A novel Babesia protein was found to be a homologue of a Plasmodium heparin-binding merozoite protein.
In our earlier study on heparin-binding merozoite proteins of P. falciparum, a novel protein, PfTrx-mero protein, containing a conserved domain of PfTrx-like-mero was identified [26, 33]. In the present study, we report the finding of a novel protein encoded by XM_021482140.1 in B. microti (Bmi_XP_021338692.1) (Fig. 1a), which was homologous to PfTrx-like-mero of P. falciparum. The amino acid sequence of this Babesia protein contains two Trx-like domains (Fig. 1c); thus, we named it as B. microti (Bmi) Trx-like protein. The ‘-CXXC-‘ motif, which is essential for antioxidant function, was identified within the sequence of most of the Trx-like proteins. However, the ‘-CXXC-‘ motif was missing in the sequence of both, PfTrx-mero protein and BmiTrx-like protein (Fig. 1b). The sequence of BmiTrx-like protein contains a 17-aa long signal peptide (Fig. 1c), indicating that the protein may have a role outside of the organism.
Recombinant BmiTrx-like protein could be identified by anti-Babesia serum from B. microti-infected mice.
Recombinant BmiTrx-like protein with a His-tag or a Glutathione S-transferase (GST)-tag were expressed, respectively, in E. coli and subsequently purified. The molecular weight is 46 kDa for recombinant His-tagged BmiTrx-like protein (rBmiTrx-like protein) and 72 kDa for the GST-tagged 72 protein (Fig. 2a). Anti-B. microti serum, obtained from mice 21 days post-infection of B. microti, was used to detect the rBmiTrx-like protein by western blotting. As shown in the western blot analyses, recombinant rBmiTrx-like protein could be identified by infected serum but not by serum from uninfected mice (Fig. 2b).
Dynamic transcriptional and protein expression of BmiTrx-like protein in vivo.
Expression of mRNA from BmiTrx-like protein-encoding gene XM_021482140.1 was detected by real-time quantitative PCR (RT-qPCR) at 1, 3, 5, 7, 9, 11, and 13 days post-infection. Specific gene expression was higher on days 5, 7, and 9 post-infection, peaking on the 7th day and then decreasing gradually (Fig. 3a). Rabbit anti-BmiTrx-like protein serum was used to detect native protein; specific bands could be detected in parasite lysates at 1, 3, 5, 7, 9, 11, and 13 days post-infection (Fig. 3b). Again, a protein band with an apparent molecular weight of 46 kDa was observed. The protein expression gradually increased and peaked on the 11th day; a sharp decline of the expression was observed on the 13th day.
BmiTrx-like protein adhered to heparin and host erythrocytes.
To elucidate the role of BmiTrx-like protein in the invasion process, GST-tagged recombinant proteins were separately incubated with heparin-sepharose, human erythrocytes, or mouse erythrocytes, with GST used as the control. As shown in Fig. 4, GST-tagged rBmiTrx-like protein could bind both heparin and host erythrocytes, while GST protein did not show any binding to heparin or erythrocytes.
Immunization with recombinant BmiTrx-like protein protected mice against B. microti infection.
Immunization with PfTrx-mero protein has been proven to provide significant protection against Plasmodium infection [33]; thus, we studied whether this is also the case for BmiTrx-like protein against B. microti infection in vivo. His-tagged BmiTrx-like protein was used to immunize BALB/C mice. After successful immunization (Fig. 5a), a challenge with 1 × 106 B. microti-infected red blood cells (iRBCs) was implemented. The parasitemia was significantly lower in the immunized groups; especially at day 9 post-infection, the parasitemia decreased more than 50%, compared to that in the control group (immune group vs control group, 2.43% ± 0.33% vs 5.95% ± 0.30%, p < 0.0001, Fig. 5b). The results demonstrate that BmiTrx-like protein immunization could partially protect mice from parasite attack.
BmiTrx-like protein might locate on the surface of merozoites.
The subcellular location of BmiTrx-like protein was detected by indirect immunofluorescence, while DAPI was used to show the nuclear chromatin (Fig. 3c). BmiTrx-like protein (green fluorescence) was observed on the membrane of merozoites that were inside the iRBCs. No green fluorescence was observed when normal rabbit serum was incubated with infected erythrocytes. Likewise, only blue fluorescence was detected when the primary antibody was omitted (only the secondary antibody was incubated with the infected erythrocytes). In addition, no fluorescence was detected when uninfected erythrocytes were probed with rabbit anti-rBmiTrx-like protein serum. Results indicate that the anti-rBmiTrx-like protein serum could specifically identify BmiTrx-like protein that might be expressed on the surface of the merozoite of B. microti.