The Molecular Mechanism Of MTB Rv0309 Suppresses Host Innate Immune Responses

Mycobacterium tuberculosis is one of the leading pathogens causing tuberculosis(TB)in humans and it is one of the ten most deadly diseases in the world.Mycobacterium tuberculosis can not be eliminated by the body to a large extent due to the ability of the tuberculosis to regulate the host immune response.In Mycobacterium tuberculosis complex,M.tuberculosis and M.bovis can be pathogenic,while attenuated BCG is an anti-tuberculosis vaccine.The region of difference(RD)is between virulent and vaccine strains.In different regions,studying the differences between these bacterial genomes can provide ideas for revealing the differences between the pathogenicity of virulent and vaccine strains.Using the macrophage model and RD region protein expression library constructed in our laboratory,the Rv0309 protein that can affect the host immune response was screened,to study its biological significance and regulating cell signaling pathways will lay a foundation for further revealing the immune mechanism and pathogenesis of Mycobacterium tuberculosis.The prokaryotic expression Rv0309 protein was exposed to LPS-stimulated RAW264.7 cells.The content of IL-6 in the culture supernatant was detected by ELISA.Compared with the control group,Rv0309 inhibited LPS-induced IL-6 secretion in a dose-dependent manner.In this study,recombinant M.smegmatis and recombinant BCG strains that overexpressed Rv0309 protein were successfully constructed,and Western blot was used to verify that Rv0309 protein was successfully expressed in recombinant strains.RAW264.7 cells were infected with the Rv0309 recombinant strain and the empty strain at a MOI of 10:1,RNA samples and cell supernatants were harvested.Real-time fluorescence quantitative RT-PCR and ELISA showed that the recombinant strain could inhibit the expression and production of macrophage inflammatory cytokines compared with the control group,and the statistical difference was significant.RAW264.7 cells were infected with the Rv0309 recombinant strain and the empty strain at a MOI of 10:1 and the protein samples were collected.Western blot analysis showed that the recombinant strain could inhibit the expression of TLR2 and the phosphorylation of MAPKs signaling molecules ERK and JNK compared with the control group.RAW264.7 cells were infected with Rv0309 recombinant strain and empty strain at a MOI of 10:1.The anti-phagocytic capacity and intracellular viability of the recombinant strain were detected at 37°C.The results demonstrate that Rv0309 can significantly enhance the anti-phagocytic ability and intracellular viability of the recombinant strain.The adhesive ability of the recombinant strain was detected at 4°C,and it was confirmed that Rv0309 can significantly enhance the adhesion ability of the recombinant strain.In this study,allelic exchange plasmids containing the homologous arms upstream and downstream of Rv0309 were constructed.This plasmid was ligated to the vector phAE159 containing the mycobacterial thermo-sensitive phage element.Screening and identification of positive clones using resistant plates,the results of identification showed that the BCG knockout strain of Rv0309 gene was successfully constructed.