Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion

Background: The thrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate in several apicomplexan parasites. However, this protein and members of this family have not been characterized yet in Babesia bigemina , one of the most prevalent species causing bovine babesiosis. Methods: The Babesia bigemina TRAP-1 ( BbiTRAP-1 ) gene of 3186 bp was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through the search of the TSP-1 adhesive domain, the hallmark motif in both proteins. Structural modelling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at ~107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody test (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by ELISA and invasion assays. Results: TRAP family has 3 members in B. bigemina (BbiTRAP-1-3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1) and cytoplasmic C-terminus domains along with transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site (MIDAS) for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units is present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed expression of BbiTRAP-1 by blood stage parasites. Partial recognition by a panel of sera from B. bigemina infected cattle in ELISA using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were capable of neutralizing merozoite invasion in vitro . Conclusions: We identified the TRAP and TRP gene families in several Babesia and Theileria species, and characterized BbiTRAP-1 as a novel antigen of B. bigemina . The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in future vaccine candidates against B. bigemina .