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Elucidation of receptor binding mechanism of Enterotoxigenic Escherichia coli colonization factor CS6

Research Project

Project/Area Number 22K15466
Research Category

Grant-in-Aid for Early-Career Scientists

Allocation TypeMulti-year Fund
Review Section Basic Section 49050:Bacteriology-related
Research InstitutionNational Center for Global Health and Medicine

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)

Project Period (FY) 2022-04-01 – 2025-03-31
Project Status Granted (Fiscal Year 2023)
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)
KeywordsEnterotoxigenic E. coli / CS6 / Cell adhesion / Cell invasion / Receptor / Colonization factor / Cell receptor
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.

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.

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.

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.

Report

(2 results)
  • 2023 Research-status Report
  • 2022 Research-status Report
  • Research Products

    (4 results)

All 2024 2023

All Journal Article (2 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 2 results) Presentation (2 results) (of which Int'l Joint Research: 1 results)

  • [Journal Article] The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion2024

    • Author(s)
      Ayibieke Alafate、Wajima Takeaki、Kano Shigeyuki、Chatterjee Nabendu Sekhar、Hamabata Takashi
    • Journal Title

      Microbial Pathogenesis

      Volume: 190 Pages: 106636-106636

    • DOI

      10.1016/j.micpath.2024.106636

    • Related Report
      2023 Research-status Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Gene expression analysis during the conversion from a viable but nonculturable to culturable state in Vibrio cholerae2023

    • Author(s)
      Ayibieke Alafate、Nishiyama Ayae、Senoh Mitsutoshi、Hamabata Takashi
    • Journal Title

      Gene

      Volume: 863 Pages: 147289-147289

    • DOI

      10.1016/j.gene.2023.147289

    • Related Report
      2022 Research-status Report
    • Peer Reviewed / Int'l Joint Research
  • [Presentation] Gene expression analysis during the conversion from a viable but nonculturable to culturable state in Vibrio cholerae2023

    • Author(s)
      Alafate Ayibieke, Ayae Nishiyama, Mitsutoshi Senoh, Takashi Hamabata
    • Organizer
      57th United States-Japan Cooperative Medical Science Program Joint Panel Conference on Cholera and Other Bacterial Enteric Infections
    • Related Report
      2023 Research-status Report
    • Int'l Joint Research
  • [Presentation] Gene expression analysis during the conversion from a VBNC to culturable state in Vibrio cholerae2023

    • Author(s)
      Alafate Ayibieke, Ayae Nishiyama, Mitsutoshi Senoh, Takashi Hamabata
    • Organizer
      第96回日本細菌学会総会
    • Related Report
      2022 Research-status Report

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Published: 2022-04-19   Modified: 2024-12-25  

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