2019 Fiscal Year Research-status Report
Functional characterization of Babesia bovis proteins expressed on the surface of infected erythrocytes- Toward identification of novel vaccine and therapeutic candidates
| Project/Area Number |
19K15983
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| Research Institution | Nagasaki University |
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Principal Investigator |
晴希生 ハッサン 長崎大学, 熱帯医学研究所, 助教 (80745183)
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| Project Period (FY) |
2019-04-01 – 2021-03-31
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| Keywords | Babesia bovis / Surface proteins / Vaccine / Cattle |
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| Outline of Annual Research Achievements |
Using proteomics we identified nearly 100 proteins as Babesia bovis protein candidates exported to infected red blood cell (iRBC). Immunofluorescence microscopy confirmed the export of 3 novel proteins. Two proteins (Bb60 and Bb11920) with 10 transmembrane regions that were expressed in spherical bodies and on the surface of iRBCs. Further BLAST (Basic Local Alignment Search Tool) searches of PiroplasmaDB clarified that these proteins belong to a novel multigene family with 44 copies which we named as "mtm". Because another RBC-infecting parasite Plasmodium falciparum was shown to become blasticidin S (BSD) resistance by decreasing anion channel activity on iRBC, we generated BSD-resistant B. bovis to confirm whether this protein may serve as a channel. Development of BSD resistance resulted in downregulation of one major expressing gene of mtm, suggesting an association with BSD uptake.
Gene deletion of third exported protein, Bb4280, using CRISPR/Cas9 system was unsuccessful indicating the essentiality of this gene for the parasite growth in vitro. Induced knockdown using the glmS riboswitch system resulted in decreased growth rate, reduced ridge numbers on the erythrocyte surface, mislocalized VESA1 (Variant Erythrocyte Surface Antigen 1) as a ligand for cytoadhesion, and abrogated cytoadhesion to endothelial cells, suggesting that this protein is a novel virulence factor for B. bovis. We named this protein VESA1-export associated protein (BbVEAP).
These advances in characterizing the B. bovis exported proteins will aid in the development of new therapeutic strategies.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
We initially tried to express mtm in Xenopus laevis oocytes to check the transporter activity. Xenopus laevis was shown to be a promising heterologous expression system to characterize transporters. This experiment was done in a collaboration with a research group in UK. Using glucose as substrate, we performed the uptake assay but we did not see any uptake activity by the oocytes expressing mtm. Additional experiments are needed to gain more evidences for choosing an appropriate substrate in the future studies.
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| Strategy for Future Research Activity |
The future plan consist of two parts:
1. Our experiments showed that there is a link between BSD resistance and mtm expression. Further experiments needed to verify this link. We are planning to overexpress the downregulated mtm in BSD resistant parasites and calculate IC50 for BSD. If over-expression of mtm increases BSD sensitivity, it suggests that mtm are functioning as transporter and responsible for BSD uptake.
2. We showed that BbVEAP is a novel virulence factor contributing to cytoadhesion of parasites and possibly cerebral babesiosis. The next plan is to find the interacting proteins with BbVEAP and clarify why the parasite need this protein for survival. To gain insights into immunogenicity of BbVEAP, field samples will be screened.
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| Causes of Carryover |
I am planning to perform immuno precipitation and mass spectrometry to find the interacting proteins with BbVEAP. The protein samples will be sent to W. M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia, USA.
Initially, I was planning to perform one set of mass spectrometry experiment this fiscal year but due to the problem in GIT medium (Wako) production line which is used for B. bovis in vitro culture, I was not able to prepare the samples. Thus, 196,383 yen remained to be transferred and used in the next fiscal year to perform this experiment.
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