Novel Strategy for In Vitro Validation of Babesia orientalis Heat Shock Proteins Chaperone Activity and Thermostability.

BACKGROUND: Babesia orientalis is an intra-erythrocytic protozoan parasite that causes babesiosis in water buffalo. The genome of B. orientalis has been reported and various genes have been accurately annotated, including heat shock proteins (HSP). Three B. orientalis HSPs (HSP90, HSP70 and HSP20) have been previously identified as potential antigenic targets. Here, a new validation strategy for the chaperone activities and cell protection characteristics of the three HSPs was developed in vitro. METHODS: BoHSP20, BoHSP70 and BoHSP90B were amplified from cDNA, followed by cloning them into the pEGFP-N1 vector and transfecting the vector plasmid separately into 293T and Hela mammalian cells. Their expression and localization were determined by fluorescence microscopy. The biological functions and protein stability were testified through an analysis of the fluorescence intensity duration. Their role in the protection of cell viability from heat-shock treatments was examined by MTT assay (cell proliferation assay based on thiazolyl blue tetrazolium bromide). RESULTS: Fusion proteins pEGFP-N1-BoHSP20, pEGFP-N1-BoHSP70, and pEGFP-N1-BoHSP90B (pBoHSPs: pBoHSP20; pBoHSP70 and pBoHSP90B) were identified as 47 kDa/97 kDa/118 kDa with a 27 kDa GFP tag, respectively. Prolonged fluorescent protein half-time was observed specifically in pBoHSPs under heat shock treatment at 55 °C, and BoHSP20 showed relatively better thermotolerance than BoHSP70 and BoHSP90B. Significant difference was found between pBoHSPs and controls in the cell survival curve after 2 h of 45 °C heat shock. CONCLUSION: Significant biological properties of heat stress-associated genes of B. orientalis were identified in eukaryote by a new strategy. Fusion proteins pBoHSP20, pBoHSP70 and pBoHSP90B showed good chaperone activity and thermo-stability in this study, implying that BoHSPs played a key role in protecting B. orientalis against heat-stress environment during parasite life cycle. In conclusion, the in vitro model explored in this study provides a new way to investigate the biological functions of B. orientalis proteins during the host-parasite interaction.

Identification and characterization of a novel 34 kDa merozoite protein in Babesia orientalis.

A novel Babesia orientalis 34 kDa protein (designated BoP34) was obtained by immunoscreening of a cDNA expression library using B. orientalis infected water buffalo serum. The complete nucleotide sequence of the BoP34 was 1088 bp, which contained one open reading frame (ORF), two untranslated regions (UTRs) and a poly (A) tail. The length of ORF was 933 bp, encoding a polypeptide of 310 aa with a predicted size of 34 kDa. BLAST analysis showed that the nucleotide sequence of BoP34 had 71% similarity with that of the Babesia bovis gene XM_001611335, which encodes a nuclear movement family protein. This suggested that BoP34 is a homologous of the movement family protein. Structural analysis of the BoP34 protein indicated a CS domain which may interact with the ATPase domain of the heat shock protein 90. A truncated version of BoP34 was cloned into the expression vector pET-32a and subsequently expressed and purified from the Escherichia coli Rosetta™ (DE3) pLysS stain as a Trx-fusion (designated rBoP34-T). Antibodies in the serum of a B. orientalis-infected water buffalo were able to recognize this protein in immune-bloting analysis. Rabbit antibodies raised against rBoP34-T could detecte native BoP34 (34 kDa) in B. orientalis-infected water buffalo erythrocytes. These results suggested that BoP34 might be a good diagnostic antigen for the specific detection of anti-B. orientalis antibody in water buffalo. Further research is required to explore the biological function and diagnostic potential of this molecule.

Characterisation of a Babesia orientalis apical membrane antigen, and comparison of its orthologues among selected apicomplexans.

In the present study, we identified and characterised the complete coding sequence of Babesia orientalis apical membrane antigen 1 (designated Bo-ama1); it is 1803bp in length and encodes a polypeptide of 601 amino acids (aa). The Bo-ama-1 gene product (Bo-AMA1) is predicted to be 67kDa in size and contains a signal peptide. Mature Bo-AMA1 is predicted to have one transmembrane region and a short cytoplasmic tail (C-terminal domain). The extracellular part of Bo-AMA1 has three functional domains (DI, DII and DIII) with 14 conserved cysteine residues. A Bo-AMA1 fragment containing all three of these domains (designated Bo-AMA1-DI/II/III) was cloned into the plasmid vector pET-28a and expressed as a recombinant (His-fusion) protein of 53kDa. Antibodies in the serum from a B. orientalis-infected water buffalo specifically recognised this protein in immunoblotting analysis. Rabbit antibodies raised against the recombinant protein were able to detect native Bo-AMA1 (67kDa) from erythrocytes of B. orientalis-infected water buffalo. Bo-AMA1 is a new member of the AMA1 family and might be a good antigen for the specific detection of antibodies produced in B. orientalis infected cattle. This protein is likely to play critical roles during host cell adherence and invasion by B. orientalis, as the AMA1s reported in other organisms such as Plasmodium falciparum and Toxoplasma gondii. Further research is required to explore the biological functions of this protein and to determine whether its immunisation can induce protective effects in water buffalo against B. orientalis infection.

Molecular cloning and characterization of Babesia orientalis rhoptry-associated protein 1.

The rhoptry-associated protein 1 (RAP-1) gene of Babesia orientalis was obtained from a cDNA expression library by immunoscreening with B. orientalis-infected water buffalo sera. The nucleotide sequence of the cDNA was 1732 bp with an open reading frame (ORF) of 1434 bp, encoding a polypeptide of 478 amino acid residues with a predicted size of 52.5 kDa. The ORF was cloned into a pGEX-KG plasmid and subsequently expressed as a GST-fusion protein. The recombinant RAP-1 of B. orientalis (rBoRAP-1) was purified and evaluated as an antigen using Western blotting. The native BoRAP-1 was recognized by the antibodies raised in rabbits against rBoRAP-1. Strong immunofluorescence signals were observed in erythrocytes infected with B. orientalis. Phylogentic analysis revealed that B. orientalis fell into a Babesia clade and most closely related to Babesia bovis and Babesia ovis, which was similar to the previous reported trees based on 18S rRNA and HSP70 genes. The present study suggests that the BoRAP-1 might be a potential diagnostic antigen, and the RAP-1 genes can aid in the classification of Babesia and Theileria species.

Molecular cloning and characterization of a novel heat shock protein 20 of Babesia orientalis.

The heat shock protein 20 (HSP20) gene of Babesia orientalis (BoHSP20) was identified from both genomic DNA and cDNA. The full-length BoHSP20 gene was 690bp with one intron from position 88-243bp. The amplicon obtained from cDNA corresponded to a full-length open reading frame (ORF) with a length of 534bp, encoding a polypeptide of 178 amino acid residues with a predicted size of 20kDa. The ORF was cloned into a pET-28a plasmid and subsequently expressed as a His-fusion protein. The recombinant HSP20 of B. orientalis (rBoHSP20) was purified and evaluated as an antigen using Western blotting. Anti-B. orientalis water buffalo serum reacted with rBoHSP20, indicating that this protein was an immunodominant antigen and could be a useful diagnostic reagent to detect antibodies against B. orientalis in water buffalo. The native BoHSP20 was recognized by polyclonal antibody from the serum of rabbit immunized with rBoHSP20. Strong immunofluorescence signals were observed from B. orientalis in blood smears by fluorescence microscopy. Bacterial survival experiments indicated that HSP20 can significantly increase the viability of bacteria when the culture is exposed to thermal stress. The results suggest that BoHSP20 might play an important role during B. orientalis transmission from tick to host animal, given the sudden shifts in temperature involved. Phylogenetic analysis revealed that B. orientalis is in the Babesia clade and most closely related to Babesia bovis. Similar topologies were obtained from trees based on 18S rRNA and the HSP70 gene. The present study suggests that BoHSP20 might be a potential diagnostic antigen and that the HSP20 genes can aid in the classification of Babesia and Theileria species.