Fasciolosis, caused by liver fluke parasites of genus Fasciola, is responsible for reducing global agricultural yield in livestock animals and is a neglected tropical disease of humans. A limited portfolio of flukicide drugs are available, however treatment of the highly pathogenic acute stage is unique to the benzimidazole, triclabendazole (TCBZ). Reports of TCBZ-resistant Fasciola hepatica populations in cattle, sheep and humans, could undermine fluke control and necessitates the exploration of new treatments. The availability of genomic/transcriptomic datasets complemented by an in vitro culture platform and the amenability of F. hepatica to RNA interference (RNAi)-induced gene silencing permits the interrogation of putative novel drug targets. This thesis herein explores avenues for overcoming TCBZ-resistant phenotypes in juvenile F. hepatica, through impeding detoxification and regenerative processes. ATP-binding cassette (ABC) transporters are implicated in multidrug resistant cancers through the active export of drugs from cells. Twenty-four putative ABC transporter sequences were identified in the F. hepatica genome. Pharmacological inhibition and RNAi-induced silencing of selected ABC transporters potentiated the efficacy of TCBZ and TCBZ metabolites, which was shown to overcome resistance in vitro. The previous development of the in vitro culture platform highlighted neoblast-like cells to be the drivers for fluke growth. Here, it was shown that these neoblast-like cells exhibit stem cell-like features, including the capacity for self-renewal and asymmetric division. Through irradiation and silencing of the cell-cycle regulator histone-2b, it was shown that these cells could be selectively depleted, which undermined juvenile growth. Furthermore, it was shown that TCBZ and TCBZ metabolites hinder juvenile growth and development through the inhibition of neoblast-like cells – furthering our understanding of TCBZ’s mode-of-action and calling into question the relevance of stem-cell mediated regeneration in the resistance phenotype. Corroborating this hypothesis, TCBZ-resistant juveniles were found to possess increased proliferative potentials in comparison to susceptible fluke. Subsequently, it was shown that the TCBZ-susceptibility status of juveniles could be altered through changes in neoblastlike cell dynamics – highlighting neoblast-like cells as a likely source of drug targets. Finally, the F. hepatica kinome was explored for genes implicated in growth and development. This highlighted an orthologue of polo-like kinase 1, which was vital for maintaining the neoblast-like cell population. Overall, this thesis contributes to our understanding of TCBZ-resistance mechanisms and emphasises interring with detoxification and stem-cell mediated development as favourable candidates for drug intervention.
|Date of Award||Dec 2020|
- Queen's University Belfast
|Sponsors||Northern Ireland Department for the Economy|
|Supervisor||Nikki Marks (Supervisor), Aaron Maule (Supervisor) & James McKenna (Supervisor)|
- drug resistance