| Project/Area Number |
22K15466
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 49050:Bacteriology-related
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| Research Institution | National Center for Global Health and Medicine |
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Principal Investigator |
アイビエケ アラファテ 国立研究開発法人国立国際医療研究センター, 研究所, 感染症制御研究部 細菌感染研究室 研究員 (80933647)
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2024: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2023: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2022: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Keywords | Enterotoxigenic E. coli / CS6 / Cell adhesion / Cell invasion / Receptor / Colonization factor / Cell receptor |
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| Outline of Research at the Start |
Enterotoxigenic E. coli (ETEC) has significant morbidity and mortality especially among children in developing nations. We aim to identify the host receptor for a major ETEC colonization factor, CS6, mainly by using the difference in the binding affinity of CS6 to different cell lines.
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| Outline of Annual Research Achievements |
To identify the receptor protein for CS6, we examined different protein-protein interaction methods, including co-immunoprecipitation, far-western blotting, and pulldown assay. Among these assays, the pulldown assay, in which we His-tagged the CssB subunit and used it as bait to pull down receptor candidates from whole cell lysates of INT407 and Caco-2 cells, was informative. SDS-PAGE analysis of the pulled-down proteins revealed two protein bands that may interact with CS6. These proteins were further identified as MYH9 and ACTB through LC-MS/MS.
In addition, our previous research has shown that CS6 also has cell invasive properties. We further elucidated the involvement of major cell signaling pathways in CS6-mediated cell invasion by using cell signal transduction inhibitors.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
By the application of pulldown assay, we were able to establish two new proteins as possible receptor candidates for the CS6, and confirmation of these receptor candidates are still ongoing. Moreover, we were able to finalize our research on the invasive capability of the CS6 and published it Microbial Pathogenesis journal.
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| Strategy for Future Research Activity |
A single amino acid mutation, K118N, on the CssB subunit of the CS6 has shown a significant reduction in the cell adhesive capability of CS6 in vitro. We are planning to His-tag both the wildtype and mutated CssB proteins and perform pulldown experiments using whole cell lysates to investigate if there will be a difference in the pulled-down proteins between the wildtype and the mutant. In addition, the results from our previous co-immunoprecipitation experiment were not informative due to high antibody background. We are planning to use a covalent antibody coupling method to eliminate the final co-elution of the antibody, thereby reducing background, and compare the results with the pulldown assay for further confirmation of the receptor candidates.
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