ABSTRACT
Host cell invasion by apicomplexan parasites driven by gliding motility and empowered by actin-based movement is essential for parasite survival and pathogenicity. The parasites share a conserved invasion process: actin-based motility led by the coordination of adhesin-cytoskeleton via aldolase. A number of studies of host cell invasion in the Plasmodium species and Toxoplasma gondii have been performed. However, the mechanisms of host cell invasion by Babesia species have not yet been studied. Here, we show that Babesia gibsoni aldolase (BgALD) forms a complex with B. gibsoni thrombospondin-related anonymous protein (BgTRAP) and B. gibsoni actin (BgACT), depending on tryptophan-734 (W-734) in BgTRAP. In addition, actin polymerization is mediated by BgALD. Moreover, cytochalasin D, which disrupts actin polymerization, suppressed B. gibsoni parasite growth and inhibited the host cell invasion by parasites, indicating that actin dynamics are essential for erythrocyte invasion by B. gibsoni. This study is the first molecular approach to determine the invasion mechanisms of Babesia species.
Subject(s)
Actins/metabolism , Babesia/enzymology , Babesia/physiology , Erythrocytes/parasitology , Fructose-Bisphosphate Aldolase/metabolism , Actins/chemistry , Animals , Babesia/drug effects , Cytochalasin D/pharmacology , DNA, Complementary/isolation & purification , DNA, Protozoan/isolation & purification , Female , Fructose-Bisphosphate Aldolase/chemistry , Fructose-Bisphosphate Aldolase/genetics , Kinetics , Mice , Mice, Inbred ICR , Nucleic Acid Synthesis Inhibitors/pharmacology , Open Reading Frames/genetics , Polymerization , Protozoan Proteins/metabolismABSTRACT
The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260microM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6microM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10mg/kg of body weight on days 8-10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.
Subject(s)
Antiprotozoal Agents/therapeutic use , Artemisinins/therapeutic use , Babesia microti/drug effects , Babesiosis/drug therapy , Animals , Antiprotozoal Agents/administration & dosage , Antiprotozoal Agents/pharmacology , Artemisinins/administration & dosage , Artemisinins/pharmacology , Artesunate , Babesia/drug effects , Babesia/growth & development , Babesia bovis/drug effects , Babesia bovis/growth & development , Babesiosis/parasitology , Humans , Mice , Parasitic Sensitivity Tests , Treatment OutcomeABSTRACT
A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced an anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 days post-infection, even when the dog became chronically infected and showed a low level of parasitemia. Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be an immunodominant antigen for B. gibsoni infection that could be used as a diagnostic antigen for B. gibsoni infection with high specificity and sensitivity.
Subject(s)
Antigens, Protozoan , Babesia/immunology , Babesiosis/veterinary , Dog Diseases/diagnosis , Protozoan Proteins , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Babesia/classification , Babesia/growth & development , Babesiosis/diagnosis , Babesiosis/immunology , Babesiosis/parasitology , Base Sequence , Cloning, Molecular , Dog Diseases/immunology , Dog Diseases/parasitology , Dogs , Enzyme-Linked Immunosorbent Assay , Molecular Sequence Data , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Analysis, DNA , Serologic TestsABSTRACT
The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.
