Characterisation of the Fasciola hepatica miRNome and an evaluation of its role in the host-parasite relationship

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The liver fluke, Fasciola hepatica, is recognised as one of the most successful parasites worldwide due to its remarkable capacity to infect every mammal it encounters. For this reason, liver fluke disease, or fasciolosis, has the widest geographical spread of any parasite disease and contributes to significant animal loss, particularly within the agricultural sector. Since the discovery of the post-transcriptional regulation of genes by micro(mi)RNA in the free-living worm Caenorhabditis elegans, a myriad of processes within worm biology are now linked to miRNAs. These concepts have catalysed interest in the contribution that miRNAs have on the dynamic shifts of the F. hepatica transcriptome and parasite survival within the host. In Chapter 1, the miRnome assemblies of early miRNA discovery projects were compared to determine knowledge to date. Examination of 38 miRbase miRNAs revealed that the revised miRNome was highly associated to the regulation of inflammatory events and innate mechanisms of pathogen recognition and expulsion by the host. These preliminary explorations were experimentally challenged in Chapter 2. Sequencing of miRNAs isolated from the peritoneal macrophages of F. hepatica infected mice revealed that specific Fasciola miRNAs were internalised by host macrophages. In particular, fhe-miR-125b was uncovered as a potent immune regulator due to its capacity to suppress the expression of a central signal transduction molecule Traf6 within the host, after functionalisation by mammalian Ago. The realisation of the complete F. hepatica miRnome in Chapter 3 expanded the number of F. hepatica miRNAs to 124 within intra-mammalian life stages; newly excysted juveniles (NEJs), immature and adult fluke, exposing a wider collection of isomiRs, life stage specific novel miRNAs and genomic clustering. By integrating the life stage miRnomes with their predicted targets within the transcriptomes of each life stage identified the key biological processes in metabolism, parasitism, and growth that were systematically targeted during parasite development within the host. With the expanded miRnome, the utility of parasite miRNAs as biomarkers of fasciolosis was explored, with diagnostic capabilities examined through RT-qPCR analysis of sera from infected sheep (Chapter 4). The collective outcomes of this research project have fostered new perspectives in F. hepatica research. These include, evolving methods of miRNA discovery; re-thinking the biogenesis of microRNAs; mapping the molecular events of parasite development; unveiling new mechanisms of host-parasite interplay, and advancing diagnostic techniques.
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