| Summary: | Immunocytochemistry revealed that Fasciola hepatica cathepsin L (FheCL) is widely expressed in nerves and putative myocytons in adult worms. Significantly, some FheCL immunostaining co-localised with that for FMRFamide-like peptides (FLPs) which are known to modulate neuromuscular function in flatworms. These observations drove subsequent efforts towards investigating the link between FheCL and FLPs. Next, high performance liquid chromatography and mass spectrometric methods were used to demonstrate that recombinant FheCL could efficiently cleave the flatworm neuropeptides GYIRFamide and GNFFRFamide, generating inactive peptide fragments. Further efforts examined the effects of the cathepsin L inhibitor, ZPhe- Ala-diazomethylketone (DMK) on the excitatory actions of GYIRFamide in F. hepatica muscle strips. . Although GYIRFamide was found to have excitatory actions, these were not enhanced in the presence of DMK. Subsequent efforts focussed on the development ofRNA interference (RNAi) in F. hepatica as a tool to interrogate gene function. These efforts resulted in the effective silencing of bOtll FheCL and cathepsin B (FheCB) in newly excysted juveniles (NEls) - tlle first demonstration of gene silencing in liver fluke. Furthermore, the effects of silencing on phenotype were monitored by assessing the ability of NEls to penetrate rat gut sacs. This behaviour was severely disrupted in both FheCL- and FheCB-silenced NEls, implicating both in gut penetration by the infective stage. Finally, efforts focussed on exploiting the RNAi breakthrough to investigate the proposed link between cathepsin L and FLP signalling in fluke. RNAi was used to silence FheCLI and the levels of fluke FaRP were monitored by quantitative immunocytochemistry. The silencing of FheCLl resulted in a significant elevation of FLP-immunostaining, indicating that FLPs were accumulating in fluke tissue following cathepsin L-RNAi. These observations provide compelling evidence to link cathepsin Land neuropeptide signal termination in F. hepatica - the first report of neuropeptidase activity being associated with a cathepsin protease.
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