The Inhibitory Effects Of The Virulence Factor Secretory Acid Phosphatase Of Mycobacterium Tuberculosis (SapM) On The Autophagy Of Murine Macrophages

Objective:To establish a mice macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3(RFP-GFP-LC3) reporter protein, which provide materials for studying autophagy. To investigate the effects of the virulence factor secretory acid phosphatase of Mycobacterium tuberculosis (SapM) on the autophagy of murine macrophages. To study the role of Rab7 in the blockage of autophagosome-lysosome fusion induced by SapM, a virulence factor of mycobacterium tuberculosis.Methods:1.A lentiviral vector containing RFP-GFP-LC3 gene was constructed by general molecule clone methods.Then the constructed plasmind and the packaging plasmids were packaged in HEK293T cells. The viral supernatant was collected and transfered into RAW264.7 cells. The RAW264.7 cell line with stable expression of RFP-GFP-LC3 was screened by puromycin. Fluorescence ratio was analyzed with flow cytometry and fluorescent microscopy separately. Observing the number of RFP-GFP-LC3 puncta inside cells under florescence microscopy after starvation treatment.2.GFP-LC3-RAW264.7 cells were treated with SapM, wortmannin, or starvation. Then the formation of autophagosomes was observed by fluorescence microscope, and the level of microtubule-associated proteins light chain 3Ⅱ (LC3Ⅱ) was detected using western blotting.3.The RAW264.7 cells were transfected with siRab7, and the P62 were detected using western blot. After transfected with mCherry-SapM, RAW264.7 cells were used to the co-localization detection of SapM with Rab7 using immunohischemistry and the interaction of SapM with Rab7 using co-immunoprecipitation. SapM-mutants including SapM△areA, SapM△pReD and SapM△CT were used to transfect RAW264.7 cells, and their relationship with Rab7 was analyzed.Results:1.The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were constructed after transduced by viral supernatant and screened by purocymin. Fluorescent microscopy and flow cytometry demonstrated the ratio of red fluorescent and green fluorescent are reach 100 percent. The number of autophagic puncta was significantly increased after starvation treatment.2.Both starvation and SapM resulted in an increased numbers of GFP-LC3 puncta and LC3Ⅱ. The level of LC3Ⅱ was not increased by adding chloroquine into SapM-treated cells.3.The treatment of siRab7 induced a significantly increase of P62 in these cells. Intracellular co-localization of SapM and Rab7 was found in immunohischemistry assay. Co-immunoprecipitation showed that SapM and Rab7 can be precipitated by each other. Only SapMA△CT failed to interact with Rab7 among the three SapM-mutants.Conclusion:The RAW264.7 cell line with stable expression of RFP-GFP-LC3 was successfully constructed, which provided a reliable cell platform for detecting autophagy. SapM can induce the blockage of autophagosome-lysosome fusion, by which the autophagy of murine macrophages was inhibited. The blockage of autophagosome-lysosome fusion induced by SapM is dependent on the interaction between SapM and Rab7.