Identification of a novel thrombospondin-related anonymous protein (BoTRAP2) from Babesia orientalis

Abstract

Background The thrombospondin-related anonymous protein (TRAP) was first discovered in the sporozoite of Plasmodium falciparum and TRAP family proteins are secreted by micronemes and transported to the parasite surface to participate in the invasion process. Various TRAP proteins have been identified in apicomplexan protozoans, but there have been few reports about TRAP proteins in Babesia orientalis . Methods The functional domain of TRAP2 in B. orientalis was cloned, sequenced, characterized and compared to the TRAP sequences of related apicomplexan parasites. The functional domain of BoTRAP2 was truncated, named BoTRAP2-1, and then cloned into the pET-28a expression vector. Rabbit anti-rBoTRAP2-1 polyclonal antibody was produced by immunizing three rabbits. Western blot analysis was used to identify the native form and immunogenicity of BoTRAP2. The localization of BoTRAP2 was identified by indirect fluorescence assay (IFA). Results The amplified genes of BoTRAP2 are 2817 bp in length, encoding a functional domain of about 938 aa with two vWFA domains, one TSP domain and one transmembrane domain. The amino acid sequence of BoTRAP2 has a high similarity with that of B. bovis and B. gibsoni . The predicted tertiary structure of truncated BoTRAP2-1 confirmed that BoTRAP2 contains two vWFA domains and a TSP domain, the main functional areas of the protein. The native BoTRAP2 was identified from B. orientalis lysate by using rabbit polyclonal anti-rBoTRAP2-1. A band corresponding to rBoTRAP2-1 was detected by reaction with serum from a B. orientalis -infected water buffalo, indicating that the protein has a high immunogenicity. IFA showed that BoTRAP2 is mainly localized on the apical end of parasites by rabbit anti-rBoTRAP2-1 polyclonal serum. Conclusions The rBoTRAP2 could differentiate serum from B. orientalis -infected water buffalo and normal water buffalo, implicating that BoTRAP2 has high immunogenicity and could serve as a candidate antigen for diagnosis of B. orientalis infection in buffalo. Electronic supplementary material The online version of this article (10.1186/s13071-019-3457-0) contains supplementary material, which is available to authorized users.

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

“…The involvement of members of the TRAP family during the erythrocyte invasion by merozoites has been previously demonstrated in B. bovis (4,25) and in other Babesia species (6)(7)(8). Our results of an important reduction in parasite invasion assays with anti-TRAP-1 antibodies support the hypothesis of the presence of surface exposed neutralizing B-cell epitopes, which is in accordance to the abovementioned reports.…”

“…Expression of TRAP-2 in merozoites had been already reported only for B. bovis and B. orientalis (7,25) and up to our knowledge, no other study has described the presence of these proteins in other members of Piroplasmida. In this sense, our data provides additional information on this gene family for future characterization studies not only in Babesia but in Theileria sp.…”

“…This protein was directly involved in both processes of recognition and invasion of bovine erythrocytes. Recently, other TRAP proteins have also been reported in B. orientalis and B. gibsoni (6)(7)(8).…”

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

“…Expression of TRAP-2 in merozoites has been reported previously, but only for B. bovis and B. orientalis [9,27]; to our knowledge, no other study has described the presence of these proteins in other members of Piroplasmida. In this context, our data provide additional information on this gene family for future characterization studies not only in Babesia but in Theileria sp.…”

“…This protein was found to be directly involved in both the recognition and invasion processes of bovine erythrocytes. Other TRAPs have also been recently reported in Babesia orientalis and B. gibsoni [8][9][10].…”