The Effect Of Pks12 Knockout On The Survival Of Mycobacterium Tuberculosis And Its Mechanism

Objective: To investigate the effect of pks12(Rv2048c)gene of Mycobacterium tuberculosis on the survival of Mycobacterium tuberculosis in macrophages and understand its mechanism.Methods: pks12 gene in H37 Rv was knocked out by using phage-mediated Mycobacterium tuberculosis gene knockout technique.The morphological changes of the knockout strains were observed by performing acid fast staining and solid culture,and the growth of the knockout strains was observed by measuring the growth curve.Comparing the influence of H37Rv?Rv2048c and H37Rv-WT(wild type)on the survivability of Mycobacterium tuberculosis in macrophages.Investigating the effect of pks12 on the expression levels of inflammatory cytokines in infected macrophages via real-time PCR and Elisa at m RNA and protein levels,and then Western blot was performed to further understand the relevant inflammatory signaling pathways of regulating the expression of inflammatory cytokines,in order to further explore the pathogenesis of tuberculosis.Results: The pks12 gene was successfully knocked out in H37 Rv strain.The knockout of pks12 gene in H37 Rv showed a slowed growth rate of Mycobacterium tuberculosis,and the colony form changed from R-type to S-type.The survival ability of H37Rv?Rv2048c strain in macrophages was significantly lower than that of H37Rv-WT.The results from m RNA and protein level indicated that H37Rv?Rv2048c promoted the expression of TNF-? and IL-6 in the macrophages infected by Mycobacterium tuberculosis significantly,and the difference was statistically significant.The phosphorylation levels of p65 protein and I?B? in NF-?B signaling pathway were significantly up-regulated.Conclusion: The deletion of pks12 could promote the secretion of cytokines TNF-? and IL-6,which is beneficial to the host cells to clear M.tuberculosis,but it is not conducive for M.tuberculosis to escape host cell' immune attack and to survive in macrophages.The mechanism of M.tuberculosis survival may relate to pks12 through its regulating the expression of inflammatory cytokines in macrophages via NF-?B signaling pathway.These findings could help to more understand the pathogenesis of tuberculosis and provide a new target for drug research and development.