| Project/Area Number |
19K16661
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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 | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
Cheung Martin 沖縄科学技術大学院大学, 量子波光学顕微鏡ユニット, 技術員 (90832163)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Project Status |
Completed (Fiscal Year 2021)
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| Budget Amount *help |
¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2020: ¥520,000 (Direct Cost: ¥400,000、Indirect Cost: ¥120,000)
Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
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| Keywords | Shigella flexneri / Type III secretion / Tip complex / Cryo-EM / Dysentery / T3SS / Infection / Shigelle flexneri / Shigella / Bacteria / Electron microscopy / Microbiology / Protein structure |
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| Outline of Research at the Start |
T3SSs will be purified from Shigella flexneri using an affinity purification protocol. Purified T3SS will then be embedded in suitably thin (nanometre thick) layers of vitreous ice films, which will allow for purified T3SSs to be maintained in fully hydrated, near- physiological states. High-resolution images of ice-embedded T3SSs will be collected using a FEI Talos Arctica transmission EM (TEM) operating at 200 kV. The single particle cryo-EM approach will be used to reconstruct three-dimensional electron density maps of the needle/tip complex appendage, with a targeted resolution of 4 - 6 A.
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| Outline of Final Research Achievements |
Utilizing cryo-EM and mass spectrometry to study the type III secretion system tip complex (TC), two key findings were made: i) the TC comprises three distinct subunits with heterogeneous morphologies. Prior to this study, the TC had never been visualized to better than 20 A resolution and was believed to be a homopentameric complex. By visualizing, for the first time, the TC to sub-nanometer resolution (app. 9 A) by cryo-EM, this work revealed the TC to be a trimeric complex. Furthermore, each subunit within the trimer assumes a distinct orientation; ii) a hitherto unknown auxiliary complex binds to the TC upon activation of the T3SS during infection. A pore contiguous with the TC is known to be inserted into target cell membranes during infection. We believe this newly identified complex to be a pre-pore complex and may explain how the TC is able to interact with target cells.
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| Academic Significance and Societal Importance of the Research Achievements |
These results could be highly significant not only for our fundamental understanding of type III secretion system function but also for the development of novel antimicrobials. The newly complex may serve as an ideal drug target to prevent the infectivity of Shigella.
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