单增李斯特菌谷氧还蛋白Grx介导的氧化应激耐受及感染机制研究

单增李斯特菌为人畜共患食源性胞内寄生菌，在感染过程中会遇到来自宿主体内外环境中的各种氧化应激，但该菌可依赖硫氧还蛋白（Thioredoxin, Trx）系统对巯基和二硫键进行抗应激修复。我们在前期研究中发现，单增李斯特菌还存在一套在多数革兰氏阳性菌中不存在的谷氧还蛋白（Glutaredoxin, Grx）系统，但生物学功能未知。因此，在本项目中我们将利用应激生物学和感染生物学相关研究手段，探索李斯特菌Grx介导氧化应激调控及修饰细菌关键毒力因子的生物学机制；利用荧光标记、细胞定位和蛋白互作等研究技术，阐明Grx在糖源利用及代谢过程中对磷酸转移酶系统相关蛋白的修饰机制；利用转录组测序、基因敲除和报告基因等方法，解析Grx与Trx应激修复系统维持细胞内氧化还原稳态的协同机制。本研究对于揭示革兰氏阳性食源性病原菌Grx系统介导的蛋白修饰、抗应激生存及致病机制具有重要意义。

Listeria monocytogenes is an intracellular foodborne bacterium, and is well-adapted to various physiological environments, employing different strategies to counteract hostile oxidative stress conditions present in the environment and within the vacuolar compartment of phagocytic cells. There are two critical antioxidant systems present in bacteria, the thioredoxin (Trx) and glutaredoxin (Grx) systems. Our previous work has collectively determined that L. monocytogenes thioredoxin A as a vital cellular reductase is essential for maintaining a highly reducing environment in the bacterial cytosol, which provides a favorable condition for protein correct-folding, and therefore contributes to bacterial antioxidant and virulence. We also found L. monocytogenes encodes a functional glutaredoxin (Grx) which is not found in most Gram-positive bacteria. However, the biological roles of Listeria Grx and the underlying molecular mechanisms involved in bacterial oxidative tolerance remain unknown. In this study, we aim to illustrate: (I) the redox interaction and regulation mechanisms of Grx on bacterial key virulence factors that include the central regulator PrfA, listeriolysin O (LLO), and phospholipase (PlcB) during bacterial intracellular infections by stress, cell and infection biology strategies. (II) the biological roles of L. monocytogenes Grx involved in oxidative tolerance, and in bacterial sugar source utilization and energy metabolism by regulating the phosphotransferase system-related proteins by fluorescent labeling, localization and protein to protein interaction tools. (III) the synergistic mechanism between Grx and Trx under various oxidant conditions to maintain proper cellular redox homeostasis by transcriptome sequencing, knock out and reporter genes incorporating technologies. These results will provide a valuable model for clarifying the pathways associated with the potential roles of glutaredoxins from Gram-positive foodborne pathogens in maintaining redox homeostasis, and contributing to sugar metabolism and bacterial pathogenicity, and more importantly, successfully establishing infection within the host.