| In recent years, studies have confirmed that ESX-1protein secretion system is animportant virulence factor of Mycobacterium tuberculosis and closely related to the survival,growth，reproduction and virulence of Mycobacterium tuberculosis in macrophages. It hasbeen acknowledged, that a co-secreted phenomenon exists in multiple ESX-1secreted proteinsof Mycobacterium tuberculosis, the loss of a ESX-1secreted protein can affect the secretion ofother ESX-1proteins and led to a significant decrease in virulence and pathogenicity ofMycobacterium tuberculosis. Therefore, it was so difficult to study separately a ESX-1secreted protein function through gene knockout and overexpression, and it was also not clearor had different opinions for each ESX-1protein is directly involved in the pathogenesis ofMycobacterium tuberculosis and what its molecular mechanism is. This paper was mainly totake two ESX-1secretory protein ESAT-6and EspB as the research objects, to establish stablytransfected cell lines and recombinant BCG as the principal means, to examine the effects ofthe two proteins in the phagocytosis, the apoptosis and the phagosome maturation ofmacrophage, to analyze their regulation mechanism and explore their role in the pathogenesisof Mycobacterium tuberculosis. The studies will enhance the understanding for the pathogenicmechanism of ESX-1secretion system and provide new targets for TB drugs and newvaccines.First, we constructed series of mouse macrophage cell lines stably expressing ESAT-6orEspB. The research abroad showed that EspB could be cleavaged by intracellular MycP1enzyme in the secretion process and only the N terminal (EspBN) of EspB can secrete fromMycobacterium tuberculosis to bacteria outside and could be invoved in the interaction withmacrophage. In the previous research, we had established stably transfected cell linesseparately expressing EGFP-ESAT-6or EGFP-EspB fusion protein, such as RAW-E6andRAW-EspB. Taking the plasmid expressing EGFP-EspB fusion protein as a template in thestudy, we had constructed recombinant plasmid pEGFP-EspBN expressing of EGFP-EspBNfusion protein by the reverse PCR technology. By liposome mediated method, the recombinantplasmid had been transfected into mouse RAW264.7cells. We obtained RAW-EspBN cell linestably expressing EGFP-EspBN by the selection with G418and the identification at geneslevel, transcription level and protein expression level. Because of EGFP as a larger molecularweight protein and the effects of the fusion expression unclear on affecting the structure andfunction of ESAT-6and EspB, we constructed macrophage cell lines separately stablyexpressing flag-ESAT-6or flag-EspBN fusion protein with a small molecular weight flag tag.Based on previous studies, we constructed the recombinant expression plasmidspFLAG-ESAT-6, pFLAG-EspBN and pFLAG-EGFP by reverse PCR technology. After celltransfection, screening and identification had been performed, macrophage cell lines，RAW-flag-E6, RAW-flag-EspBN and RAW-flag-EGFP separately stable expressingflag-ESAT-6, flag-EspBN and flag-EGFP fusion protein，had been obtained. Further, westernblot analysis showed that EGFP-EspBN and flag-EspBN were both distributed in the cytoplasm and cell membrane, flag-ESAT-6and flag-EGFP were mainly expressed in thecytoplasm. These cell lines provide tools for studying ESAT-6and EspB regulating thefunction of macrophage and exploring molecular mechanism of the interactions between themand macrophages.Secondly, we constructed series of recombinant BCG expressing ESAT-6and EspB. Asthe only listed tuberculosis vaccine, BCG without RD-1regions in the genome is attenuatedMycobacterium bovis. It can not expressing ESAT-6and can express EspB without secretion.To eliminate the interference of other ESX-1secretion proteins, a strategy besides stablytransfection cell lines is to build recombinant BCG expressing secreted ESAT-6or EspB. TheESAT-6gene and sESAT-6gene (with Ag85B signal peptide sequence) were separately clonedinto shuttle plasmid pLYG206, to construct expression plasmids pLYG-ESAT-6andpLYG-SESAT-6. By electroporation method, the two expression vectors were transformed intoBCG. And two recombinant BCG were obtained by selection with zeocin, colony PCRanalysis and Western blot identification, such as rBCG-E6expressing intracellular ESAT-6andrBCG-SE6expressing secreted ESAT-6. Subsequently, EspB gene and EspBN gene weredirectly cloned into pLYG206to construct expression plasmids pLYG-EspB andpLYG-EspBN. With the same methods, two recombinant BCG were respectively obtained,such as rBCG-EB expressing EspB and rBCG-EN expressing EspBN. Further,western blotanalysis showed that rBCG-EN can secret EspBN to bacteria outside by an unknown waywhile rBCG-EB can express EspB in cytoplasm. These recombinant BCG provide the basis tobuild infection model of Mycobacterium tuberculosis, to explore the role of ESX-1secretionin regulating macrophage function, to construct new vaccines.We studied the effects of ESAT-6on phagocytosis of macrophages. The phagocyticfunction of macrophages is an early defensive response against pathogen invasion, andESAT-6as a early secretion virulence protein of Mycobacterium tuberculosis has beenconfirmed in modulating macrophage function through a variety of ways. So, we speculatedthat ESAT-6may also affect the phagocytosis of macrophage and investigated the effects ofESAT-6on phagocytosis of macrophages respectively by means of stably transfectedmacrophage cell lines, recombinant protein and recombinant BCG. Firstly, the analysis fromflow cytometry, confocal microscopy and colony counting showed that the phagocytosis ofRAW-E6(expressing EGFP-ESAT-6fusion protein) and RAW-flag-E6(expressingflag-ESAT-6fusion protein) for fluorescent microspheres or E.coli was significantly enhanced.Secondly, the analysis from flow cytometry showed the phagocytosis of RAW264.7cellstreated with recombinant ESAT-6protein for fluorescent microspheres was enhanced.Thirdly,the analysis from similar methods showed that the phagocytosis of RAW264.7cells infectedwith rBCG-SE6was enhanced, but rBCG-E6had no significant effects on the phagocytosis ofmacrophages. These results showed that ESAT-6can enhance the phagocytosis ofmacrophages in mice. Further, we investigated the effects of p38, IL-12and ESAT-6membrane perforation on ESAT-6improving the phagocytosis of macrophage. We found thatit does not depend on p38pathway and cell factor IL-12that ESAT-6enhanced macrophagephagocytic function but may be associated with ESAT-6membrane perforation effect.We studied the effect of ESAT-6on macrophage apoptosis. Many research reportedMycobacterium tuberculosis was able to induce macrophages and epithelial cell apoptosis. Butthere was different interpretation for the induced composition and the mechanism of macrophages apoptosis induced by Mycobacterium tuberculosis. In order to know the roles ofESAT-6on the process and explore the possible molecular mechanism, by means of flowcytometry, we investigated the apoptosis of stably transfected macrophage cell lines,macrophages treated by recombinant ESAT-6and macrophages infected with recombinantBCG. Firstly, we found that RAW-E6only cultured for48h showed apoptosis rate significantlyincreased, but cultured for24h. RAW-flag-E6cultured for48h also showed cell apoptosissignificantly increased, while the concentration of the intracellular flag-ESAT-6is about500ng/mL. Secondly,10μg/mL rESAT-6was able to induce the apoptosis of RAW264.7cells, but5μg/mL rESAT-6wasn’t. These data showed that ESAT-6induces macrophages apoptosis in adose dependent manner and macrophages apoptosis could be induced by low concentration ofESAT-6in cytoplasm. Thirdly, there was no significant difference in the apoptosis ofRAW264.7cells infected separately by rBCG-SE6，rBCG-E6and BCG for3days. Butincreasing apoptosis rate of macrophages infected by rBCG-SE6in time dependence could beobserved. Further, we found caspase-3activity of RAW-E6and RAW-flag-E6wassignificantly increased. The data showed that ESAT-6depending on Caspase-3signalingpathway at low levels in cytoplasm could induce the apoptosis of RAW264.7cells.We also studied the effects of EspB on the phagosome maturation of macrophages. Therewere some evidences that a ESX-1secreted protein except CFP-10and ESAT-6is required forthe inhibition of phagosome maturation. We presume EspB is likely to be the key molecule.By confocal microscopy and count of survival E.coli, we analyzed respectively the phagosomematuration of stably transfected cell lines expressing EspBN and macrophages infected withrecombinant BCG. Firstly, the results showed that colocalization rate of lysosomes andphagosomes in RAW-EspBN and RAW-flag-EspBN was significantly reduced while E.colisurvival rate in RAW-flag-EspBN was significantly higher than wild type RAW264.7andRAW-EGFP. Secondly, RAW-flag-E6cells infected by rBCG-EspBN showed a significantinhibition of phagosome maturation, but RAW264.7cells infected by rBCG-EspBN didn’t. Bebased on the data, we guess N terminus of EspB in cytoplasm could inhibit phagosomematuration of macrophages. But the synergistic effects of ESAT-6were required for theinhibition of EspBN in Mycobacterium tuberculosis infection process, as ESAT-6perforationeffect against phagosome membrane for helping EspBN entrying into the cytoplasm.In summary, this study successfully established series of macrophage cell linesseparately stably expressing ESAT-6or EspB and constructed series of recombinant BCGseparately overexpressing ESAT-6or EspB. They provide research tools to explore ESAT-6and EspB regulating the function of macrophage and molecular mechanism of the interactionof them with macrophages. The results showed that ESAT-6can significantly enhance thephagocytosis of macrophage, which may be related to ESAT-6membrane perforated effect.And low levels of ESAT-6can also induce the apoptosis of macrophages in mice, which isdependent on the caspase-3way. EspB through its N terminus could suppress macrophagephagosome maturation with the synergistic effect of ESAT-6, which may play an importantrole in Mycobacterium tuberculosis survival in macrophage. |
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