Journal of Shandong University (Health Sciences) ›› 2023, Vol. 61 ›› Issue (6): 10-21.doi: 10.6040/j.issn.1671-7554.0.2023.0167

• 基础医学 • Previous Articles    

SQSTM1 in Legionella pneumophila infected RAW264.7 cells mechanism of autophagy

ZHENG Ronghui1, Li Pan2, CAO Xiuqin3, HE Ruixia1, CHEN Minjia1, CHEN Haixia1, YANG Zhiwei1   

  1. 1. Department of Pathogenic Biology and Immunology, School of Basic Medical Science, Ningxia Medical University, Yinchuan 750004, Ningxia, China;
    2. Laboratory Department of Yinchuan Third Peoples Hospital, Yinchuan 750001, Ningxia, China;
    3. Ministry of Education Key Laboratory of Fertility Preservation and Maintenance, School of Basic Medical Science, Ningxia Medical University, Yinchuan 750004, Ningxia, China
  • Published:2023-06-06

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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

CLC Number: 

  • R574
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