Molecular Characterization of a New Babesia bovis Thrombospondin-Related Anonymous Protein (BbTRAP2)

Abstract

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite’s growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

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.…”

Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules

“…The apicomplexan TRAP proteins are micronemal and surface proteins containing a signal peptide, a transmembrane region and a short cytoplasmic tail. Protein length and structure is also far from other TRAPs which having non-typical architectures [ 12 – 14 ]. For example, The TRAP-related proteins in P .…”

“…The cellular localization of BmP53 was determined by an Immunofluorescence antibody assay (IFA) according to [ 14 ]. Briefly, thin blood smears of B .…”

“…In apicomplexans, thrombospondin-related adhesive proteins (TRAPs) are the most well studied proteins presenting a TSP1 domain. TRAPs are micronemal and surface proteins containing a signal peptide, a von Willebrand factor A domain (VWA), a thrombospondin type 1 domain (TSP1), a transmembrane region and a short cytoplasmic tail containing a penultimate amino acid residue tryptophan (W) [ 12 – 14 ] which have been implicated in gliding motility [ 15 , 16 – 18 ]. Therefore, TRAPs are essential for apicomplexan gliding motility and cell invasion [ 12 , 14 , 19 ].…”