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

Abstract:

[Objective] To investigate the effect of Legionella pneumophila recombinant immunogenic protein (IP) on RAW264.7 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 (IC₅₀) of the IP on RAW264.7 cells with cell counting kit-8(CCK-8) assay.We co-cultured RAW264.7 cells with different concentration (0.05×IC₅₀,0.1×IC₅₀,0.2×IC₅₀) 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 IC₅₀ was calculated to be 0.26 μg/μL.When RAW264.7 cells were co-cultured with medium-concentration (0.026 μg/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 blotting 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.pneumophila recombinant IP can inhibit autophagy flux in macrophages.Moreover,IP may affect the autophagosome-lysosome fusion pathway and interfere with the formation and maturation of autophagosome and autophagolysosome.