SQSTM1蛋白在嗜肺军团菌感染RAW264.7细胞自噬中的作用机制

doi:10.6040/j.issn.1671-7554.0.2023.0167

摘要/Abstract

摘要： 目的以SQSTM1蛋白(P62)基因敲除及过表达的RAW264.7小鼠巨噬细胞为研究对象,建立嗜肺军团菌感染巨噬细胞模型,观察细胞内细菌增殖情况以及自噬流和自噬小体的变化,检测自噬相关因子表达水平变化,探讨P62在嗜肺军团菌感染RAW264.7巨噬细胞自噬中的作用机制。方法嗜肺军团菌以感染复数10、50和100感染RAW264.7巨噬细胞组、KO-P62细胞组、OE-P62细胞组,同时以未加嗜肺军团菌感染作为对照组; 细菌增殖实验观察嗜肺军团菌在巨噬细胞内的增殖情况; 透射电镜观察嗜肺军团菌与细胞共培养12 h时RAW264.7巨噬细胞组、KO-P62细胞组、OE-P62细胞组的自噬小体、自噬溶酶体及相关细胞器等超微结构; pmCherry-C1-EGFP-LC3B双荧光指示系统检测巨噬细胞自噬流的变化;采用免疫印迹试验(Western blotting)及实时荧光定量(RT-qPCR)法检测RAW264.7巨噬细胞组、KO-P62细胞组、OE-P62细胞组P62、自噬相关基因AMBRA1、溶酶体关联膜蛋白2(LAMP2)、自噬相关蛋白5(Atg5)、自噬效应蛋白1(Beclin1)、自噬微管相关蛋白轻链3(LC3B)的表达水平。结果细菌增殖实验检测在RAW264.7细胞组内嗜肺军团菌的数量随着时间的增长逐渐增长,在KO-P62细胞组及OE-P62细胞组内嗜肺军团菌的数量随着时间的增长均逐渐减少; 透射电镜可观察到嗜肺军团菌感染RAW264.7细胞组后,自噬小体及自噬溶酶体减少,与RAW264.7细胞组相比,KO-P62细胞组及OE-P62细胞组自噬小体及自噬溶酶体均增多;pmCherry-C1-EGFP-LC3B双荧光指示系统检测自噬流结果显示,嗜肺军团菌感染RAW264.7 细胞组,自噬流均减弱,KO-P62细胞组及OE-P62细胞组自噬流增加; Western blotting及RT-qPCR结果显示,与RAW264.7细胞组相比:KO-P62细胞组Beclin1先升高后降低,P62基本不表达,LC3Ⅱ/Ⅰ比值、AMBRA1、LAMP2及Atg5蛋白表达均明显升高,差异有统计学意义(P<0.05);OE-P62细胞组Beclin1先降低后升高,P62、AMBRA1、LAMP2、Atg5及LC3Ⅱ/Ⅰ比值均明显上调,差异有统计学意义(P<0.05)。结论敲除、过表达P62均可抑制嗜肺军团菌在RAW264.7巨噬细胞内的增殖,促进自噬,其机制可能与Atg5-P62-AMBRA1信号通路有关。

关键词: 嗜肺军团菌, 感染, 巨噬细胞, SQSTM1, 自噬

Abstract: Objective To explore the role and mechanism of P62 in the autophagy of RAW264.7 macrophages infected with Legionella pneumophila. Methods RAW264.7 macrophages, KO-P62 cells and OE-P62 cells were infected with Legionella pneumophila at multiplicities of infection(MOI)of 10, 50, and 100; unaffected groups were set as controls. The proliferation of Legionella pneumophila in macrophages was observed with bacterial proliferation assay. After co-culture with Legionella pneumophila for 12 h, the ultrastructure of autophagic vesicles, autophagic lysosomes and related organelles in the RAW264.7 group, KO-P62 group and OE-P62 group were observed with transmission electron microscopy. The changes in the autophagic flow of macrophages were detected with pmCherry-C1-EGFP-LC3 B dual fluorescence indicator system. The expression levels of autophagy-related factors, P62, AMBRA1, LAMP2, Atg5, Beclin1 and LC3 B in each group were detected with Western blotting and RT-qPCR. Results Bacterial proliferation assay detected that the number of Legionella pneumophila in RAW264.7 cells gradually increased over time, but decreased in KO-P62 and OE-P62 cells. Transmission electron microscopy showed that after Legionella pneumophila infected RAW264.7 cells, the autophagosomes and autolysosomes decreased; compared with the RAW264.7 group, the KO-P62 and OE-P62 groups had increased autophagosomes and autolysosomes. The results of pmCherry-C1-EGFP-LC3B dual fluorescence indicator system showed that the autophagic flux decreased in the RAW264.7 group infected with Legionella pneumophila, but increased in KO-P62 and OE-P62 groups. Western blotting and RT-qPCR showed that, compared with the RAW264.7 group, the KO-P62 group had firstly increased and then decreased Beclin1, P62 was basically not expressed, and the levels of LC3II/I, AMBRA1, LAMP2 and Atg5 were significantly increased(P<0.05); the OE-P62 group had firstly decreased and then increased Beclin1, and the levels of P62, AMBRA1, LAMP2, Atg5 and LC3Ⅱ/Ⅰ were significantly increased(P<0.05). Conclusion Knockout and overexpression of P62 can inhibit the proliferation of Legionella pneumophila in RAW264.7 macrophages and promote autophagy. The mechanism may be related to the Atg5-P62-AMBRA1 signaling pathway.

Key words: Legionella pneumophila, Infection, Macrophage, Sequestosome1, Autophagy

中图分类号:

R574

郑荣慧,李攀,曹秀琴,贺瑞霞,陈民佳,陈海霞,杨志伟. SQSTM1蛋白在嗜肺军团菌感染RAW264.7细胞自噬中的作用机制[J]. 山东大学学报 (医学版), 2023, 61(6): 10-21.

ZHENG Ronghui, Li Pan, CAO Xiuqin, HE Ruixia, CHEN Minjia, CHEN Haixia, YANG Zhiwei. SQSTM1 in Legionella pneumophila infected RAW264.7 cells mechanism of autophagy[J]. Journal of Shandong University (Health Sciences), 2023, 61(6): 10-21.

参考文献

[1] Xi L, Peng M, Liu S, et al. Hypoxia-stimulated ATM activation regulates autophagy-associated exosome release from cancer-associated fibroblasts to promote cancer cell invasion [J]. Extracell Vesicles, 2021, 10(11): e12146.
[2] Chauhan D, Shames SR. Pathogenicity and Virulence of Legionella: Intracellular replication and host response [J]. Virulence, 2021, 12(1): 1122-1144.
[3] Omotade TO, Roy CR. Legionella pneumophila excludes autophagy adaptors from the ubiquitin-labeled vacuole in which it resides [J]. Infect Immun, 2020, 88(8): e00793-e00719.
[4] Thomas DR, Newton P, Lau N, et al. Interfering with Autophagy: the opposing strategies deployed by and effector proteins [J]. Front Cell Infect Microbiol, 2020, 10: 599762. doi: 10.3389/fcimb.2020.599762.
[5] Brown AS, Yang C, Hartland EL, et al.The regulation of acute immune responses to the bacterial lung pathogen Legionella pneumophila [J]. J Leukoc Biol, 2017, 101(4): 875-886.
[6] Liu X, Shin S. Viewing Legionella pneumophila pathogenesis through an immunological lens [J]. J Mol Biol, 2019, 431(21): 4321-4344.
[7] Deng ZQ, Lim J, Wang Q, et al. ALS-FTLD-linked mutations of SQSTM1/p62 disrupt selective autophagy and NFE2L2/NRF2 anti-oxidative stress pathway [J]. Autophagy, 2020, 16(5): 917-931.
[8] Berkamp S, Mostafavi S, Sachse C. Structure and function of p62/SQSTM1 in the emerging framework of phase separation [J]. Febs J, 2021, 288(24): 6927-6941.
[9] Strappazzon F, Di Rita A, Peschiaroli A, et al. HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy [J]. Cell Death Differ, 2020, 27(4): 1155-1168.
[10] Kellermann M, Scharte F, Hensel M. Manipulation of host cell organelles by intracellular pathogens [J]. Int J MolSci, 2021, 22(12): 6484.
[11] Emanuele S, Lauricella M, D'Anneo A, et al. p62: friend or foe? Evidences for oncoJanus and neuroJanus roles [J]. Int J Mol Sci, 2020, 21(14): E5029.
[12] Abd El Maksoud AI, Elebeedy D, Abass NH, et al. Methylomic changes of autophagy-related genes by Legionella effector Lpg2936 in infected macrophages [J]. Front Cell Dev Biol, 2019, 7: 390. doi: 10.3389/fcell.2019.00390.
[13] Tu W, Wang H, Li S, et al. The anti-inflammatory and anti-oxidant mechanisms of the Keap1/Nrf2/ARE signaling pathway in chronic diseases [J]. Aging Dis, 2019, 10(3): 637-651.
[14] Su H, Yang F, Fu R, et al. Cancer cells escape autophagy inhibition via NRF2-induced micropinocytosis [J]. Cancer Cell, 2021, 39(5): 678-693.
[15] Wang F, Zhang Y, Shen J, et al. The ubiquitin E3 ligase TRIM21 promotes hepatocarcinogenesis by suppressing the p62-Keap1-Nrf2 antioxidant pathway [J]. Cell Mol Gastroenterol Hepatol, 2021, 11(5): 1369-1385.
[16] He F, Huang Y, Song Z, et al. Mitophagy-mediated adipose inflammation contributes to type 2 diabetes with hepatic insulin resistance [J]. J Exp Med, 2021, 218(3): e20201416.
[17] Fan P, Xie XH, Chen CH, et al. Molecular regulation mechanisms and interactions between reactive oxygen species and mitophagy [J]. DNA Cell Biol, 2019, 38(1): 10-22.
[18] Luo J, Wang L, Song L, et al. Exploitation of the host ubiquitin system: means by Legionella pneumophila [J]. Front Microbiol, 2021, 12: 790442. doi: 10.3389/fmicb.2021.790442.
[19] Baechler BL, Bloemberg D, Quadrilatero J. Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation [J]. Autophagy, 2019, 15(9): 1606-1619.
[20] Qin QF, Li XJ, Li YS, et al. AMPK-ERK/CARM1 signaling pathways affect autophagy of hepatic cells in samples of liver cancer patients [J]. Front Oncol, 2019, 9: 1247. doi:10.3389/fonc.2019.01247.
[21] Berkamp S, Mostafavi S, Sachse C. Structure and function of p62/SQSTM1 in the emerging framework of phase separation [J]. Febs J, 2021, 288(24): 6927-6941.
[22] Jin JQ, Zhang L, Li XY, et al. Oxidative stress-CBP axis modulates MOB1 acetylation and activates the Hippo signaling pathway [J]. Nucleic Acids Res, 2022, 50(7): 3817-3834.
[23] Galati S, Boni C, Gerra MC, et al. Autophagy: a player in response to oxidative stress and DNA damage[J]. Oxid Med Cell Longev, 2019, 2019: 5692958. doi: 10.1155/2019/5692958.
[24] Jo H, Shim K, Jeoung D. Targeting HDAC6 to overcome autophagy-promoted anti-cancer drug resistance [J]. Int J Mol Sci, 2022, 23(17): 9592.
[25] Casassa AF, Vanrell MC, Colombo MI, et al. Autophagy plays a protective role against Trypanosoma cruzi infection in mice [J]. Virulence, 2019, 10(1): 151-165.
[26] Xu YF, Wan W. Acetylation in the regulation of autophagy [J]. Autophagy, 2023, 19(2): 379-387.
[27] Feng X, Zhang H, Meng LB, et al. Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5 [J]. Autophagy, 2021, 17(3): 723-742.

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