中国科学院海洋研究所机构知识库

由鳗弧菌（Vibrio anguillarum）引起的弧菌病是海水养殖业的顽疾，给渔业生产造成了严重的经济损失。前期研究发现，铁的运输和利用在鳗弧菌775致病过程中发挥着重要作用，其中以铁载体鳗弧菌素（anguibactin）介导的铁获取系统作用最为显著。该系统主要分为鳗弧菌素的合成、释放、铁(III)-铁载体复合物的转运、体内铁离子的释放以及铁运输的调节五个方面。相较于其他四个方面，关于铁释放的分子机制研究几近空白。

通过基因序列比对发现，在鳗弧菌775基因组中存在一个编码铁载体相互作用蛋白SIP（siderophore-interacting protein）的基因序列。在其他致病菌，如大肠杆菌中，SIP能够将铁载体中结合的三价铁还原成二价铁，从而降低铁与铁载体的结合能力来释放铁离子以满足自身需要。因此，对于鳗弧菌775 SIP（VaSIP）结构与功能的研究有助于理解铁(III)-鳗弧菌素复合物中铁释放的机制。本论文的主要工作和研究结果如下：

通过大肠杆菌表达系统获得了重组蛋白VaSIP；分子筛层析色谱法分析表明VaSIP在溶液中以单体形式存在；光谱扫描分析显示VaSIP重组蛋白携带黄素腺嘌呤二核苷酸（FAD）。利用分子置换法成功解析了分辨率为1.11 Å的VaSIP晶体结构，这是目前SIP蛋白家族中分辨率最高的结构。晶体结构显示VaSIP由5个α螺旋和12个β折叠组成，可分成三个结构部分：N端的β桶、α螺旋-β折叠-α螺旋构成的"三明治"结构和C端的一个α螺旋。同时发现，VaSIP主要通过氢键和苯环堆叠与FAD相互作用，在SIP家族中具有保守性。整体比对SIP家族结构已知蛋白，发现它们的折叠方式基本一致，但是存在多处序列插入和缺失变化。其中VaSIP的C端具有新颖的α螺旋结构，与其所识别的特异性底物相适应。

为验证VaSIP在鳗弧菌775铁释放过程中的作用，我们利用基因敲除技术获得了鳗弧菌775 sip的缺失株△sip，△sip与野生型的生长曲线测定结果显示，在低铁环境下sip的缺失并未影响鳗弧菌775的生长；同时斑马鱼侵染实验结果表明，突变株的毒性也没有降低，这都暗示着在鳗弧菌775中可能还存在着其他的铁释放途径。

Vibriosis caused by Vibrio anguillarumis a chronic illness in the marine aquaculture industry, leading to serious economic losses to fishery production. Previous studies have found that transport and utilization of iron plays an important role in the pathogenic process of V. anguillarum775. Among them, the siderophore-mediated iron acquisition system plays the most significant role. The system is mainly divided into five aspects: siderophore biosynthesis, export, ferrisiderophore import, iron release from ferrisiderophores and regulation of iron transport. Compared with the other four aspects, the molecular mechanism of iron release is little known.

In the genome of V. anguillarum775, a gene encoding the siderophore-interacting protein SIP was identified. In other pathogenic bacteria, such as Escherichia coli, SIP can reduce the ferric iron into ferrous iron in the ferrisiderophore complex, thereby reducing the affinity of iron binding to the siderophore and releasing the iron to meet its own needs. Therefore, the study on the structural and functional characteristics of SIP (VaSIP) of V. anguillarum775 would help to understand the mechanism of iron release in ferric-anguibactin complex. The main work and research results of this thesis are shown as follows:

Recombinant protein VaSIP was obtained byE. coliexpression system; analysis by the size-exclusion chromatograph indicated that the protein exists as a monomer in solution; by the UV-visible spectrum, it was suggested that VaSIP is probably combined with flavine adenine dinucleotide (FAD). The VaSIP crystal structure was successfully resolved by molecular replacement method with a resolution of 1.11 Å, which is the highest resolution in the SIP family. The crystal structure shows that VaSIP consists of 5 α-helices and 12 β-sheets. It can be divided into three structural parts: β-barrel at N terminus, α-helix-β-fold-α-helix “sandwich” structure, and an alpha helix at C terminus. In addition, we found that VaSIP can interact with FAD through hydrogen bonding and benzene ring stacking, which is conserved in the SIP family. Comparison of the known structures in SIP family revealed that their folding patterns are similar, but that there are multiple sequence changes, including insertions and deletion. The C-terminus of VaSIP has a novel alpha helix structure, which adapts to the specific substrate it recognizes.

In order to elucidate the role of VaSIP in the release of iron in V. anguillarum775, we used gene knockout techniques to obtain a sip deletion strain Δsip. The growth of the mutant strain and the wild-type showed similar under iron limiting conditions, implying the deletion of sipdid not affect the growth. The zebrafish infection experiment results exhibited that the toxicity of the mutant strain did not decrease. The results suggest other iron release pathways might be present in V. anguillarum775.

摘 要

ABSTRACT

第一章 绪论

1.1 鳗弧菌的致病机制

1.2 鳗弧菌中铁载体介导的铁获取系统

1.2.1 铁载体的合成

1.2.2 铁载体的分泌

1.2.3 铁(III)-铁载体复合物的转运

1.2.4 铁载体上铁的释放

1.2.5 铁运输的调节

1.3 铁载体相互作用蛋白的研究现状

1.3.1 基因水平

1.3.2 蛋白活性研究

1.3.3 晶体结构

1.4 本课题的研究目的与意义

第二章 VaSIP蛋白的重组表达与纯化

2.1 材料与方法

2.1.1 实验材料

2.1.2 实验方法

2.2 实验结果

2.2.1 VaSIP蛋白的序列分析

2.2.2 VaSIP蛋白的表达与纯化

2.2.3 VaSIP重组蛋白的性质分析

2.3 小结与讨论

第三章 VaSIP蛋白晶体结构分析

3.1 材料与方法

3.1.1 实验材料

3.1.2 实验方法

3.2 实验结果

3.2.1 VaSIP结晶条件的初筛与优化

3.2.2 VaSIP晶体结构解析

3.2.3 VaSIP的晶体结构分析

3.3 小结与讨论

第四章 鳗弧菌775基因sip的体内功能分析

4.1 材料与方法

4.1.1 实验材料

4.1.2 实验方法

4.2 实验结果

4.2.1 突变株的验证

4.2.2 sip缺失对生长的影响

4.2.3 sip缺失对宿主致死率的影响

4.3 小结与讨论

第五章 结论

5.1 全文总结

5.2 创新点

5.3 展望

参考文献

致 谢

作者简历及攻读学位期间发表的学术论文与研究成果