AbstractFasciolosis, resulting from infection with the digenetic trematode (flatworm) Fasciola hepatica, is of major global economic importance in ruminants, such as cattle and sheep, and is now recognised as an important zoonosis. Following ingestion by the mammalian host, the parasite migrates through the intestinal wall and develops within the host liver parenchyma and then the bile ducts. We found that the parasites express an array of temporally regulated cysteine peptidase inhibitors – cystatins – during this migration. The aim of this thesis was to characterise these cystatins and explore the biochemical and biological functions during infection of the mammalian host. Genome and associated stage-specific transcriptome/proteome analysis identified three single domain cystatin inhibitors in this parasite – FhStf-1, FhStf-2 and FhStf-3. Phylogenetic analysis of the phylum platyhelminthes revealed that these cystatins are Type 1 cystatins (stefins); however, uniquely, FhStf-2 and FhStf-3 also possess signal peptides - a characteristic more commonly associated with Type 2 cystatins – that would facilitate their secretion by the parasite into the mammalian host. Transcriptomic analysis revealed that the FhStf genes are differentially expressed throughout all life stages of the parasite from the metacercariae to the adult which we suggest relate to their function.
Extensive characterisation of recombinant FhStf-1, FhStf-2 and FhStf-3 showed that they are potent inhibitors of the major F. hepatica secreted Cathepsin L peptidases (Ki <1 nM – 6 nM) and of human cysteine peptidases L, K, S and B (Ki <1 nM – 6 nM). Immunoblot analysis showed the presence of FhStf-1, FhStf-2 and FhStf-3 in the excretion/secretion (ES) so all three inhibitors could be delivered into the host tissues. However, FhStf1 was also present within adult worm extracellular vesicle (EVs) and, therefore, we propose that this inhibitor could be delivered directly into host cells via EV-host cell communication. These studies correlate with immunolocalisation studies on newly excysted juveniles (NEJs) that identified FhStf1 on the tegument of the parasite and in the gut and FhStf-2 and FhStf-3 also in the gut and ‘secretory channels’. Therefore, the potent inhibition of the parasite-secreted and host cysteine peptidases, their localisation in tissue that interface with the host, and presence in the parasite EVs and secretions suggests a primary role for these F. hepatica stefins in the regulation of F. hepatica and host cysteine proteases. The protease/anti-protease balance may be critical in the regulation of parasite processes including penetration, feeding, development and immune evasion, as well as modulation of host innate cell proteases involved in antigen processing and presentation. The potential of up-setting the balance of this protease/anti-protease function was assessed in sheep vaccine trials. Our vaccination studies revealed that animals vaccinated with recombinant FhStf-1, FhStf-2, FhStf-3 and FhKT1 and orally challenged with F. hepatica metacercariae had a statistically significant weight gain when compared to non-vaccinated animals; a novel and economically important result warranting further analysis of this antigen combination in future vaccine trials.
Given that anti-allergic and anti-inflammatory potential has been seen in other helminth cystatins, assessing the immunomodulatory properties of FhStf-1, FhStf-2 and FhStf-3 would be an exciting future study and could lead to the discovery of novel therapeutics for the treatment of immune-related conditions like asthma.
|Date of Award
|Angela Mousley (Supervisor) & John Dalton (Supervisor)