensively examined the role of EVs, and their miRNA cargo, on (fibroblast/endothelial/macrophage) cells representative of the testicular GCT (TGCT) tumour microenvironment (TME). Small RNA next-generation-sequencing was performed on 34 samples, comprising representative malignant GCT cell lines/EVs and controls (testis fibroblast [Hs1.Tes] cell-line/EVs and testis/ovary samples). TME cells received TGCT co-culture, TGCT-derived EVs, and a miRNA overexpression system (miR-371a-OE) to assess functional relevance. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were overexpressed in all TGCT cells/subtypes compared with control cells and were highly abundant in TGCT-derived EVs, with miR-371a-3p/miR-371a-5p the most abundant. TGCT co-culture resulted in increased levels of miRNAs from the miR-371~373 and miR-302/367 clusters in TME (fibroblast) cells. Next, fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME (fibroblast/endothelial/macrophage) cells. TME (fibroblast/endothelial) cell treatment with EVs derived from different TGCT subtypes resulted in increased miR-371~373 and miR-302/367 miRNA levels, and other generic (eg, miR-205-5p/miR-148-3p) and subtype-specific (seminoma, eg, miR-203a-3p; yolk-sac-tumour, eg, miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), via direct mRNA downregulation and alteration of relevant pathways. TGCT cells communicate with nontumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, potentially contributing to tumour progression.

Dataset Information

The surface proteome of extracellular vesicles from Fasciola hepatica

Publications

Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells.

Similar Datasets

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