寡发酵链球菌抗氧胁迫的分子机制

Antioxidant system helps bacteria to defense against oxidative stress, which is generated by exposure to reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and hydroxyl radical. The expression of oxidative stress resistance genes are regulated by redox-sensing transcriptional factors. Streptococcus oligofermentans is an oral commensal. It produces copious amounts of hydrogen peroxide (H2O2), and also endures high concentration of H2O2, predicting that it may possess unique resistance mechanisms to oxidative damage caused by ROS. Our preliminary study showed that mutation of putative perR (peroxide responsive repressor) gene enhanced S. oligofermentans' resistance to H2O2, suggesting that PerR may act as an oxidative stress responsive repressor. However, its regulons and regulatory mechanism are still unclear. To clarify the molecular mechanism of PerR in oxidative stress resistance, the following three aspects are included in this study: 1. Explore the key oxidative stress resistance genes regulated by PerR. 2. Clarify the role and mechanisms of PerR regulons and other related genes in defense against oxidative stress. 3. Analyze the molecular mechanism of PerR regulation in S. oligofermentans. Through exploring the global responses to oxidative stress in S. oligofermentans, a novel mechanism of antioxidant defense is expected to be discovered. Since S. oligofermentans can specifically inhibit the growth of caries pathogen Streptococcus mutans by producing large amount of H2O2, this study will provide insights into the prevention of dental caries.

细菌抗氧化系统可保护细胞免受活性氧自由基（ROS）的伤害。抗氧化基因的诱导表达受氧化还原应答转录因子的全局调控。寡发酵链球菌是口腔牙菌斑生物膜中的有益菌，它能产生、且耐受高浓度的过氧化氢，暗示它有强的抗氧能力或独特的抗氧胁迫机制。前期研究显示失活氧化还原应答转录因子PerR的同源基因，寡发酵链球菌对过氧化氢的耐受能力显著提高。这表明PerR参与过氧化氢抗性应答，但是其作用机制尚不清楚。本课题拟开展PerR在寡发酵链球菌抗氧胁迫中的作用机制研究，包括:发现PerR调控的抗氧胁迫关键基因；探讨PerR调控的功能基因在抗氧胁迫中的作用及机制；分析PerR对关键功能基因调控的分子机制。目的是阐明寡发酵链球菌的抗氧胁迫应答机制，从而发现生物新的ROS抵御系统。由于寡发酵链球菌能特异地抑制致龋菌-变形链球菌生长，该研究还将对实现龋病的生态防治具有重要指导意义。

细菌抗氧化系统可保护细胞免受活性氧自由基（ROS）的伤害。抗氧化基因的诱导表达受氧化还原应答转录因子的全局调控。寡发酵链球菌（Streptococcus oligofermentans）是口腔牙菌斑生物膜中的有益菌，它产生、且耐受高浓度的过氧化氢，暗示它有独特的抗氧胁迫机制。本实验室前期研究显示失活过氧化物响应抑制蛋白PerR（peroxide - responsive repressor）显著提高了寡发酵链球菌对过氧化氢的耐受，表明PerR参与过氧化氢抗性应答。本课题主要研究PerR在寡发酵链球菌抗氧胁迫中的作用机制，取得了如下结果：1.在转录组水平鉴定PerR调控的抗氧胁迫关键基因：利用Microarray技术对比野生型菌株和perR突变株在有、无过氧化氢胁迫时全局性基因的差异表达，发现473个H2O2响应基因中，56%的基因受PerR调控。Binomial test对PerR调控的H2O2响应基因参与的生物学过程进行富集分析，发现这些基因主要参与金属离子平衡及氨基酸代谢，表明PerR蛋白在寡发酵链球菌抗H2O2胁迫中发挥重要作用。2.发现PerR调控锰转运蛋白表达及锰代谢的抗氧胁迫新机制：PerR调控的抗氧胁迫关键基因谱提示，不同于枯草芽孢杆菌，PerR可能调控锰平衡而发挥抗氧保护作用。研究表明内源性或外源性H2O2失活PerR，解除对锰转运蛋白mntABC及Fe2+螯合蛋白dpr基因的抑制。MntABC的上调表达使胞内的Mn2+量增加。Mn2+不仅作为超氧化物歧化酶SOD的辅因子，使SOD发挥功能，它还具有无机触酶的作用，分解H2O2，从而抵抗氧胁迫。3.阐明PerR与锰转运抑制蛋白MntR利用氧还调节机制，感应H2O2调控锰摄取而抵抗氧胁迫的新机制：我们的研究表明传统的Mn2+感应蛋白MntR还能感应细胞内的H2O2，而介导对H2O2的抗性。生物化学分析，点突变及生理、遗传学实验表明H2O2氧化MntR的半胱氨酸，使其形成二硫键交联的二聚体而失活，并自Mn2+ 转运蛋白mntABC的启动子区解离；H2O2压力使MntR形成二硫键交联的聚体及胞内降解，因此导致mntABC表达及胞内Mn2+量上调,从而增加了H2O2抗性。.综上所述，通过研究寡发酵链球菌的抗氧胁迫应答机制，我们发现了新的ROS防御策略。该研究还将有助于龋病的生态防治。