Bone Morphogenic Proteins of Fasciola hepatica: Potential Roles in Fluke Physiology and Resistance to Triclabendazole

Abstract

Fasciola hepatica, the liver fluke, is a globally-distributed trematode parasite infecting multiple mammalian species, particularly ruminants, as well as humans. As such, it represents a significant economic burden in livestock, and is classified as a (re-) emerging neglected tropical disease by the World Health Organization. No commercial vaccine currently exists to control fluke, and resistance against the main anthelminthic treatment (triclabendazole, TCBZ) is on the increase. Bone morphogenic proteins (BMPs) are the largest subfamily of the transforming growth factor-β (TGF-β) group of molecules. They may have roles in dorsal-ventral patterning, organogenesis and cell differentiation in the parasite, with putative developmental and immune functions that allow a shift towards immunotolerance by the host. As such, they might be suitable targets for immunoprophylaxis and could be involved in the mechanism of TCBZ resistance. Two BMP genes are encoded in the F. hepatica genome, FhBMP3 and FhBMP15, as well as a gene encoding the TGF-β-like protein FhTLM, which has already been characterised. The expression of BMP genes throughout the life cycle of F. hepatica and the closely related Fasciola gigantica was analysed from publicly available RNA-Seq datasets. Generally, expression was found to be low in eggs and miracidia but increased in juvenile stages before a steep decline in adults; qRT-PCR assays revealed that FhBMP3 is expressed at a higher level than FhBMP15 in the mature parasites. Experiments using human TGF-β signalling phosphorylation arrays demonstrated that fluke proteins could be detected successfully, but incubation of adult flukes with recombinant FhBMPs did not affect TGF-β signaling. However, adult flukes from cattle vaccinated with FhTLM were significantly smaller than control flukes and exhibited elevated levels of several phosphorylated TGF-β signaling pathway proteins. In comparisons of archived TCBZ-resistant or -susceptible adult flukes treated with TCBZ during experimental infections of sheep, consistent changes in FhBMPs were not observed at the RNA level, and the proteins could not be detected by mass spectrometry. However, global shotgun proteomic analysis revealed significant regulation of pathways involved in endoplasmic reticulum stress, the unfolded protein response and iron binding following TCBZ exposure in susceptible compared with resistant isolates, but not in proteins encoded by genes in the resistance locus recently identified from the same in vivo study by bulked segregant analysis.