Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism

Abstract

The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host's blood. Despite their importance in the host-parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze-thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite.

Keywords: Fasciola hepatica; extracellular vesicle; helminth; proteome; secretion; trematode.

Figure 1

Different functional groups of proteins identified in the laser microdissected gut and tegument. (A) The percentage distribution of F. hepatica gastrodermal cell proteins within different functional groups based on their relative abundance. The number of matches corresponds to the number of different proteins that were detected in each category. (B) The number of proteins in gastrodermal cells associated with KEGG pathways. (C) The percentage distribution of F. hepatica laser microdissected tegument proteins within different functional groups based on their relative abundance. (D) The number of proteins in the LMD tegument associated with KEGG pathways.

Figure 2

Individual gastrodermal cells of adult F. hepatica undergo progressive disintegration. (A–D) Toluidine blue staining showing gastrodermal cells at different stages of disintegration. Arrows point to the rupturing cells, and arrowheads indicate the disintegrating apical cell membrane (A,B) and residual material (C,D). (E) Immunostaining against FhRal-A (green) and tyrosinated α-tubulin (magenta) in the region proximal to a ruptured gastrodermal cell. Cell nuclei were counterstained with DAPI (cyan). Tyrosinated α-tubulin localises to vesicular structures in the parenchyma (dashed arrows) and packed within a specific cell (asterisk). FhRal-A localises to discrete puncta within gastrodermal cells which are concentrated at the cell apices (arrowheads), as well as groupings of vesicular structures (V) dispersedly packed within the sub-gastrodermal parenchyma. FhRal-A does not localise to the same cell labelled by the anti-tyrosinated α-tubulin antibody (asterisk). (F) Toluidine blue staining of the section shown in E shows that the structures positive for FhRal-A (V) stain dark blue (displaying basophilia), whilst those positive for tyrosinated α-tubulin (dashed arrows, asterisk) stain a lighter blue (displaying less basophilia). G, gut; L, lumen. Scale bars: 7.5 µm (A,B,D,E), 25 µm (C,F).

Figure 3

Transmission electron micrographs showing progressive breakdown of the gastrodermal cells of adult F. hepatica. (A) The apex of a columnar gastrodermal cell. The lamellae (La) are spaced far apart, and the apical plasma membrane (PM) appears very thin. (B) The apical plasma membrane is broken (arrows) and the cell has begun to leak apical cytoplasm (Cy) into the gut lumen. (C) High-power image of a break (arrow) in the apical plasma membrane. (D) An array of membrane fragments, mitochondria, and vesicles are released into the gut lumen. GER, granular endoplasmic reticulum; M, mitochondria. Scale bars: 5 µm (A,B), 2.5 µm (C), and 10 µm (D).