The solution structure of the adhesion protein Bd37 from Babesia divergens reveals structural homology with eukaryotic proteins involved in membrane trafficking.

Babesia divergens is the Apicomplexa agent of the bovine babesiosis in Europe: this infection leads to growth and lactation decrease, so that economical losses due to this parasite are sufficient to require the development of a vaccine. The major surface antigen of B. divergens has been described as a 37 kDa protein glycosyl phosphatidyl inositol (GPI)-anchored at the surface of the merozoite. The immuno-prophylactic potential of Bd37 has been demonstrated, and we present here the high-resolution solution structure of the 27 kDa structured core of Bd37 (Delta-Bd37) using NMR spectroscopy. A model for the whole protein has been obtained using additional small angle X-ray scattering (SAXS) data. The knowledge of the 3D structure of Bd37 allowed the precise epitope mapping of antibodies on its surface. Interestingly, the geometry of Delta-Bd37 reveals an intriguing similarity with the exocyst subunit Exo84p C-terminal region, an eukaryotic protein that has a direct implication in vesicle trafficking. This strongly suggests that Apicomplexa have developed in parallel molecular machines similar in structure and function to the ones used for endo- and exocytosis in eukaryotic cells.

Identification of common antigens in Babesia bovis, B. bigemina, and B. divergens.

Bovine babesiosis, caused by Babesia bovis, B. bigemina, and B. divergens, is a significant impediment to livestock production in countries with tropical/subtropical and temperate climates. Previous studies conducted on the immunoprophylaxis against the disease and diagnosis of these parasites has demonstrated the presence of similar antigens. The objective of this article was to identify and partially characterize antigens conserved among these three species. Immunochemical analysis using sera from cattle immunized individually with antigens from these three Babesia species revealed a number of antigens recognized by heterologous antisera. Cross-reactions were more evident in sera from cattle immunized with B. bovis/B. bigemina which recognized several antigens (15 kDa to >200 kDa) in B. divergens. Immunoscreening of a B. divergens cDNA library with bovine serum to B. bigemina allowed the isolation of five clones and DNA sequencing of plasmid BdJF5 showed a 680 bp cDNA insert. Basic Local Alignment Search Tool (BLAST) analysis of the predicted amino acid sequence revealed 47% identity with a protein identified as alphaNAC. Serum from mice immunized with a recombinant Glutathione S-Transferase-BdJF5 fusion protein immunoprecipitated a 20 kDa B. bovis antigen. However, 30 kDa and 18 kDa antigens were immunoprecipitated from B. divergens and immunoblotting analysis revealed the recognition of a 35 kDa B. bigemina antigen. An indirect fluorescence antibody assay on merozoites showed strong reaction with B. divergens and weak recognition of B. bovis and B. bigemina. Despite the existent antigenic polymorphism among the Babesia spp., these results demonstrated that common antigens occur between European B. divergens and Mexican B. bovis/B. bigemina.

Potent antihematozoan activity of novel bisthiazolium drug T16: evidence for inhibition of phosphatidylcholine metabolism in erythrocytes infected with Babesia and Plasmodium spp.

A leading bisthiazolium drug, T16, designed to mimic choline, was shown to exert potent antibabesial activity, with 50% inhibitory concentrations of 28 and 7 nM against Babesia divergens and B. canis, respectively. T16 accumulated inside Babesia-infected erythrocytes (cellular accumulation ratio, >60) by a saturable process with an apparent K(m) of 0.65 microM. Subcellular fractionation of Babesia parasites revealed the accumulation of T16 into a low-density fraction, while in malaria-infected erythrocytes a significant fraction of the drug was associated with heme malaria pigment. T16 exerts an early and specific inhibition of the de novo biosynthesis of phosphatidylcholine both in B. divergens- and Plasmodium falciparum-infected erythrocytes. Choline accumulation into isolated Babesia parasites was highly sensitive to inhibition by T16. These data are consistent with the hypothesis that bisthiazolium drugs target the de novo phosphatidylcholine biosynthesis of intraerythrocytic hematozoan parasites. In malaria parasites, which generate ferriprotoporphyrin IX during hemoglobin digestion, T16 binding to heme may enhance the accumulation and activity of the drug. The selectivity of accumulation and potent activity of this class of drug into parasite-infected erythrocytes offers unique advantages over more traditional antihematozoan drugs.

Genetic basis for GPI-anchor merozoite surface antigen polymorphism of Babesia and resulting antigenic diversity.

Glycosyl-phosphatidylinositol anchor merozoite surface antigens (GPI-anchor MSA) are proposed to act in the invasion process of infective merozoites of Babesia into host erythrocytes. Because of their essential function in the survival of Babesia parasites, they constitute good candidates for the development of vaccines against babesiosis and they have been extensively analyzed. These include Babesia bovis variable MSA (VMSA) and Babesia bigemina gp45/gp55 proteins of the agents of bovine babesiosis from tropical and subtropical countries, and the Babesia divergens Bd37 and Babesia canis Bc28 proteins of the main agents of bovine and canine babesiosis in Europe, respectively. However, these are very polymorphic antigens and Babesia parasites have evolved molecular mechanisms that enable these antigens to evade the host immune system as a survival strategy. This review focuses on the genetic basis of GPI-anchor MSA polymorphism and the antigenic diversity of B-cell epitopes that might be generated in each of these Babesia species. The picture is incomplete and no Babesia genome sequence is yet available. However, the available sequences suggest that two distinct, non cross-reactive GPI-anchor MSA (i.e., with unique B-cell epitopes) may be required by all Babesia species for invasion, and that these two distinct GPI-anchor MSA would be encoded by a multigene family. Furthermore, the data are consistent with the ability of biological clones from Babesia to use these multigene families for the expression of GPI-anchor MSA, either conserved (B. canis and B. bovis) or polymorphic (B. divergens and B. bigemina) in their amino acid sequence. Moreover, as a consequence for successful parasitism, the data suggest that both conserved and polymorphic GPI-anchor MSA would present unique B-cell epitopes.

Hydrophobic moeties in recombinant proteins are crucial to generate efficient saponin-based vaccine against Apicomplexan Babesia divergens.

Throughout Europe, bovine babesiosis is mainly caused by Babesia divergens, an Apicomplexan parasite transmitted by tick bites. The intra-erythrocytic development of B. divergens merozoites leads to haemolytic anaemia, and bovine babesiosis is responsible for economic losses in the agro-business industry. A totally efficient recombinant vaccine based on the merozoite surface protein Bd37 and saponin QuilA was recently described. In the present study we determined that protective immunity elicited by the Bd37 recombinant protein was related to the presence of hydrophobic residues in the protein. Using polymeric fusion of Bd37 as well as cell-free in vitro protein expression, we successfully expressed recombinant proteins containing hydrophobic sequences without the need of GST fusion. We used different hydrophobic sequences and different recombinant Bd37 proteins to demonstrate that antigen hydrophobicity affects the immune system, turning an inefficient protein into a 100% protective vaccine. Some hypotheses about the hydrophobic effect and its potential application to other parasitic protozoa vaccine are also discussed.

Identification of a coronin-like protein in Babesia species.

The present study was designed to immunochemically identify a coronin-like protein in Babesia bovis, B. bigemina, B. divergens, and B. canis. A 2-kbp cDNA insert of B. bovis carried by plasmid BvN9 was sequenced by the dideoxichain-termination method on both strands. The cDNA insert contained a 1719-bp long open reading frame coding for a deduced protein sequence of 61.7 kDa. Sequence analysis using the PSI-BLAST program revealed about 30% protein sequence identity with a coronin-like protein of Plasmodium falciparum. The encoding sequence of the cDNA insert lacking 70 amino acids at the N-terminal was subcloned in frame into pGEX 4T-3 to produce a recombinant glutathione S-transferase (GST)-pBv fusion protein. Polyclonal antibodies prepared in rabbits immunized with the purified GST-fusion protein recognized a Babesia-specific component of approximately 60 kDa by immunoprecipitation with [35S]methionine-labeled parasites. However, two molecules with relative sizes of 60 and 70 kDa were recognized in Babesia-infected erythrocyte extracts by immunobloting analysis. The 70-kDa component was apparently of host erythrocyte origin. In an indirect fluorescent antibody test, the rabbit serum strongly reacted with the merozoite stage of the four Babesia species, but also, although weakly, with the host erythrocyte. A cosedimentation assay performed with GST-pBv fusion protein and exogenous actin from rabbit liver showed that the GST-pBv fusion protein, but not the GST protein, was associated to actin. From these results, we conclude that the protein present in the four Babesia species analyzed here may be considered as a novel coronin-like, actin-binding protein.

Babesia divergens: cloning of a Ran binding protein 1 homologue.

Babesia divergens is an Apicomplexa transmitted to bovines by its acarian vector, the tick I. ricinus. Babesia divergens merozoites have an intraerythrocytic development in the blood of infected mammals. The nucleocytoplasmic transport system in this parasite is not yet characterized and no protein involvement in such transport has been described. In this report, we describe the cloning of a protein that shares important homologies with Ran binding protein 1. This protein in Eukaryote belongs to the nucleocytoplasmic transport system.