ABSTRACT
Transcriptome data supports the notion of a Platyhelminthes-specific protein family that is characterized by combination of two N-terminal EF-hands and a C-terminal dynein light chain-like domain. Family members in schistosomes induce an IgE response that has been connected with resistance to reinfection in schistosomiasis and is considered as a marker of protection. In the present study, we have compared three homologs of the liver fluke Fasciola gigantica for their immunological properties in mouse. Antisera raised against the recombinant proteins detected the native proteins in tegumental type tissues and epithelial linings of excretory system and intestinal tract. The recombinant EF-hand domains induced strong IgG and IgE responses in immunised mice while only weak to moderate responses were observed against the complete recombinant proteins and their DLC-like domains. Parasite crude worm and tegumental extract antisera reacted predominantly with one isoform and its EF-hand domain. Sera of F. gigantica infected mice did not react with the recombinant proteins. The RNA products of the three genes were detected from the metacercarial up to the adult stage. These observations indicate that the investigated EF-hand proteins are not at the frontier of humoral host/parasite interaction in acute fascioliasis gigantica in mouse but are acting as intracellular proteins in tissues that interface with the parasite's environment or tubular tracts.
Subject(s)
Calcium-Binding Proteins/immunology , EF Hand Motifs/immunology , Fasciola/chemistry , Amino Acid Sequence , Animals , Antibodies, Helminth/biosynthesis , Antibodies, Helminth/blood , Blotting, Western , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Cattle , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fasciola/immunology , Fasciola/metabolism , Female , Immune Sera/immunology , Immunoglobulins/biosynthesis , Immunoglobulins/blood , Immunohistochemistry , Mice , Mice, Inbred ICR , Molecular Sequence Data , Phylogeny , Sequence AlignmentABSTRACT
A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.
