Preliminary Study Of M. Tuberculosis Secreted Proteins ESAT-6 And EspB In Modulating Macrophage Functions

ESX-1 secretion system is a special protein secretion system in M. tuberculosis and critical for its virulence. ESX-1 secretion system is also named Type VII secretion system. More than five proteins have been confirmed to be secreted by ESX-1 secretion system including ESAT-6,CFP-10,EspA,EspB and EspR. ESAT-6 is one of secreted proteins found earliest, however, there is still controversy about its functions.Firstly, it is an important T-cell antigen and has protective effects on animal model infected by Mtb. Moreover, it is an important compotent of candidate vaccines and is extensively applied to the design of new Mtb vaccines and diagnostic antigen of Mtb. Secondly, ESAT-6 can affect the functions of macrophage and dentritic cell and is related to the virulence of Mtb. There is another new protein named EspB which can be co-secreted with ESAT-6 and CFP-10. The co-secretion of EspB and ESAT-6 is important for Mtb intracellular growth in macrophages and is required for inhibiting phagosome maturation. In this study, we make full use of the techniques and methods of molecular biology, Cellular Biology and Immunology to preliminaryly investigate ESX-1 system secreted proteins ESAT-6 and EspB in modulating macrophage functions.In this research, ESAT-6 and EspB genes were first seperately amplified by PCR from the genome of Mycobacterium tuberculosis H37Rv, and then cloned into pET21a(+) plasmid. The recombinant plasmids pET21a(+)-ESAT-6 and pET21a(+)-EspB that were successfully constructed were individually transformed into E.coli BL21(DE3). After induced with IPTG, the expressed recombinant proteins were confirmed by SDS-PAGE and Western blot. This vectors yielded satisfactory levels of recombinant proteins expressed as soluble proteins in E. coli. After ultrasonication, the recombinant ESAT-6 protein or EspB protein was firstly purified by a column packed with Ni-NTA Resin and then a column packed with DEAE-SepharoseTM Fast Flow matrix. The purity of the purified proteins were about 95%. The purified ESAT-6 protein was incubated with RAW264.7, and the result got by Immunofluorescence showed that ESAT-6 could directly bind to the macrophage membrane. In order to faciliate the following research work, The deleted C terminus mutant of EspB was also expressed and purified. The purified proteins ESAT-6 and EspB were seperately used to immunize BALB/c mice. Twelve stably anti-ESAT-6 specific mAb cell strains and eleven stably anti-EspB apecific mAb cell strains were obtained by hybridoma cell fusion technique.The five strains of anti-ESAT-6 cell strains and five strains of anti-EspB cell strains were seperately identified and used to prepare the ascite fluids.And the monocolonal antibodies were purified by Protein G.For studying the effects of ESAT-6 and EspB on the related functions of macrophages, RAW264.7 cell was transfected with pEGFP-C1-ESAT-6,pEGFP-C1-EspB and pEGFP-C1 by liposome respectively. After screening with a high level of G418, the macrophage cell lines that stably expressed EGFP-ESAT-6,EGFP-EspB or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and Western Blot. The results indicated that the EGFP-ESAT6 or EGFP-EspB fusion gene was integrated into the chromosome and the proteins can be stably expressed in the selected macrophage cell lines. These results gave us a tool for the future study in the mechanisms of ESAT-6 or EspB protein in modulating the macrophage cells.In order to investigate ESAT-6 and EspB in modulating macrophage phagocytosis, the macrophage cells were incubated with fluorescent beads for 2h at 5% CO2,37℃,then the samples were detected by flow cytometry. The results of data analysis indicated that cellularly-expressed ESAT-6 can remarkably enhance the macrophage phagocytosis. On the contrary, cellularly-expressed EspB have no effect on macrophage phagocytosis. The results got by the technique of confocal microscopy was consistent with the results of flow cytometry which further proved the results. In this research, the situation of cell apoptosis was also researched after cultivation for 48 h.The results got by flow cytometry showed that cellularly-expressed ESAT-6 can significantly increase macrophage apoptosis.In this reseach, we expressed and purified ESAT-6 and EspB recombinant proteins in E.coli expression system. The result got by immunofluorescence showed that ESAT-6 could directly bind to the macrophage membrane. Using the purified proteins ESAT-6 and EspB seperately to immunize BALB/c mice, several ESAT-6 and EspB monoclonal antibodies were produced; The macrophage cell lines that stably expressed EGFP-ESAT-6 or EGFP-EspB fusion protein were established; At the basis of establishment of macrophage cell lines, we have demonstrated that ESAT-6 protein can significantly enhance the phagocytosis ability of the macrophage, however, EspB have no effect on macrophage phagocytosis. Moreover, cellularly-expressed ESAT-6 can induce the macrophage apoptosis. And these results gave us tools for the future study in the interaction of the M.tuberculosis and the host and the molecular mechanisms of secreted proteins in modulating macrophage cells.