硫氧还蛋白TrxA介导的单增李斯特菌运动性和抗氧化应激机制研究

项目名称： 硫氧还蛋白TrxA介导的单增李斯特菌运动性和抗氧化应激机制研究

项目编号： No.31470179

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 微生物学

项目作者： 宋厚辉

作者单位： 浙江农林大学

项目金额： 83万元

中文摘要： 单核细胞增多性李斯特菌（Lm）为食源性胞内致病菌，可以突破消化道上皮、血脑和胎盘屏障。Lm在感染过程中是如何通过硫氧还蛋白（TrxA）应对氧化应激的呢？我们在前期工作中发现，缺失TrxA的Lm运动性和鞭毛形成能力减弱、群体感应信号分子AI-2分泌能力和毒力下降。本研究将从影响鞭毛组装的二硫键蛋白互作以及群体感应信息素AI-2入手，通过缺失株构建、蛋白突变和互作、巯基化和烷基化修饰、酶学活性分析等手段，利用质谱、生物膜干涉、共聚焦等技术深入解析TrxA介导的抗氧化应激机制。通过构建trx启动子与荧光素酶报告系统，检测Lm的trx如何在体内外不同应激条件下被激活的。本项目将为揭示Lm等革兰氏阳性胞内菌在消化道酸性环境中的抗氧化应激以及维持巯基/二硫键稳态机制奠定实验证据。

中文关键词： 单增李斯特菌；硫氧还蛋白；运动性；氧化应激；群体感应

英文摘要： Listeria monocytogenes (Lm) is an invasive food-borne intracellular bacterium that breaks the intestinal, the blood-brain, and the placental barriers. It is still elusive how circulating bacteria escape the killing of reactive oxygen species (ROS) in the niche host cells. In exposure to oxidative stress, changes in the thiol-disulfide redox status alter functions of exported proteins and result in rapid protein degradation. In our previous studies, we found that thioredoxin TrxA was essential in Lm. Deletion of trxA compromised the motility, decreased secretion of the quorum sensing molecule autoinducer-2 (AI-2), impaired integrity of flagella and attenuated the virulence of Lm in mice. To further investigate the anti-oxidant mechanism of TrxA, the disulfide forming protein MogR and GamR, key components in flagella assembly, and the quorum sensing molecule AI-2 will be exploited in Lm by mutagenesis analysis, protein-protein interaction and allyl methyl sulfide (AMS) modification approaches. To this end, the GC-MS, confocal imaging, bio-layer interferometry and fluorescence-activated cell sorting (FACS) techniques will be integrated to identify Trx modulated proteins and redox-sensitive signaling pathways. Meanwhile, infection of Lm towards epithelial cells, macrophages and mice will be carried out to evaluate the activation dynamics of gfp and luc reporter genes driven by trxA/B promoters under various stress conditions. The expression of trx triggered by ROS, released by mitochondria and endoplasmic reticulum (ER), under thapsigargin (TG) and dithiothreitol (DTT) stresses will be investigated to uncover the network-derived features. Contribution of listerial TrxA in response to oxidative damage initiated by ROS will be characterized by intracellular bacteria survival assay. This will shed light on novel anti-oxidative mechanisms of Lm during times of environmental stress through thioredoxin related proteins, leading to identify new pathways for sulfur/disulfide homeostasis in intracellular gram-positive bacteria.

英文关键词： Listeria monocytogenes;Thioredoxin;Motility;Oxidative stress;Quorum sensing