ESX-1 secreted protein ESAT-6 of Mycobacterium tuberculosis enhances the phagocytosis of RAW264.7 macrophages

Abstract:

Abstract:［Objective］ To investigate the effects of Mycobacterium tuberculosis ESX-1 protein early secreted antigenic target of 6 kDa(ESAT-6) in modulating phagocytosis of RAW264. 7 cells.［Methods］RAW264.7 cells were transfected with recombinant plasmids pFLAG-ESAT-6 and pFLAG-EGFP by liposome． After screening with a high level of G418，the macrophage cell lines stably expressing flag-ESAT-6 or flag-EGFP proteins were obtained．The cell lines were further identified by PCR，RT-PCR and western blot．The phagocytosis of those cell lines was analyzed for ingested fluorescent beads by flow cytometry and for phagocytized Escherichia coli(E.coli) by colony count and confocalmicroscopy.［Results］We established successfully RAW-E6 cell line stably expressing flag-ESAT-6 and RAW-EGFP cell line stably expressing flag-EGFP． Flow cytometric analysis shows that the percentage of phagocytosis of RAW-E6 was higher than that of RAW264.7 and RAW-EGFP. Colony count and confocal microscopy test also show that RAW-E6 had higher phagocytosis ability than RAW264.7 and RAW-EGFP.［Conclusion］The secreted protein ESAT-6 can enhance the phagocytosis of macrophages，which provides new evidence to understand the pathogenesis of Mycobacterium tuberculosis.