细胞膜微结构域中胆固醇对小鼠巨噬细胞摄取土拉弗朗西斯菌的作用

Role of cholesterol of membrane microdomain in the uptake of Francisella tularensis by mouse macrophages

本文旨在探讨细胞膜微结构域中胆固醇在小鼠巨噬细胞摄取土拉弗朗西斯菌LVS株中的作用。用电穿孔法将穿梭质粒pFNLTP6gro-gfp导入土拉弗朗西斯菌LVS株;细胞胆固醇用菲律平Ⅲ染色,结合单克隆抗体的小窝蛋白-1用键合了Alexa594的羊抗鼠二抗显色;用Z轴电动聚焦的荧光显微镜分析土拉弗朗西斯菌LVS株感染;用甲基-β-环糊精干扰富含脂质的胞膜,损耗胆固醇,以评估膜微结构域的损伤对土拉弗朗西斯菌入侵的影响,菲律平Ⅲ用于检测胆固醇损耗的效果。结果显示,细胞胆固醇在早期与摄取的土拉弗朗西斯菌疫苗株共定位,膜微结构域相关成分小窝蛋白-1与两者接触密切;土拉弗朗西斯菌入侵巨噬细胞需要胆固醇丰富的膜结构域。结果提示,胆固醇丰富的膜微结构域和小窝蛋白-1在土拉弗朗西斯菌早期进入巨噬细胞过程中发挥重要作用。

The purpose of the current study was to evaluate the role of cholesterol of the membrane microdomain in the uptake of Francisella tularensis （ F. tularensis） LVS by mouse macrophages. A shuttle plasmid, pFNLTP6 gro-gfp, was transformed by electroporation to F. tularensis LVS. Cell cholesterol was stained with Filipin Ⅲ, and caveolin-1 was detected with monoclonal antibody and visualized with Alexa 594 conjugated goat anti-mouse antibodies. F. tularensis LVS infection was analyzed using a fluorescence microscope equipped with a motorized Z-focus. In order to evaluate the effect of depletion of the membrane microdomain on F. tularensis entry into macrophages, interference with lipid-rich plasma membrane through the depletion of cholesterol was performed by methyl-β-cyclodextrin. The cholesterol-binding agent, Filipin Ⅲ, was used to detect the effect of cholesterol depletion. The results showed that cell cholesterol was co-localized with F. tularensis live vaccine strain in the early uptake stage, and both had close contact with the membrane microdomain-associated components, such as caveolin-1. F. tularensis requires cholesterol-rich membrane microdomains for entry into macrophages. These findings suggest that cholesterol-rich membrane microdomains and caveolin-1 play an important role in F. tularensis early entry into macrophages.