The Effect And Mechanism Of PknG On Promoting The Survival Of Mycobacterium In Macrophages

Objective: To understand the effect and mechanism of PknG promoting the survival of mycobacteria in macrophages through Mycobacterium smegmatis(MS)overexpressing the PknG protein of Mycobacterium tuberculosis.Methods: In this study,the PknG gene of Mycobacterium tuberculosis was amplified by PCR to construct the p MV261-PknG plasmid and the prokaryotic expression plasmid p ET28a-PknG,and the p MV261-PknG plasmid was electrotransformed to MS competence to construct Mycobacterium smegmatis(MS::PknG)overexpressing Mycobacterium tuberculosis PknG.We used PCR and Western blot technology to verify the successful construction of MS::PknG.Liquid culture method was used to determine the in vitro growth curve.At the same time,MS::PknG and MS::Vector were used to infect RAW264.7at MOI=20,and the intracellular viability of the recombinant strain was detected by counting colony forming units.RAW264.7 was infected with Mycobacterium smegmatis(MS)strain(MS::PknG)and empty strain(MS::Vector)at MOI=10,and detected the expression of related cytokines by q PCR.And then,the plasmid p ET28a-PknG was transformed into the competence E.coli BL21,and the PknG protein of Mycobacterium tuberculosis was expressed by IPTG induction and purified by nickel column.Coomassie brilliant blue staining and Western blot were used to verify the expression of PknG protein.The PknG protein was applied to LPS-stimulated macrophages RAW264.7,then we used q PCR to detect the effect of Mycobacterium tuberculosis PknG protein on inflammatory cytokines.Results: The pMV261-PknG and p ET28a-PknG plasmids were successfully constructed.We used PCR and Western blot to verify the successful construction of the Mycobacterium smegmatis strain(MS::PknG)overexpressing Mycobacterium tuberculosis PknG.There is no significant difference between the MS::PknG and MS::Vector in the in vitro growth rate.After the recombinant strain infected the macrophages,the colony forming unit count results suggested that PknG can enhance the survival ability of the recombinant strain in the macrophages.The inflammatory cytokines of infected macrophages were measured from the m RNA level,and the results showed that MS::PknG significantly reduced the expression levels of cytokines TNF-?,IL-6,IL-12,and IL-1? in infected macrophages.Then we transformed the prokaryotic expression plasmid p ET28a-PknG into BL21 competence.After IPTG induced expression and nickel column purification,PknG protein was successfully obtained.The q PCR results suggested that the PknG protein can inhibit the expression of cytokines IL-6 and TNF-? induced by LPS,and as the concentration of PknG increased,the inhibitory effect became more significant.Conclusion: The growth rate of Mycobacterium smegmatis in vitro is not affected after overexpression of PknG,but its intracellular survival ability is significantly enhanced after infection of macrophages,indicating that Mycobacterium tuberculosis PknG can promote the survival of mycobacteria in macrophages.Mycobacterium smegmatis that overexpressed and prokaryoticly expressed PknG protein can inhibit the release of IL-6and TNF-? in infected macrophages.It is speculated that PknG may escape the host immune attack by inhibiting the expression of inflammatory cytokines,thereby promoting the survival of Mycobacterium tuberculosis in macrophages.This study further explored the pathogenic mechanism of tuberculosis and provided a basis for targeted therapy.