嗜肺军团菌重组免疫原蛋白(IP)对RAW264.7细胞自噬的影响  

Effect of Legionella pneumophila recombinant immunogenic protein(IP)on autophagy in RAW264.7 cells

作　　者：贺瑞霞 曹秀琴[2] 陈民佳 郑荣慧 杨志伟 HE Ruixia;CAO Xiuqin;CHEN Minjia;ZHENG Ronghui;YANG Zhiwei(I Department of Pathogenic Biology and Medical Immunology,School of Basic Medical Science,Ningxia Medical University,Yinchuan 750004,Ningxia,China;Ministry-of-Educatin Key Laboratory of Fertility Preservation and Maintenance,School of Basic Medical Science,Ningxia Medical University,Yinchuan 750004,Ningxia,China)

机构地区：[1]宁夏医科大学基础医学院病原生物学与医学免疫学系,宁夏银川750004 [2]宁夏医科大学生育力保持省部级共建教育部重点实验室,宁夏银川750004

出　　处：《微生物学报》2022年第10期3886-3898,共13页Acta Microbiologica Sinica

基　　金：国家自然科学基金(82060362)。

摘　　要：【目的】观察嗜肺军团菌重组免疫原蛋白(immunogenic protein,IP)对RAW264.7细胞自噬流及自噬相关因子表达水平的影响并探讨其作用机制。【方法】采用His标签蛋白纯化试剂盒纯化嗜肺军团菌重组免疫原蛋白(IP),并用CCK-8法检测IP对RAW264.7细胞半数抑制浓度(half maximal inhibitory concentration,IC_(50))。采用低浓度(0.05×IC_(50))、中浓度(0.1×IC_(50))、高浓度(0.2×IC_(50))IP与RAW264.7细胞进行体外共培养1、3、6、12 h,并设细胞对照组,应用自噬双标质粒pmCherry-C1-EGFP-LC3B检测巨噬细胞自噬流的变化,筛选最佳浓度进行后续实验;最佳浓度IP与RAW264.7细胞进行体外共培养6、12、24 h,并设细胞对照组,RT-qPCR检测各组自噬相关蛋白Beclin1、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、SQSTM 1(sequestosome 1,p62)及组蛋白去乙酰化酶6(histone deacetylase 6,HDAC6)的mRNA表达水平;Western blotting法检测各组自噬相关因子的蛋白表达水平;免疫荧光检测各组自噬相关因子的表达。【结果】根据CCK-8结果计算IC_(50)为0.26μg/μL。自噬双标质粒pmCherry-C1-EGFP-LC3B检测自噬流结果显示,与对照组相比,中浓度(0.026μg/μL)IP与RAW264.7细胞进行体外共培养后自噬流抑制显著。RT-qPCR及Western blotting检测结果显示,与对照组相比,IP与RAW264.7细胞共培养6 h,P62表达水平升高,LC3B、HDAC6、Beclin1表达水平均降低,P<0.05;与6 h组相比,12 h组LC3B表达水平降低,P62、HDAC6及Beclin1表达水平均升高,P<0.05;与12 h组相比,24 h组Beclin1表达水平升高,P<0.05。IP一定程度上以时间依赖的方式降低了LC3-Ⅱ/LC3-Ⅰ比值并升高了P62蛋白的表达水平,P<0.05。免疫荧光结果基本与RT-qPCR及Western blotting检测结果一致。【结论】嗜肺军团菌IP抑制巨噬细胞自噬,可能与通过影响自噬小体-溶酶体融合途径,干扰自噬小体及自噬溶酶体的形成、成熟等过程有关。[Objective]To investigate the effect of Legionella pneumophila recombinant immunogenic protein(IP)on RA W264.7 cell autophagy and the mechanism.[Methods]We purified the L.pneumophila IP using the His-tag protein purification kit and determined the half maximal inhibitory concentration(IC_(50))of the IP on RA W264.7 cells with cell counting kit-8(CCK-8)assay.We co-cultured RAW264.7 cells with different concentration(0.05×IC_(50),0.1×IC_(50),0.2×IC_(50))of IP for 1,3,6,and 12 h,respectively,and set up a cell control group.In addition,we used the pmCherry-GFP-LC3B tandem fluorescent protein to monitor changes in autophagy flux in RAW264.7 cells,and chose the optimal concentration for subsequent experiments.We co-cultured RAW264.7 cells with the optimal concentration of IP for 6,12,and 24 h,separately,and established a cell control group.Apart from that,the mRNA and protein expression levels of autophagy-related factors Beclin1,microtubule-associated protein 1 light chain 3B(LC3B),SQSTM1(sequestosome 1,p62),and histone deacetylase 6(HDAC6)were detected by RT-qPCR,Western blotting,and immunofluorescence staining.[Results]The IC_(50) was calculated to be 0.26μg/μL.When RAW264.7 cells were co-cultured with medium-concentration(0.026μg/μL)IP,the autophagy flux was clearly inhibited,as monitored by pmCherry-C1-EGFP-LC3B.As revealed by the results of RT-qPCR and Western blotting analysis,when RAW264.7 cells were co-cultured with IP for 6 h,the expression level of P62 increased,but the expression levels of LC3B,HDAC6,and Beclin1 decreased(P<0.05).The expression of LC3B reduced and the expression of P62,HDAC6,and Beclin1 rose at 12 h compared with those at 6 h(P<0.05).Beclin1 expression was higher at 24 h than that at 12 h(P<0.05).According to the RT-qPCR and Western blting results,IP decreased the LC3-Ⅱ/LC3-Ⅰ ratio and increased the expression level of P62 in a time-dependent manner(P<0.05).The immunofluorescence results were essentially consistent with the RT-qPCR and Western blotting results.[Conclusion]L.pneum

关 键 词：嗜肺军团菌 免疫原蛋白 巨噬细胞 自噬 

分 类 号：R378[医药卫生—病原生物学]