Effects Of Virulence-related Factors PhoP And EsxH Of Mycobacterium Avium On Apoptosis And Construction Of Its Mutants Harboring Defective Phop And Esxh Gene

BACKGROUND & OBJECTIVE: Mycobacterium avium is the most important pathogen in non-tuberculosis mycobacteria.The infection of Mycobacterium avium often associated with patients suffered from AIDS.In recent years,the infection rates of Mycobacterium avium is increasing with the increased of AIDS.PhoP is an effect modulator of the PhoP-PhoR two-component system,with a range of regulatory functions that playing an important role in the virulence of Mycobacterium avium.EsxH,a member of the Esat-6 family proteins,is secreted in the early culture of mycobacteria and have strong immunological activities.Studies have shown that Esat-6 family proteins are closely related to the survival,reproduction and pathogenesis of Mycobacterium tuberculosis in macrophages.In order to study the role of PhoP and EsxH in Mycobacterium avium,PhoP and EsxH protein was expressed inprokaryotic expression system and they effect on U937 cells apoptosis was investigated.In addition,PhoP and EsxH gene deletion mutant strains were constructed which laid the fundamental for further study of they regulatory mechanism and provided a new target for development of new drugs.METHODS: To investigate the effect of PhoP and EsxH on cell apoptosis,the PhoP and EsxH coding sequences were amplified by PCR.The recombinant vectors p GEX-PhoP and p GEX-EsxH were constructed by ligated PhoP and EsxH fragment with the expression vector pGEX-4T-3.The recombinant expression vector pGEX-PhoP and pGEX-EsxH were transformed into E.coli BL21(DE3),and the target proteins were induced by IPTG.The GST-PhoP and GST-EsxH fusion proteins were purified by glutathione-agarose gel column after the cells were disrupted by ultrasonic disruption.The purified PhoP and EsxH proteins were culture with U937 cells and then the apoptotic rates were measured by flow cytometry.To further study the function of PhoP and EsxH,the PhoP and EsxH mutant strains was constructed by designed two pairs of specific primers according to the PhoP gene and the EsxH gene sequence individually.And the upper and down-stream fragments of the PhoP gene and the EsxH gene were amplified by PCR.Then the upstream and downstream fragments were ligated to constructed PhoP and EsxH gene deletion homologous fragments individually.The homologous fragments were then cloned into pGMB151 suicide plasmid to constructed recombinant suicide plasmid.The recombinant suicide plasmid was transformed into Mycobacterium avium by electroporation.Because the pGMB151 suicide plasmid has sacB activity,it could be induced to“suicided” and led to homologous recombination under 5% sucrose.And then the mutant strains were screened by PCR.RESULTS: The PhoP and EsxH genes PCR products were measured by agarose gel electrophoresis and sequencing,results showed that the size of PhoP fragment was 720 bp and the size of EsxH fragment was 294 bp.These fragments were cloned into the expression vector pGEX-4T-3 and induced by IPTG for expressions.10% SDS-PAGE results showed that the molecular weight of GST-PhoP fusion protein was 52 kDa and the GST-EsxH fusion protein was 36.5 kDa.Flow cytometry results showed that the apoptotic rate of the treated group was higher than that of the control group,with statistical significance(P <0.05).The results indicated that PhoP and EsxH could promote the apoptosis of U937 cells,and the apoptotic rate increased with the increase of PhoP and EsxH protein concentration.The upstream homologous fragment F1(408 bp)and downstream homologous fragment F2(618 bp)of PhoP,and the upstream homologous fragment F3(501 bp)and downstream homologous fragment F4(516 bp)of the EsxH gene were amplified by PCR.F1 and F2 were ligated to constructed PhoP gene deletion homologous fragment F1-F2(1032 bp),and F3 and F4 wereligated to constructed EsxH gene deletion homologous fragment F3-F4(1023 bp)under T4 DNA ligase.The recombinant pGMB151 suicide plasmid harboring PhoP gene and EsxH gene defective homologous fragment was successfully constructed by inserting the fragment into pGMB151 suicide plasmid.And verified by PCR and BamHI digestion.The recombinant plasmids were transformed into Mycobacterium avium for homologous recombination.After homologous recombination,a deletion of 309 bp of the defective PhoP gene and a deletion of 294 bp of the defective EsxH gene were confirmed by PCR and DNA sequencing.The deletion site Which is replaced by six bases of EcoRI site GAATTC.CONCLUSION: PhoP and EsxH proteins were expressed and purified,and PhoP and EsxH could promote the apoptosis of U937 cells.The PhoP and EsxH gene deletion mutants of Mycobacterium avium were constructed,which laid the foundation for further study of its function.