摘要
目的了解问号钩端螺旋体（简称钩体）MazEF毒素-抗毒素系统中MazF蛋白对巨噬细胞的细胞毒性及其机制。方法采用原核表达系统表达问号钩体赖型赖株MazEF毒素-抗毒素系统中的MazE和MazF蛋白（rMazE和rMazF）。采用DNA和RNA降解试验确定rMazF的DNA或RNA酶活性以及rMazE抑制rMazF酶活性的作用。采用实时荧光定量RT-PCT、Westernblot和激光共聚焦显微镜法，分别检测问号钩体赖株感染THP-1单核细胞时mazE和mazF基因转录、表达及分泌情况。采用CCK-8法和流式细胞术检测mazF基因产物对THP-1细胞的细胞毒性及死亡方式。结果rMazF能水解问号钩体赖株和THP-1细胞的RNA，但无DNA酶活性，rMazE能抑制rMazF的RNA酶活性。感染THP-1细胞后，mazE-mRNA和mazF-mRNA及MazE和MazF蛋白表达水平显著升高，但仅有MazF蛋白外分泌。mazF基因产物对THP-1细胞有细胞毒性并引起细胞坏死。结论MazEF毒素-抗毒素系统中具有RNA酶活性的MazF蛋白是问号钩体的毒力因子，可引起感染的巨噬细胞坏死。
Objective To investigate the cytolytic effects of MazF protein on macrophages and its mechanism mediated by MazEF toxin-antitoxin system in Leptospira interrogans. Methods Prokaryotic ex- pression system was established to express MazE and MazF proteins （rMazE and rMazF） of MazEF toxin-an- titoxin system in L. interrogans serovar Lai strain. The DNase or RNase activity of rMazF as well as the in- hibitory effects of rMazE on the enzymatic activity of rMazF was determined by DNA or RNA cleavage test. Human THP-1 macrophages were infected with L. interrogans Lai strain and laser confocal microscopy was performed to measure the secretion of Maze and MazF in cytoplasm. The transcription and expression of mazE and mazF were detected by real-time fluorescent quantitative RT-PCT and Western blot assay respec- tively. CCK-8 assay and flow cytometry were applied to analyze the cytotoxicity of mazF gene products on THP-1 cells and apoptosis/necrosis of the macrophages. Results rMazF hydrolyzed the RNAs but not the DNAs from L. interrogans Lai strain and THP-1 cells, which could be inhibited by rMazE. After infection of THP-1 cells with L. interrogans Lai strain, expressions of mazE and mazF at mRNA and protein levels were significantly elevated, but only the MazF protein was found in cytoplasma. The products of mazF gene had cytolytic effects on THP-1 cells and resulted in cell necrosis. Conclusion MazF protein with RNase activity is a virulence factor of L. interrogans in the MazEF toxin-antitoxin system, which could cause necrosis of the infected maerophage.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2013年第8期561-567,共7页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目（81171534,81271781）
浙江省卫生高层次创新人才培养工程项目（[2012]241）
