沙门氏菌VI型分泌系统对其在巨噬细胞内生存的影响及机制

Salmonella is an intensely investigated intracellular bacterial pathogen that causes gastroenteritis in humans and lethal typhoid fever in mice. Macrophages are an essential vehicle for the pathogenesis of S. Typhimurium, which utilizes them to disseminate from the intestine to the spleen and liver. An additional type of protein secretion system involved in protein translocation known as a type VI secretion system (T6SS) has been found to contribute to internal replication in macrophage. We found that the Salmonella T6SS mutant strain shows a higher sencitivity to the intracellular ROS than the wild type. We also discovered that the Salmnella T6SS could supress the expression of NADPH oxidase NOX2 in macrophage cell line.. These findings indicate that the T6SS in Salmonella may protect the intracellular bacteria survival in macrophage by decreasing the NADPH oxidase NOX2 derived ROS. Using defined salmonella T6SS mutant strain, we are trying to discover the role of NADPH oxidase in macrophage response to salmonella infection. Gentamicin protection assays, Western Blotting and immunofluorescence technique will be performed to screen the candidate effector protein that plays an important role in such infection. We will further validate the function of important effectors playing role in this process. This study will lay the foundation for the mechanism study of interaction between pathogen and host.

沙门氏菌是重要的致病菌，全球每年有上百万人死于各种类型沙门氏菌感染。然而，其致病机制至今尚不明确。VI型分泌系统（T6SS）是革兰阴性菌中新发现的重要分泌系统。研究显示，沙门氏菌T6SS与其在巨噬细胞内的生存相关，而其分子机制未知。我们的前期研究发现，鼠伤寒沙门氏菌T6SS的表达与巨噬细胞内ROS的产生密切相关；鼠伤寒沙门氏菌T6SS可调控巨噬细胞NADPH氧化酶NOX2的表达。因此，我们推测沙门氏菌T6SS是通过调控NOX2及其亚蛋白的表达和亚细胞分布来逃避ROS的杀伤作用。在本项目中，我们拟利用巨噬细胞及NOX2基因敲除小鼠，通过庆大霉素保护实验、免疫印记、免疫荧光等技术从体内及体外水平分别印证以上假设。此外，我们还将制备T6SS效应分子突变和补充菌株以筛选与胞内生存相关的T6SS关键因子。本研究将为深入了解沙门氏菌的致病因素及其在巨噬细胞内生存相关的致病机提供新的信息和视角。

1沙门氏菌是重要的致病菌，全球每年有上百万人死于各种类型沙门氏菌感染。然而，其致病机制至今尚不明确。VI型分泌系统（T6SS）是革兰阴性菌中新发现的重要分泌系统。研究显示，沙门氏菌T6SS与其在巨噬细胞内的生存相关，而其分子机制未知。该研究中，我们通过庆大霉素保护实验等证实了沙门氏菌T6SS与其在巨噬细胞内的生存相关，ROS清除剂预处理后的保护实验及NBT还原实验等明确了T6SS介导的沙门氏菌在巨噬细胞内生存与ROS相关。免疫印记证实T6SS可调控NOX2的表达且NOX2抑制剂可消除野生株与突变株在巨噬细胞内生存能力的差别。野生株和突变株感染后的巨噬细胞转录组测序进一步揭示了更多参与这一过程的相关因子。最后我们通过动物实验进一步证实了T6SS通过调控NOX2来介导对机体的感染。我们的研究为人们进一步了解T6SS介导的沙门氏菌致病机制提供了数据支持。