Abstract
BackgroundDeficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.MethodsIn this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.ResultsOur results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.ConclusionsThese data indicate that the Ca2+-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1324-9) contains supplementary material, which is available to authorized users.
Highlights
Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites
0.01; FhCaM2 = 1.5 ± 1.5 %, n = 5, P < 0.001; FhCaM3 = 4.0 ± 1.3 %, n = 5, P < 0.001; Fig. 3f ). These data showed that CaM transcript knockdown persists throughout this maintenance period; this persistence occurs even in the absence of dsRNA during maintenance, since worms were only exposed to dsRNA during the initial 4 h exposure soak. Such long-term maintenance of knockdown is consistent with our previous observations on RNA interference (RNAi) of liver fluke cathepsin B, L and glutathione transferase genes, in which we reported the persistence of knockdown for 21 days following a similar RNAi protocol [21]
We have demonstrated that CaMs are expressed widely in the parenchyma of the invasive juvenile life stage of F. hepatica
Summary
Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. Triclabendazole is the only flukicide with significant activity against juvenile liver fluke [6], the life-cycle stage that causes host tissue damage during its migration from the gut lumen, through the intestinal mucosa and hepatic parenchyma to the bile ducts. Novel flukicides, especially those with activity against juvenile fluke, are a pressing need. Longer acting responses, effector proteins cannot bind Ca2+ directly and require an intermediate Ca2+ sensing protein in order to initiate a biological response
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