Molecular characterisation of EmTFP250: a novel member of the TRAP protein family in Eimeria maxima.

We have previously described a high molecular mass, asexual stage antigen from Eimeria maxima (EmTFP250), implicated as a target of maternal antibodies produced by breeding hens infected with this protozoan parasite. Following partial purification of the protein by ion exchange chromatography, N-terminal and internal peptide sequences were generated and used in the design of degenerate PCR primers. Using a rapid amplification of cDNA ends PCR-based strategy, the cDNA encoding EmTFP250 has been cloned and sequenced. Translation predicts a mature polypeptide with a molecular mass of 246kDa and an isoelectric point of 4.2. Analysis of the amino acid sequence has revealed a novel member of the TRAP (thrombospondin-related anonymous protein) family, containing 16 thrombospondin type-1 repeats and 31 epidermal growth factor-like calcium binding domains. EmTFP250 also contains two low complex, hydrophilic regions rich in glutamic acid and glycine residues, and a transmembrane domain/cytosolic tail associated with parasite gliding motility that is highly conserved within apicomplexan microneme proteins. The protein has 61% identity (71% similarity) with EtMIC4, a 218kDa microneme protein of Eimeria tenella also rich in epidermal growth factor-like and thrombospondin type-1 domains. Using Southern blotting, the gene encoding EmTFP250 has been determined to be present as a single copy within the genome, and reverse transcriptase-PCR has shown that expression is confined to the asexual stages of development. By employing a PCR-based method, a region of the E. maxima Houghton strain EmTFP250 gene was found conserved in Australian isolates of several (at least four) Eimeria species that parasitise chickens. The characterisation of EmTFP250 adds to the expanding apicomplexan TRAP family and suggests a functional significance for the protein.