Construction Of Mycobacterium Tuberculosis H37Ra Heat Shock Protein 16.3 Knockout Strain And Its Effect On Mouse Bone Marrow-derived Macrophages Function

Objective:To construct the H37Ra Hsp16.3 knockout strain and the replacement strain strain,further to study the function of Hsp16.3 in the H37Ra strain and detect the expression level of inflammatory cytokines,chemotactic function and antigen presentation function in mouse bone marrow-derived macrophages infected with H37Ra Hsp16.3 knockout strainMethods:1.Constructing the HspX gene knockout strain(MUT)by homologous recombination;Electroporating pMV261-HspX plasmid into MUT strain to construct HspX gene knockout replacement strain(COMP).2.The effects of Hsp16.3 knock out on H37Ra strains were observed by comparing the colony characteristics and growth rate of wild-type strains(WT),COMP strains and MUT strains.3.The WT,COMP and MUT strains were used to infect mouse bone marrow-derived macrophages(BMDMs)in vitro to construct a latent infection model.When this model was constructed successfully and then macrophages were cultured 24h or 48h,the mRNA expression levels of inflammatory cytokines iNOS,IL-6 and TNF-?in macrophages were detected by Real-time PCR,the expression levels of iNOS were detected by Western-blot,the expression levels of IL-6 and TNF-?were detected by ELISA.4.The effect of Hsp16.3 on the chemotaxis function of BMDM was detected by transwell experiment.The expression levels of chemokines CCL2,CCL3,CCL4,CCL5and CCL12 were determined by Real-time PCR,and the number of F4/80~+CCR1~+,F4/80~+CCR2~+and F4/80~+CCR5~+cells was detected by FCM.5.BMDMs were infected with WT,COMP and MUT strain respectively at an MOI of2 at 1h,and then the effect of the strain on phagosomes were captured by transmission electron microscope;after 24h infection,the effect of strains on phagolysosomal membranes were captured by transmission electron microscope;After the latent infection model was constructed successfully,and then co-cultured with(or without CD4~+T cells),the the number of F4/80~+MHC?~+cells was detected by FCM,the expression levels of MHC?and CIITA were determined by Western-blot,the mRNA expression levels of PI,PIII and PIV were detected by Real-time PCR,and the expression levels of IL-10 and IFN-?were detected by ELISA.After the latent infection model was constructed successfully,IFN-?was added at a final concentration of100ng/ml and cultured for 48h,and then the the number of F4/80~+MHC?~+cells was detected by FCM,the mRNA expression levels of PI,PIII,and PIV were detected by Real-time PCR.BMDMs were infected with WT strain,COMP strain and MUT strain at an MOI of 2 respectively for 3h,and then co-cultured with CD4~+T cells or stimulated with IFN-?for 30 min,60 min,and 3h and the expression levels of p-STAT1 and p-STAT3 were determined by Western-blot.6.After the latent infection model was constructed successfully,and then co-cultured with or without CD4~+T cells for 72 h,the intracellular killing function of macrophages were examined.Results:1.It was found that the homologous recombination region of the MUT strain was constructed successfully.The pMV261-HspX plasmid was successfully transformed into the MUT strain.The results of Western-blot revealed that the MUT strain did not express Hsp16.3,while the COMP strain expressed Hsp16.3.2.There was no difference in the colony size of WT,COMP and MUT strains.WT and COMP strains had more cord-like protrusions than MUT strains and grew faster in the early stages of culture.In addition,there was no difference in macrophages phagocytizing WT,COMP and MUT strains.3.The deletion of Hsp16.3 increased the mRNA expression levels of IL-6 and TNF-?genes in macrophages at 24h,and there was no difference at 48h.There was no difference in iNOS at 24h,and compared with WT and COMP strain,the mRNA expression level of iNOS in MUT strain infected group was lower at 48h(P<0.05).There was no difference in protein expression levels of IL-6,TNF-?and iNOS among the infected groups.4.The deletion of Hsp16.3 caused more macrophages moving to the transwell lower room.Compared with WT and COMP strain infected group,MUT strain infected group macrophage up-regulated the mRNA expression level of chemokines CCL2,CCL3,CCL4,CCL5 and CCL12(P<0.05)and increased the number of F4/80~+CCR5~+cells(P<0.05)and F4/80~+CCR2~+cells,and there was no difference in the number of F4/80~+CCR1~+cells among all groups.5.The deletion of Hsp16.3 had no effect on the phagolysosomal membrane of macrophages,and after 24h infection,the MUT strain had less interference on the phagolysosomal membrane and the phagolysosomal membrane was more complete.The WT,COMP and MUT strains all down-regulated the number of F4/80~+MHC?~+cells,but the expression of CIITA remained unchanged.When co-cultured with CD4~+T cells,Hsp16.3 suppressed the increasing of the number of F4/80~+MHC?~+cells,suppressed the up-regulating of the protein expression levels of CIITA and the mRNA expression level of PIV in macrophages.The expression of IL-10 in MUT strains infected group was lower than that in the WT and COMP strain infection groups(P<0.05),while IFN-?was higher(P<0.05).When IFN-?was added,Hsp16.3 also suppressed the increasing of the number of F4/80~+MHC?~+cells and suppressed the up-regulating of the mRNA expression level of PIV(P<0.05).After co-cultured with CD4~+T cells,Hsp16.3 down-regulated the expression level of p-STAT1(P<0.05),while the expression of p-STAT3 was not affected;However,after the stimulation of IFN-?,Hsp16.3 down-regulated the expression level of p-STAT1 and p-STAT3(P<0.05).6.After the latent infection model was constructed successfully,the BMDMs were cultured for 72 h,and there was no difference in the number of CFU among the infection groups.When this model co-cultured with CD4~+T cells,the number of CFU in MUT strain infection group was lower than the WT strain infection group(P<0.001)and COMP strain infection group(P<0.01).Conclusion:1.H37Ra HspX gene knockout strain and H37Ra HspX gene replacement strain were constructed successfully.2.MTB Hsp16.3 is an important protein that maintains the growth of H37Ra strain and resists macrophage killing.3.MTB Hsp16.3 can regulate BMDMs the expression of IL-6,TNF-?and iNOS at the mRNA level,but has no effect on their protein expression.4.MTB Hsp16.3 can inhibit the chemotactic function of BMDMs,possibly through the CCR5/CCL3_CCL4_CCL5 and CCR2/CCL2_CCL12 signal axis to inhibit the chemotactic function.5.MTB Hsp16.3 may inhibit BMDMs the expression levels of MHC class?induced by IFN-?by down-regulating the phosphorylation of STAT1 and STAT3.