RESUMO
Objective To explore the interaction between 6 kD early secretory antigenic target (ESAT6) and autophagy in order to provide an experimental basis for the immune evasion mediated by ESAT6. Methods RAW264.7 cell lines (mouse macrophages) were treated with Earle's balanced salt solution (EBSS), and another cells were transfected by control plasmid pCMV-HA or recombinant plasmid pCMV-HA-ESAT6. Then we observed the growth of Bacillus Calmette-Guerin (BCG) in macrophages. Western blotting was used to detect the LC3 levels in RAW264.7 cells at 0, 8, 12, 32 hours after transducted by pCMV-HA-ESAT6. In RAW264.7 cells transfected with PCMV-HA, PCMV-HA-ESAT6, and treated with chloroquine (CQ) and CQ combined with pCMV-HA-ESAT6, which were lysed and cultured in Lowenstein-Jensen culture medium for BCG counting, LC3 was detected by Western blot analysis, and the number and size of lysosomes were observed by LysoTracker Red staining. Results Compared with control plasmid pCMV-HA transfected RAW264.7 cells, the number of BCG significantly increased in PCMV-HA-ESAT6-transfected cells, while decreased in EBSS-treated cells. PCMV-HA-ESAT6 transfection resulted in the increased transition of LC3 I to LC3 II in a time-depended manner. Compared with the controls, LysoTracker Red staining showed PCMV-HA-ESAT6, CQ and CQ plus PCMV-HA-ESAT6 transfections resulted in the increased number and size of lysosomes, and there were no differences among the three groups. Moreover, the growth potential of BCG was strong in the three transfection groups. Conclusion ESAT6 can inhibit the autophagy and promote the growth of BCG in RAW264.7 cells.
Assuntos
Antígenos de Bactérias/imunologia , Autofagia , Proteínas de Bactérias/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Tuberculose/microbiologia , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Mycobacterium tuberculosis/genética , Células RAW 264.7 , Tuberculose/imunologiaRESUMO
Objective To study the role of Rab7 in the blockage of autophagosome-lysosome fusion induced by secretory acid phosphatase (SapM), a virulence factor of mycobacterium tuberculosis. Methods The Raw264.7 cells were transfected with siRab7, and the P62 was detected using Western blotting. After transfected with mCherry-SapM, the co-localization of SapM and Rab7 in Raw264.7 cells was detected by immunofluorescence cytochemistry and the interaction of SapM with Rab7 was determined by co-immunoprecipitation. SapM mutants including SapM(δ ARCA), SapM(δ FRED) and SapM(δ CT) were used to transfect Raw264.7 cells, and their associations with Rab7 were analyzed. Results The treatment of siRab7 induced a significant increase of P62 in these cells. Immunofluorescence cytochemistry showed the intracellular co-localization of SapM and Rab7. Co-immunoprecipitation showed that SapM and Rab7 were precipitated by each other. Only SapM(δ CT) failed to interact with Rab7 among the three SapM mutants. Conclusion The inhibition of autophagosome-lysosome fusion induced by SapM is dependent on the interaction between SapM and Rab7.
Assuntos
Fosfatase Ácida/metabolismo , Autofagossomos/enzimologia , Proteínas de Bactérias/metabolismo , Lisossomos/enzimologia , Mycobacterium tuberculosis/enzimologia , Tuberculose/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Fosfatase Ácida/genética , Animais , Proteínas de Bactérias/genética , Humanos , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos , Mycobacterium tuberculosis/genética , Ligação Proteica , Células RAW 264.7 , Tuberculose/genética , Tuberculose/microbiologia , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
Objective To investigate the effect of secretory acid phosphatase as a virulence factor of Mycobacterium tuberculosis (SapM) on the autophagy of murine macrophages. Methods GFP-LC3-Raw264.7 cells were treated with SapM, wortmannin, or starvation. Then the formation of autophagosomes was observed under a fluorescence microscope. The level of microtubule-associated protein 1 light chain 3 (LC3) II was detected using Western blotting. After chloroquine was added in the SapM-treated cells, LC3II level was again tested by Western blotting. Results Both starvation and SapM increased the number of GFP-LC3 puncta and the level of LC3 II. There was no further increase of LC3 II level in SapM-treated cells after chloroquine addition. Conclusion SapM can block autophagosome-lysosome fusion and inhibit autophagy of murine macrophages.
Assuntos
Fosfatase Ácida/metabolismo , Autofagia/fisiologia , Proteínas de Bactérias/metabolismo , Macrófagos/metabolismo , Fosfatase Ácida/genética , Animais , Antimaláricos/farmacologia , Autofagossomos/metabolismo , Autofagia/genética , Proteínas de Bactérias/genética , Western Blotting , Linhagem Celular , Cloroquina/farmacologia , Meios de Cultura/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mycobacterium tuberculosis/enzimologiaRESUMO
OBJECTIVE: To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3). METHODS: A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. RESULTS: The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. CONCLUSION: The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.
Assuntos
Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Proteínas Associadas aos Microtúbulos/genética , Animais , Autofagia , Células Cultivadas , Humanos , Camundongos , Células RAW 264.7 , Proteína Vermelha FluorescenteRESUMO
The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis.
