Ag(I)和Hg(II)对大肠杆菌铁硫簇组装系统的作用机制

The increasing application of mercury and silver as antimicrobial agents in textile, medical and medicine have dramatically increased people’s exposure. Whereas the toxic mechanisms remain unknown. Mercury and silver have high thiolphilicity and thus defined as soft metals. Previously we have demonstrated that soft metals poison Escherichia coli by directly attacking the exposed [4Fe-4S] of dehydrratases at doses that initiate bacteriostasis. Instead of reactive oxygen species, the damage stems directly from metal thiophilicity. Results determined that those enzymes are among the primary targets, whereas also indicated primary damage might happen at multiple levels. Whether soft metals also target iron sulfur cluster assembly machinery (also rich in thiols moieties) remain unknown. In this research, E. coli will be used as the model organism to investigate the effects of mercury and silver against Fe/S assembly system. To do that, the physiological Isc function loss and Suf induction, Isc and Suf mutants sensitivity screen, the induction of Fe/S sensor and regulator IscR, Fur and their upstream OxyR will be evaluated. Accordingly, proteins showed sensitivity to metals will be purified and tested in vitro and in vivo. We aimed to clarify the targets of soft metals during toxicity and explore their action modes.

近年来汞和银在纺织、医药及医疗等诸多领域得到大规模应用，极大增加了生物在金属环境中的暴露几率，但其对细胞致毒机制仍然存在较多盲点。汞和银都具有强亲硫性属于柔性金属的范畴。前期研究结果表明柔性金属能直接破坏大肠杆菌细胞中一类关键脱水酶外露的[4Fe-4S]簇，这种破坏作用可能在多个层面同时发生。铁硫簇组装系统负责组装和运输[4Fe-4S]簇，系统本身同样富含巯基而成为金属潜在的攻击目标。本研究以大肠杆菌为模型，以亲硫性最强且毒性最强的两种柔性金属汞和银为例，通过分析金属压力下铁硫簇主系统Isc功能的缺失、备用系统Suf的诱发、感应及调控因子IscR、OxyR和Fur等的诱发和表达、排查Isc和Suf各基因缺失变异株的金属敏感度，蛋白提纯和全波长扫描等手段全面分析亲硫金属对铁硫簇组装系统的影响。旨在明晰柔性金属致毒的主要靶点，揭示亲硫性在金属致毒过程中的重要作用及其分子机制。

工业化进程导致的重金属污染已然使得金属毒理成为研究者们关注的焦点，但重金属对细胞的毒性机制仍有待阐明。其中Hg(I/II)、Ag(I)、Cu(I)和Cd(II)等柔性金属对微生物的毒性与其亲硫性强弱基本一致，暗示着金属毒性产生机制与亲硫性密切相关。前期结果已证明柔性金属能直接破坏大肠杆菌中一类关键脱水酶外露的[4Fe-4S]簇，且暗示了破坏作用可能在多个层面同时发生。.本研究选取了亲硫性及细胞毒性最强的Ag(I)和Hg(II)为代表，以大肠杆菌为模式生物，通过细菌生长曲线测定、突变株及融合株的构建和酶活分析等方法，深入分析了金属离子对细胞内铁硫簇组装系统功能的影响及金属胁迫下OxyR、IscR和 Fur三个转录因子对Suf操纵子的调控机制。.本研究得出了以下结果：①在低于抑制浓度的Ag(I)、Hg(II)作用下，大肠杆菌Fe/S簇组装常备系统Isc功能受损，备用系统Suf被诱导表达；②明确了suf操纵子的诱导表达并非主要来自氧化应激调控转录因子OxyR及IscR，表明在受测金属离子的作用下细胞并未启动氧化应激防御机制；③首次验证了Suf系统的诱发来自于细胞内的缺铁响应调控。.通过研究提出了重金属离子对细胞的一个全新作用机制：受测金属破坏Fe/S簇组装Isc系统迫使细胞启用备用系统Suf。同时金属破坏了细胞内Fe/S蛋白中外露的[4Fe-4S]簇，导致大量脱辅基酶的累积。细胞重构Fe/S簇增加了细胞对Fe的需求，使细胞呈现缺Fe状态，继而通过Fur调控并启动细胞缺铁应答下游基因的表达。Fur通过去抑制作用直接诱导suf操纵子及编码Mn-SOD的sodA。该工作揭示亲硫性在金属毒性中发挥的重要作用，将为明晰柔性金属致毒的首要靶点提供理论依据，并为金属在无氧条件下致毒提供了新的解释途径。