The Influence Of Brucella On Autophagic Lysosomal Degradation Pathway

Brucellosis is a chronic zoonotic infectious disease which caused by Brucella. Brucellosis is widespread in the world, seriously threaten public health and safety, and causes great loss of animal husbandry economy. Macrophages are the primary target cells when Brucella infects the host. Brucella infection can stimulate Atg5-independent autophagy, but evade the lysosomal degradation. The autophagy-related protein Atg5 and the domain AIR of membrane fusion-associated protein Tecpr1 play an important role in promoting the integration of autophagy and lysosome. But the role of these two proteins in autophagy which induced by Brucella is unclear.Objective: To explore the effects of Atg5 and the domain AIR in the fusion of autophagosomes and lysosomes on atypical autophagy induced by Brucella infection and intracellular survival of Brucella in murine macrophages RAW264.7.Methods: The lentiviral vectors for expression Atg5 and silence AIR were built respectively by molecular biology methods. Briefly the recombinant lentivirus was packaged by co-transfecting 293 T cells using recombinant lentivirus plasmid, packaging plasmid and envelope plasmid. After transfection of 48 h, green fluorescent was observed to check the virus packaging. The expression of Atg5 and AIR were detected by QRT-PCR after the packaged virus infection of RAW264.7. Brucella 16 M respectively infect Atg5 overexpressed and AIR silenced RAW264.7, and infect normal RAW264.7 cell for blank control. After infection of 4h, 12 h, 24 h, 48 h, the number of intracellular Brucella were detceted by CFU. After infection of 24 h, the fusion rate of intracellular autophagosome and lysosome was detected by laser confocal microscope. After infection of 24 h, the expression of Autophagy-related protein LC3Ⅰ/Ⅱ and p62 at the m RNA level were detected by QRT-PCR, the expression of LC3Ⅰ/Ⅱ and p62 at the protein level were detected by Western blot.Results: Identification of restriction endonuclease double digestion, and DNA sequencing showed that recombinant lentivirus plasmid of Atg5 and AIR were successfully constructed. Many green phosphor dots in cells were observed under the fluorescence microscope after the recombinant lentiviruses transfected cells, which demonstrated the recombinant lentiviruses knocking-down AIR gene were packaged successfully. QRT-PCR results indicated that lentiviral vector of expression of Atg5 and silent of AIR were successfully transfected. In the Brucella infected Atg5 over-expression RAW264.7 cells group, Confocal laser scanning results showed the fusion of Autophagy and lysosomes is obviously higher than the blank carrier group and the control group(p < 0.05). Laser confocal microscop scanning results showed the fusion of Autophagy and lysosomes was obviously higher than the blank carrier and the control group(p < 0.05). Compared with the control group, QRT-PCR and Western blot experiments showed that the expression of Autophagy-related protein LC3 I/II and p62 were declined in Brucella infected Atg5 over-expression and AIR knock-down RAW264.7 cells(p < 0.05). Compared with the control group, the number of intracellular bacteria(CFU) was increased in the Atg5 over-expression RAW264.7 cells(p < 0.05), also CFU was inscreased in the AIR knock-down RAW264.7 cells(p < 0.05).Conclusion: The over-expression of Atg5 and the AIR silence through promoting the fusion between autophagosome and lysosome reduced the viability of Brucella in host cells in a certain extent. This study investigate how Brucella block the fusion of autophagosomes and lysosomes which laid the foundation for survival mechanism within the host cell.