单核细胞增多性李斯特菌二硫键形成蛋白DsbA对氧化应激、鞭毛合成和毒力的调控机制研究

单核细胞增多性李斯特菌是一种存在于环境中的革兰氏阳性条件病原菌。二硫键形成蛋白（Dsb）对于该菌在宿主体内外环境中的生存、氧化应激、运动和鞭毛生成至关重要，但机理知之甚少。在本研究中，我们将对单增李斯特菌DsbA突变株和野生型的表型及各种应激条件下的转录组进行分析；研究DsbA对上皮细胞的黏附性、侵袭力以及对巨噬细胞的毒力和对小鼠致病性的作用和影响。为了进一步确定单增李斯特菌感染细胞后DsbA对线粒体完整性的影响，我们将采用荧光标记技术研究DsbA在维持细胞内氧化还原稳态中的作用。最后我们将利用ITC技术定量分析DsbA与鞭毛调节蛋白MogR 和 GamR的热动力学互作情况，同时利用iTRAQ蛋白质组学方法，挖掘DsbA的作用底物，阐明一种由二硫键形成蛋白和硫氧还蛋白共同修饰的鞭毛蛋白合成新机制。本研究对于揭示食源性病原菌抗氧化应激的适应机制具有重要意义。

Listeria monocytogenes (L. monocytogenes) is an environmental facultative, foodborne and Gram-positive bacterial pathogen. The presence of disulfide bond (Dsb) forming proteins is critical for viability, mobility and formation of folded proteins. It remains unknown how L. monocytogenes adapts redox stresses occurred intracellularly upon infection, and how the DsbA proteins participate in the biogenesis of flagella proteins when L. monocytogenes exposes to extracellular environments. In this study, the roles of DsbA will be investigated by phenotypes examination and transcriptome analysis of L. monocytogenes wild type and delta-dsbA towards various oxidative stress conditions. Survival and proliferation of L. monocytogenes mutants will be evaluated in epithelial cells, macrophages and a mouse model. To further determine the effect of DsbA to the homeostasis of the thiol redox environment when L. monocytogenes exposes intracellular compartments, the mitochondrial integrity will be analyzed in response to oxidative stresses by fluorescent labeling of mitochondrial membrane and autophagy-related proteins. The disulfide bond-formation properties of DsbA during flagella biogenesis will be characterized by thermodynamic interactions to flagella regulator MogR and GamR using a quantitative isothermal titration calorimetry (ITC) assay. Substrates of DsbA will be determined by iTRAQ quantitative proteomics mass spectrometry. This study will reveal a novel mechanism by which Gram-positive bacterial flagella proteins are co-modified by DsbA and thioredoxin protein, and also shed light on an evolutionary adaptation of foodborne pathogens towards redox stresses.