Fasciola hepatica hemoglobin: isolation, characterisation and induction of protective immune response in cattle

A hemoprotein released in vitro by Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S200 followed by ion exchange chromatography on DEAE sepharose. Absorption spectra studies characterised the molecule as hemoglobin. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica hemoglobin and other vertebrate or invertebrate hemoglobins. Immunolocalisation studies demonstrate that the hemoglobin is present in the vitelline glands and excretory tubules of mature flukes. The hemoglobin was shown to be highly immunogenic in F. hepatica infected bovines. Hemoglobin was included in a cattle vaccine trial to determine its immunoprophylactic potential. Vaccination with partially purified hemoglobin (Hf) yielded a significant level of protection (43.8%) against challenge infection. Protective immunity was increased to 72.4% when Hf was combined with the liver fluke cysteine protease CL2. Vaccination with Hf and CL2 also resulted in reduced liver damage as assessed by serum GLDH and 7GT. Furthermore, eggs recovered from both vaccine groups showed reduced viability. This anti-embryonation effect of vaccination was particularly evident in the group that received Hf / CL2 where >98% of recovered eggs did not embryonate to miracidia. Although both vaccine preparations induced high antibody titres which were boosted following the challenge infection, there was no correlation between antibody titre and protection. The results of these trials demonstrate that Hf and CL2 could form the basis of a molecular vaccine that would not only reduce parasite burden but would also prevent transmission of liver fluke disease. Using sera from cattle vaccinated with Hf to screen an adult F. hepatica cDNA library, genes encoding p tubulin and the novel antioxidant, peroxiredoxin were isolated. The presence of these proteins in the immunising fraction may have contributed towards the induction of the protective response. Peroxiredoxin activity was demonstrated in fluke extracts as the ability to protect glutamine synthetase from oxidative damage by mixed iron thiol oxidation systems. The antioxidant enzyme may play an important role in protecting against oxidative stress in the fluke

Conserved role for 14-3-3ϵ downstream of type I TGFβ receptors

AbstractSchistosoma mansoni receptor kinase-1 (SmRK1) is a divergent type I transforming growth factor β (TGFβ) receptor on the surface of adult parasites. Using the intracellular domain of SmRK1 as bait in a yeast two-hybrid screen we identified an interaction with S. mansoni 14-3-3ϵ. The interaction which is phosphorylation-dependent is not specific to schistosomes since 14-3-3ϵ also binds to TβRI, the human type I TGFβ receptor. 14-3-3ϵ enhances TGFβ-mediated signaling by TβRI and is the first TβRI-interacting non-Smad protein identified that positively regulates this receptor. The interaction of 14-3-3ϵ with schistosome and human TβRI suggests a conserved, but previously unappreciated, role for this protein in TGFβ signaling pathways

Functional expression of fasciola hepatica cathepsin l1 in saccharomyces cerevisiae

A cDNA encoding the complete precursor of a Fasciola hepatica cathepsin L protease was isolated and sequenced. Functionally active enzyme was expressed and secreted by Saccharomyces cerevisiae transformed with a plasmid carrying the complete gene. Experiments with temperature-sensitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway. Yeast transformed with a truncated gene, which lacked the pre-peptide-encoding and most of the pro-peptide-encoding sequences, did not express functionally active enzyme. The yeast-expressed enzyme exhibited physicochemical properties in common with the native enzyme including, pH optimum for activity, stability at 37 degrees C and ability to cleave gelatin and immunoglobulin. Enzyme kinetic data showed that the native and yeast-expressed cathepsin L1 have similar specificities for substrates with hydrophobic residues in the P-2 position. This is the first report of the functional expression of a cathepsin L proteinase in S. cerevisiae that did not require the use of yeast secretory signal sequences