铽荧光与荧光假单胞菌铁吸收机理

Under iron-limiting conditions, fluorescent Pseudomonads aeruginosa secrete siderophores called pyoverdine (Pvd) and pyochelin (Pch), which form complexes with iron that are then taken up by the bacteria. Siderophores can also bind other metal ions except for ferric ion. Terbium fluorescence is enhanced greatly after terbium binding to siderophores. The mechanism of ferric uptake via siderophores could be followed in vitro and in vivo in Pseudomonads aeruginosa cell by fluorescence resonance energy transfer between three partners, the tryptophans of siderophores outer membrane transporters (FpvA and FptA), siderophores (Pvd and Pch) and terbium. In the proposal the binding of terbium to siderophores (Pvd and Pch) and the complexes of terbium-siderophores to outer membrane transporters (FpvA and FptA) will be investigated by using terbium as a fluorescence probe. The mechanism of ferric uptake via siderophores will be followed in vitro and in vivo in Pseudomonads aeruginosa. The effects of a few antibiotics on terbium fluorescence in the process will be observed.

研究铁载体介导的假单胞菌铁吸收、利用、调控机理对于新型抗生素的研究、假单胞菌在农业中的应用等具有重要的理论意义和潜在的应用价值。本项目使用化学方法合成非荧光铁载体、用分子生物学方法表达荧光铁载体及铁载体受体；在生理条件下研究铽(III)与铁载体、铽(III)-铁载体配合物与铁载体受体的结合热力学、动力学性质以及一些抗生素对结合的影响；以荧光假单胞菌(Pseudomonas aeruginosa)为研究对象，通过铽(III)荧光、蛋白质荧光等的测定(铁载体与铽(III)、铁载体与铁载体受体、铽(III)与铁载体受体间的无辐射能量转移)、菌体吸收铽(III)-铁载体后的铽分布分析、一些抗生素对吸收铽(III)的影响等研究铁载体途径的荧光假单胞菌铽(铁)吸收机理，为新型抗生素的开发、荧光假单胞菌的应用、稀土的微生物学效应等提供理论依据。

本研究使用铽(III)为荧光探针，在生理条件下研究了铽(III)与铁载体(Pvd, Pch)、抗生素(CIP, TC)结合的热力学性质；测定了Tb-Pvd、TbPch2配合物与受体蛋白FpvA、FptA结合的热力学、动力学性质；观察了Tb-Pvd、TbPch2配合物与荧光假单胞菌的作用；探讨了荧光假单胞菌对TbPch2配合物的吸收及铽(III)在细胞内的分布。结果表明，铽(III)可在Pch的介导下进入荧光假单胞菌细胞，铽(III)分布于细胞外模、膜间、内膜中；抗生素(CIP, TC)对荧光假单胞菌与TbPch2的作用没有影响。本研究对非荧光铁载体介导的荧光假单胞菌铁吸收机理、稀土微生物学效应等研究具有一定的参考价值。