2020 Fiscal Year Research-status Report
Structural and functional studies of iron uptake ATP-binding cassette transporters (ABC transporters) in Gram-negative bacteria
| Project/Area Number |
20K22561
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| Research Institution | The University of Tokyo |
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Principal Investigator |
陸 鵬 東京大学, 大学院農学生命科学研究科(農学部), 助教 (20880339)
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| Project Period (FY) |
2020-09-11 – 2022-03-31
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| Keywords | VmFbpA / ABC transporter / Vibrio. spp / Membrane Protein / Iron |
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| Outline of Annual Research Achievements |
1. To find bacteriostatic compounds that inhibit the function of VmFbpA and the growth of V. metschnikovii.
The screening of VmFbpA-Fe3+ interaction inhibitors was performed using the water extracts of 20 different spices. In this study, rosemary and cinnamon extracts showed significant inhibitory activity. The UV-Vis spectral analysis showed that the inhibition of VmFbpA caused by rosemary extract is a specific reaction. Moreover, it was revealed that rosmarinic acid (RA) is the main compound responsible for the inhibition. ITC and docking simulation results demonstrated that RA binds to the VmFbpA at the Fe3+-binding site and acts as a competitive inhibitor.
In the antibacterial assay, the supplementation of citrate significantly increased the inhibitory activity of RA on the growth of V. metschnikovii. However, RA with or without the citrate showed little effect on E. coli, which is more resistant to iron restriction.
2. Structural analysis of TtFbpBC/A complexes
The interaction of TtFbpA and TtFbpBC is revealed.In X-ray crystallography, The best TtFbpBC crystals were formed in the presence of 0.2% n-fecyl-β-D-maltopyranoside (DM) and 20% PEG2000, which diffracted X-rays to ~4.0 Angstrom.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
1) In the anti-vibrio experiment.
The inhibition of V. metschnikovii, a infectious bacterium from vibrio .spp, is achieved by rosmarinic acid (RA) and citrate. However, the anti-vibrio spectrum is not tested yet. The mechanism of RA inhibiting the iron-binding protein VmFbpA is also unclear.
2) In the structural analysis of TtFbpBC/A complex
The goal for the solving TtFbpBC and TtFBC-A complex is to the resolution of ~3.0 Angstrom. However, the current resolution is ~4.0 Angstrom.
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| Strategy for Future Research Activity |
1) In the anti-vibrio experiment.
Test the antibacterial effects of RA on V. parahaemolyticus, V. vulnificus, v. alginolyticus
Solve the crystal structure of VmFbpA(Fe) and VmFbpA(RA)
2) In the structural analysis of TtFbpBC/A complex
The main strategies for solving the structure of TtFbpBC/A are to enlarge the molecular size: ① using artificial monoclonal antibodies; and/or ② cross-linking TtFbpA and FbpbBC by gradient fixation (Grafix), a method to stabilize fragile macromolecular complexes.
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| Causes of Carryover |
次年度使用額が生じた理由：
消耗品を購入する予算なんですが、少なく済んだため。
使用計画：
来年度の消耗品と試薬費として使用する。
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Research Products
(1 results)
