AbstractFasciola spp have significant impacts on human and animal health worldwide and are estimated to cost the agri-food industry billions of US$ every year. The treatment of fascioliasis/fasciolosis relies heavily on Triclabendazole (TCBZ), the only flukicide active against immature and mature stages of the liver fluke. Widespread reports of drug resistance coupled with increasing infection forecasts (due to climate change and the intensification of farming), highlight the requirement for a better understanding of drug resistance mechanisms and for novel control methods. This study integrates newly developed investigatory platforms, including bioinformatic analysis of a new Fasciola hepatica genomic resource and the application of RNA interference (RNAi) methodology, to investigate diverse aspects of liver fluke biology. In particular, new data on the diversity and expression of liver fluke ABC transporters has revealed previously unrecognised diversity/complexity of this protein superfamily and suggest a considerable expansion of subfamilies associated with multi-drug resistance (MDR). ABC transporter family members, specifically FHMDR2 and FhMRPl, were found to be responsive to TCBZ/TCBZ-metabolite exposure suggesting that they may play a role in the worms response to drug treatment. Further, RNAi interrogation of FhMDR2/FhMRPl roles in resistant fluke isolates demonstrate that silencing these ABC transporters increases drug sensitivity, which is consistent with reduced drug efflux and supports their hypothesized role in drug resistance. Additionally, an in vitro assay was developed to exemplify differential phenotypic responses to TCBZ in newly excysted juveniles (NEJs) from clonal populations of TCBZ-resistant and -susceptible fluke. Further work utilised RNAi to probe the potential of liver fluke calmodulins (CaMs) as new control targets. These efforts identified a reduced- growth phenotype in NEJs associated with CaM-specific RNAi that matched the phenotypes induced by exposure to calmodulin inhibitors, validating the specificity of both the RNAi and the inhibitory drugs. Immunocytochemical localisation data also suggested CaMs are expressed throughout the parenchyma in NEJs and are potentially located in myocytons. These data support the candidature of calmodulins as novel drug targets and further validate the use of RNAi as a tool for functional genomics in liver fluke.
|Date of Award||Dec 2014|
|Supervisor||Aaron Maule (Supervisor) & Angela Mousley (Supervisor)|
Using post-genomics technologies to advance understanding of liver fluke therapeutics
McCammick, E. (Author). Dec 2014
Student thesis: Doctoral Thesis › Doctor of Philosophy