Proteomic Study Of M.tb Infected Macrophages And Exploration Of Fast-growing M.tb Strains Construction

Tuberculosis(TB),caused by Mycobacterium tuberculosis(M.tb),is the second infectious disease in the world,because of its morbidity and mortality.However,the infection mechanism of M.tb remains to be elucidated now.In this study,we used TMT(Tandem Mass Tag)quantitative proteomics to investigate the proteome of M.tb infected macrophages.We identified some macrophage proteins that are regulated by the virulent factor of M.tb..These proteins possibly have great effect on the fate of macrophages.These findings would contribute to further illustrate how macrophages reponse to the infection of M.tb.The main results are as follows:1)In the proteomic studies of M.tb infected macrophages,6,702 proteins were identified;H37Ra and H37Rv caused 235 differentially expressed proteins in macrophages.These proteins are related to the biological processes of apoptosis,coagulation and oxidative phosphorylation and indicate four possible different mechanisms of macrophages infected by two M.tb strains respectively.2)Among the above differential expressed proteins,AHSG was negative acute-phase reactant.AHSG may help M.tb to escape from macrophage killing by knockdown experiment.The coagulation factor F10 was one factor of blood coagulation cascade,which could bind to virus surface and active immune response.F10 would take part in the M.tb infection by binding to the surface of M.tb by ELISA and immunofluorescence.This research revealed the function of AHSG and F10 during M.tb infecting macrophages for the first time.3)Interferon-y(IFN-y)is a key factor in macrophage immune response.After M.tb infection,IFN-y acitivated macrophages produced 257 differentially expressed proteins comparing with those not treated macrophages.These proteins are related to such biological processes as apoptosis,antigen presentation and coagulation.The results show that IFN-y obviously enhances the host immune response against M.tb by improving the ability of macrophage antigen presentation after infection.Additionally,as the slow growth of M.tb(about 1/50-1/70 of E.coli growth rate)significantly hinders the diagnosis,treatment and scientific research of tuberculosis,we try to construct fast-growing M.tb strains in this study.M.smegmatis is one of Mycobacterium sp with rapid growth.Therefore,we firstly constructed the cosmid library of M.smegmatis.Then transformed the library into M.tb.We considered the original metabolic network of M.tb would be changed by the heterologous expression of M.smegmatis genes in M.tb and thus create fast growing M.tb.Large amount of M.smegmatis derived genes,were lost in M.tb.We believe this project would be finished by constructing of M.tb strain,the DNA recombinant enzyme activity and DNA limited modification system of which were weak.