Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells - PubMed (original) (raw)
Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells
Eduardo de la Torre-Escudero et al. PLoS Negl Trop Dis. 2019.
Abstract
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 membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Fig 1. Qualitative and quantitative proteomic analyses of 15k EVs and 120k EVs released by adult F. hepatica suggest different intracellular origins of the EV sub-populations and identify specific surface proteins.
(A) Venn diagram showing the distribution of proteins qualitatively identified in the 15k and 120k EVs (>2 matched peptides in at least 2 out of 3 replicate samples). (B) The 180 surface proteins that were expressed by both the 15k and 120k EVs were subjected to quantitative analysis shown as a Volcano plot. The x-axis represents log2(fold-change) (15k/120k EVs) and the y-axis shows the −log10 (p value). The dashed red line indicates the significance threshold (p = 0.05). The vertical dashed black line indicates zero fold change. Green boxes represent proteins whose expression is significantly different between the two EV sub-populations whilst the orange circles represent proteins that did not significantly change. The position of proteins selected for further analysis by Western blot are shown. (C) Equal amounts (10μg total protein) of 15k and 120k EVs were analysed by Western blot using antibodies raised against F. hepatica proteins identified on the EV surface by LC-MS/MS. The relative expression patterns of the target proteins follows that of the quantitative proteomics analysis.
Fig 2. CD63 receptor-positive EVs are found in sub-gastrodermal regions of adult F. hepatica.
Tissue sections of adult F. hepatica were probed with anti-cathepsin L (A) or anti-CD63 receptor antibodies (B, D-E) or with rabbit pre-immune serum (C). A. Specific cathepsin L immunoreactivity (arrows) can be seen within the gastrodermal cells (G) that line the parasite gut. L, gut lumen. B. Strong CD63 receptor immunolabeling is observed in vesicles that occur as distinct clusters (arrows) just beneath the gastrodermal cell layer. C. No specific immunofluorescence can be seen in these areas when sections were probed pre-immune control sera. D-E. CD63 receptor-positive vesicles (V) can be seen along duct-like structures (arrows) that converge upon the vesicle clusters in sub-gastrodermal regions of the adult fluke. Scale bars 25 μm (A-C) or 10 μm (D-E).
Fig 3. RAL-A-positive EVs are found in sub-gastrodermal regions and other structures of adult F. hepatica.
Tissue sections of adult F. hepatica were probed with anti-RAL-A antibodies (A-B) or with rabbit pre-immune serum (C). A. Strong RAL-A immunolabeling is observed in vesicles that occur as distinct clusters (arrows) in extracellular locations just beneath the gastrodermal cell layer. Specific RAL-A immunoreactivity can be seen within the gastrodermal cells (G) that line the parasite gut. L, gut lumen. B. Faint immunofluorescence was observed throughout the tegumental syncytium (T) and in the underlying tegumental cell bodies (arrows). C. No specific immunofluorescence was observed when sections were probed with pre-immune control sera. Scale bars 50 μm (A) or 7.5 μm (B-C).
Fig 4. Comparison of 15k and 120k EV and tegument (Teg) mean lectin microarray responses.
Heat map and two-dimensional hierarchical clustering of scale-normalized lectin microarray profile data for all technical replicates. Data depicted in heat map was scaled to fit a 0–30,000 RFU window and clustered by average linkage, Euclidean distance method.
Fig 5. Competitive inhibition of lectin binding on the 120k EV surface.Arrowheads indicate significant (p ≤ 0.05) mean lectin microarray response changes imparted by competitive inhibition with 50 mM final concentrations of Lac, αManOMe or GlcNAc as indicated.
Fig 6. Comparison of the effects of glycosidase treatment on 120k EV lectin-binding profiles.
Heat map of 120k EV mean lectin microarray profiles imparted by treatment with _exo_-glycosidases, _endo_-glycosidases or glyco-amidase in comparison with their respective untreated, pH-adjusted controls. Mean data derived from total intensity mean adjusted replicate data (mannosidase, galactosidase (n = 5); PNGase F, Endo Tv, Endo H (n = 4); non-enzyme controls adjusted to pH 4.5, pH 5.5, pH 7.4 (n = 3)). All data subjected to two-dimensional hierarchical clustering by average linkage, Euclidean distance method.
Fig 7. De-glycosylation of F. hepatica EVs blocks their internalisation by host macrophages.
(A) RAW264.7 macrophages were incubated with PKH26-labelled 120k EVs for 3 h at 37°C and the cells were analysed by confocal microscopy. (B) Pre-treatment of labelled EVs with the glycosidases PNGase F and Endo H significantly reduced the uptake of the EVs when measured as fluorescence intensity relative to cells incubated with untreated EVs.
Fig 8. Host antiserum enhances internalisation of F. hepatica EVs by macrophages.
(A) Immunogenicity of F. hepatica EVs in F. _hepatica_-infected rats. Equal amounts (10μg total protein) of 15k and 120k EVs were analysed by Western blot using pre-infection (PI) sera and serum samples taken 7, 21 and 70 days post-infection. (B) RAW264.7 macrophages were incubated with PKH26-labelled 120k EVs for 3 h at 37°C and the cells were analysed by confocal microscopy. EVs pre-incubated with pre-infection rat serum were internalised by the macrophages as shown by the punctate red fluorescence observed throughout the cytoplasm. However, when EVs were pre-treated with 21-day rat serum the internalisation the EVs increased considerably. A similar, but less dramatic, effect was observed when EVs were pre-treated with the 70-day serum. Nuclei were stained with DAPI (blue). (C) These effects were statistically significant when measured as fluorescence intensity relative to cells treated with EVs pre-incubated with pre-infection serum.
Fig 9. Antibodies raised against specific F. hepatica EV surface proteins also influence uptake by macrophages.
(A) RAW264.7 macrophages were incubated with PKH26-labelled 120k EVs for 3 h at 37°C and the cells were analysed by confocal microscopy. Pre-treatment of labelled EVs with antibodies raised against specific F. hepatica proteins (DM9, myoferlin and CD63 receptor) resulted in a greater level of internalisation by macrophages compared to control EVs (left panels). (B) Internalisation of myoferlin- and CD63 receptor-treated EVs was statistically significant when measured as fluorescence intensity relative to cells incubated with untreated EVs.
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