| 研究課題/領域番号 |
22K15466
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| 研究機関 | 国立研究開発法人国立国際医療研究センター |
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研究代表者 |
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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| 研究期間 (年度) |
2022-04-01 – 2025-03-31
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| キーワード | Enterotoxigenic E. coli / CS6 / Cell adhesion / Cell invasion / Receptor |
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| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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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| 今後の研究の推進方策 |
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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| 次年度使用額が生じた理由 |
The cost of reagents used this fiscal year was slightly lower than expected, and no travel expenses were incurred. The remaining funds will be used for the purchase of reagents and consumables for the next year.
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