The Inhibitory Of 6 KD Early Secretory Antigen Target(ESAT6) On The Autophage Of Murine Macrophages

Objective:6 kD early secretory antigenic target through activation of the mammalian target of rapamycin inhibit autophagy,thus promoting the growth and proliferation of BCG,to explore the interaction relationship between ESAT6 and the fusion of lysosomes autophagosomes,provide experimental basis for revealing ESAT6 mediated immune escape.Methods:1.The RAW264.7 cells transfected with pCMV-HA-ESAT6 plasmid of different time,cell lysis,BCA method to measure the protein concentration and Western Blot method to detect the amount of LC3? and LC3 ?.Then,the mouse macrophage cells RAW264.7 were transfected with pCMV-HA-ESAT6 recombinant plasmid,chloroquine(CQ),pCMV-HA control plasmid and CQ combine pCMV-HA-ESAT6,Fluorescence microscopy was used to record the results,and use computer software to count;cell lysis,measure the protein concentration by BCA method and Western blot assay the amount of LC3? and LC3 ?.2.RAW264.7 cells were treated with EBSS(Earle balanced salt solution)and transfected with pCMV-HA-ESAT6 recombinant plasmid,pCMV-HA plasmid,then cell lysis,and inoculated on the culture medium,the growth of BCG was observed.Then,the mouse macrophage RAW264.7 transfected with pCMV-HA-ESAT6 recombinant plasmid,chloroquine(CQ),pCMV-HA control plasmid and CQ,pCMV-HA-ESAT6 combined treatment,under this conditions,the cells were lysised,inoculated in Lowenstein-Jenden medium,to observe the growth of BCG3.The mouse macrophage cells RAW264.7 were treated with pCMV-HA-ESAT6,then the cells were lysised,tested protein concentration,the protein level of p-mTOR,p62 LC3 ??LC3? detected by Western Blot.Then use the mTOR inhibitor Torin1 and ESAT6 treatment RAW264.7 cells,the lysosome was stained with LysoTracker Red,and the number of lysosomes was counted by computer software;the amount of p-70S6K,p62 and p-mTOR were detected by the method of protein immunoblotting;the cells were cultured for BCG after lysis.Results:1.The mouse macrophage cells RAW264.7 were transfected with ESAT6 recombined plasmid,as time goes on,the level of LC3? gradually increased;for the three groups of RAW264.7 mouse macrophage treated with ESAT6 and CQ,the amount of LC3?was similar,and there was no significant difference between the three groups,but there were all higher than the control group,the results of fluorescence microscopy showed that the number of autophagy in ESAT6 and CQ treated group was similar,but higher than that in control group.2.The growth of BCG treated by EBSS was lower than that in the control group,the growth of BCG in ESAT6 group was more than the control group,the growth of BCG treated by CQ and ESAT6 was higher than that in the control group,and the growth of the three groups were similar.3.In the ESAT6 transfection group,the result of Western Blot showing,compared to the control group the level of p-70S6K,p-mTOR,LC3 ?and p62 were increased;the mouse macrophage cells RAW264.7 were treated with ESAT6 and Torinl,Western Blot showed that p62 and p-mTOR decreased than those treated with ESAT6 alone,LysoTracker Red dye assay results showed the presence of Torinl alone or co existing with ESAT6,the dyeing recovery due to the ESAT6 treatment caused reducing;BCG shows that Torin1 alone or combined with ESAT6,the growth of BCG were reduced compared with ESAT6 treatment alone.Conclusion:This study shows that MTB secreted virulence factor ESAT6(6kD early secretory antigenic target)can inhibite the fusion of autophagy and lysosomal by induce mTOR activation,thereby inhibiting autophagy flow,.and can promote the proliferation of BCG in macrophages...