The cellular and molecular origins of extracellular vesicles secreted by Fasciola hepatica

Abstract

Parasitic helminths maintain chronic infections in the host by secreting molecules that promote parasite survival. These molecules can be packaged into extracellular vesicles (EVs) which are internalised by host cells and exert potent immunomodulatory effects. Thus, blocking helminth (h)EV release is an attractive therapeutic approach to eliminate the parasite. However, this is hindered by our limited understanding of the mechanisms that govern hEV biogenesis and release. To begin to address this, a comparative genomics approach was used to determine whether established EV biogenesis pathways could operate in helminths. This revealed that canonical EV biogenesis pathways were broadly conserved, but helminth phyla diverged in their EV RNA loading proteins. As the sites of hEV secretion could give insights into their mechanisms of production, antibodies raised against proteins enriched in F. hepatica (Fh)EVs were used to identify their cellular origins in adult flukes. This analysis revealed multiple sites of FhEV production (the parenchyma, tegument, gastrodermal cells and protonephridial system) and indicated that further subtypes of FhEVs exist within the previously characterised 15K and 120K EV populations. Additionally, ultrastructural analysis of these tissues identified several potential mechanisms of FhEV formation and release. Oral sucker and excretory pore ligation of adult flukes in vitro demonstrated that the gut is the primary source of FhEVs, but the tegument and protonephridial system also contribute FhEV sub-populations to a lesser extent. Proteomic analysis of laser microdissected gut and tegument supported these findings and revealed proteins shared with FhEVs that are associated with EV biogenesis. Of those, sphingomyelinases were targeted using a chemical inhibitor (GW4869) to try to disrupt EV biogenesis in F. hepatica. This resulted in an 80% reduction in 120K EV secretion from flukes cultured in vitro, whereas 15K EV release was only moderately impacted, indicating that various EV biogenesis pathways operate in the parasite. Ultrastructural observation of GW4869-treated F. hepatica tissue showed severe disruption to the parenchyma, indicating that this could be a source of 120K EVs. In a pilot experiment, selected F. hepatica transcripts encoding EV biogenesis proteins were successfully silenced using RNAi in juvenile flukes, with nanoparticle tracking analysis identified as a potential phenotypic readout to show the inhibition of FhEV release by RNAi. This work establishes that F. hepatica releases multiple subtypes of EVs through different mechanisms and, despite the complexities of this process, demonstrates that targeted disruption of EV biogenesis is possible. As such, this body of work provides proof-of-concept for future studies investigating hEV biogenesis as a target for fluke control.

Date of Award	Dec 2019
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Mark Robinson (Supervisor) & Aaron Maule (Supervisor)