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

Authors: MONTENEGRO, VALERIA - Instituto De Clima Y Agua (INTA); PAOLETTA, MARTINA - Instituto De Clima Y Agua (INTA); JARAMILLO ORTIZ, JOSE - Instituto De Clima Y Agua (INTA); Suarez, Carlos; WILKOWSKY, SILVINA - Instituto De Clima Y Agua (INTA)

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/5/2020
Publication Date: 12/1/2020
Citation: Montenegro, V.N., Paoletta, M.S., Jaramillo Ortiz, J.M., Suarez, C.E., Wilkowsky, S.E. 2020. Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion. Parasites & Vectors. 13. Article 602. https://doi.org/10.1186/s13071-020-04469-5.
DOI: https://doi.org/10.1186/s13071-020-04469-5

Interpretive Summary: Bovine babesiosis pose a major constrain to livestock production worldwide, and is also an important threat for the cattle industry in the US. 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. New vaccines and diagnostic methods for bovine babesiosis are needed. Members of the well conserved TRAP family have been identified as subunit vaccine candidates in other species, but were never reported in B. bigemina. Hereby we defined the members of the TRAP superfamily in B. bigemina for the first-time using bioinformatics tools. In addition, we used experimental procedures to demonstrate the expression of BbiTRAP-1 by blood stage parasites. We also show that antibodies against TRAP-1 significantly neutralize invasion of erythrocytes by the parasite in invitro assays. However, serological analysis using sera from B. bigemina infected cattle suggests that this antigen is not immunodominant and might be not suitable for developing diagnostic assays. Yet, it has potential as a candidate for subunit vaccine development against B. bigemina.

Technical Abstract: 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 was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein family (TRP) families and 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 was 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 lack the vWFA domain. 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. 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 presence of neutralization-sensitive B-cell epitopes in this antigen suggests that conserved regions of BbiTRAP-1 could be included in future vaccine candidates against B. bigemina.