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

MONTENEGRO, VALERIA NOELY; PAOLETTA M; JARAMILLO ORTIZ, JOSÉ M.; SUAREZ, C; WILKOWSKY S. E

Congreso; XXXII Reunión Anual de la Sociedad Argentina de Protozoología; 2020

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.The Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein family (TRP) families were identified in Babesia and Theileria through the search of the TSP-1 adhesive domain, the hallmark motif of TRAP proteins. Structural modelling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 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.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 lack 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 neutralize merozoite invasion in vitro. 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 suggests that BbiTRAP-1 could be included in future vaccine candidates against B. bigemina.