2020 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 | Obihiro University of Agriculture and Veterinary Medicine |
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Principal Investigator |
晴希生 ハッサン 帯広畜産大学, 原虫病研究センター, 特任研究員 (80745183)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | Babesia bovis / Surface proteins / Protein export / Vaccine / Therapeutic target |
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| Outline of Annual Research Achievements |
Using proteomics we identified of 3 novel exported proteins: The first two protein (Bb60 and Bb11920) with 10 transmembrane regions that were expressed in spherical bodies and on the surface of infected red blood cells (iRBCs). BLAST (Basic Local Alignment Search Tool) searches in PiroplasmaDB clarified that these proteins encoded by 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 (BS) resistant by decreasing anion channel activity on iRBC, we generated BS-resistant B. bovis to confirm whether this protein may serve as a channel. Development of BS resistance resulted in downregulation of one major expressing gene of mtm, suggesting an association with BS uptake. Episomal overexpression of downregulated mtm in the resistant line made these parasites more sensitive to BS, supporting our hypothesis on their role in BS uptake.
Induced knockdown of the third exported protein, Bb4280, using the glmS riboswitch system resulted in decreased growth rate, reduced ridge numbers on the erythrocyte surface, mislocalized VESA1 (Variant Erythrocyte Surface Antigen 1) that is 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). Currently, we are identifying the interacting proteins with mtm and BbVEAP to gain more insights into functions of these protein during the erythrocytic stage of B. bovis.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Last year I moved from Nagasaki University to Obihiro University of Agriculture and Veterinary Medicine. Transferring reagents, parasites, and stablishing the laboratory for performing experiments took several months which resulted in a delay of research progress.
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| Strategy for Future Research Activity |
The future plan consist of further characterization of mtm and BbVEAP:
1. Our experiments showed that there is a link between BS resistance and mtm expression. It is needed to use heterologous expression system such as proteoliposome or Xenopus laevis to find the potential substrates for mtm. We performed the uptake assay using glucose as substrate, but we did not see any uptake activity by the X. laevis oocytes expressing mtm. Further experiments using different substrates are needed to be conducted in future.
2. We showed that BbVEAP is a novel virulence factor contributing to cytoadhesion of parasites and possibly cerebral babesiosis. We performed immunoprecipitation and mass spectrometry to find the interacting proteins with BbVEAP. Current work is under progress to verify the identified proteins and dissect their function in regard to BbVEAP biology and function during parasite development in the erythrocytes.
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| Causes of Carryover |
We performed immunoprecipitations and mass spectrometry to identify the interacting proteins with mtm and BbVEAP. Preparation of protein samples were delayed due to the parasite and reagents transfer from Nagasaki University to Obihiro University of Agriculture and Veterinary Medicine. The transferred money to next fiscal year will be used for verification and functional characterization of BbVEAP and mtm interactome.
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