The Mechanism Of Mtb Conserved Hypothetical Protein Rv0790c On Potentiating The Survival Of Mycobacterium In Macrophages

Tuberculosis(Tuercolusis,TB)is a chronic infectious disease that threatens the respiratory system caused by Mycobacterium Tubercolusis(Mtb)infection.The "Global Tuberculosis Report 2019" survey shows that there are 10 million new tuberculosis cases in the world in 2018,with more than 1 million deaths,including about 30,000 deaths in our country,which seriously threatens the lives of residents in my country and the world,so the prevention and treatment of tuberculosis is urgent.Mtb is a complex aerobic bacterium composed of more than 4,000 proteins,including virulence proteins,cell wall proteins,conserved proteins and PE/PPE family proteins,etc.,involved in bacterial proliferation,infection and immune escape.In recent years,many Mtb proteins(Rv1411c,PPE32,Rv3477,Mce2E,Rv1252c,etc.)have been reported to evade host immune cell killing by inhibiting phagosome maturation,macrophage apoptosis,inflammation,and autophagy.However,the function of more Mtb Conserved Hypothetical Proteins has not been reported.Rv0790c is a conservative hypothetical protein encoded by the 790th gene of H37Rv,and its immunological function in macrophages has not been reported.Therefore,in-depth study of the function of Rv0790c in macrophages,and further understanding of the interaction between Mtb and macrophages,laid a solid theoretical foundation for the prevention and treatment of new drugs and vaccine targets.The first part of this article explores the survival of Mtb in macrophages by Rv0790c.Macrophage was infected by constructing Mycobacterium smegmatis(Ms::Rv0790c)that up-regulated expression of Rv0790c,and the survival of bacteria in macrophages was detected.It was found that compared with the control group,the bacterial load that up-regulated the expression of Rv0790c was significantly increased,suggesting that Rv0790c promoted the survival of mycobacteria in macrophages.In order to further verify this phenomenon,RAW264.7 cells were infected by packaging retroviruses,and a stable transfected cell line(RAW-Rv0790c)with up-regulated expression of Rv0790c was established.Mycobacterium smegmatis strain mc2155 was infected with RAW-Rv0790c and RAW-Vector,and the number of colonies in macrophages was detected at different time points.It was also found that up-regulating expression of Rv0790c in cells can promote the survival of mycobacteria.In addition,the establishment of an in vivo infection model in mice,compared with the empty group,Ms::Rv0790c infection of mice,the lung,spleen and liver bacterial load were significantly increased,suggesting that Rv0790c can promote macrophage smear Mycobacterium survival.In addition,by collecting clinical samples of tuberculosis patients and detecting the expression of Rv0790c in clinical strains,combined with patient disease information,it was found that Rv0790c is relevant to clinical disease,suggesting that Rv0790c protein has certain biological or immunological functions for further research on Rv0790c The foundation of the function.The second part of this article explores the mechanism by which Rv0790c regulates the survival of M.smegmatis in macrophages.After Ms::Rv0790c and Ms::Vector infection of macrophages,the changes of cell inflammation,cell autophagy and apoptosis were detected.The establishment of an in vitro Mycobacterium smegmatis infection model found that compared with the control group,the Ms::Rv0790c group could reduce the expression of LC3II protein in macrophages,but had no significant effect on the inflammation and apoptosis of macrophages.In order to further verify the effect of Rv0790c on macrophage autophagy.we stimulated RAW-Rv0790c and RAW-Vector with the exogenous drug Rapamycin and found that Rv0790c can inhibit macrophages autophagy caused by Rapamycin.At the same time.the establishmentof an in vivo infection model in mice found that Ms::Rv0790c-infected lung autophagy levels were significantly lower than the control group,suggesting that Rv0790c inhibited macrophage autophagy.In order to better study the specific stage of Rv0790c’s regulation of autophagy,exogenous addition of chloroquine(CQ)or bafilomycin(Baf.A1)inhibits the fusion of autophagosomes and lysosomes,and then through Rapamycin and Ms::Rv0790c stimulation,and found that Rv0790c still can inhibit the production of autophagy-related protein LC3II,this result suggests that Rv0790c does not affect the fusion of autophagosomes and lysosomes,may affect the initiation or extension of autophagy.In the third part of this article.I will make an in-depth study on the mechanism of Rv0790c regulating macrophage autophagy.Screening for proteins that may interact with Rv0790c by immuno-precipitation(co-IP)to help explore the sites where autophagy is inhibited.Screening of key proteins in the autophagy signaling pathway revealed that Rv0790c interacted with mTOR and p62.This suggests that Rv0790c may be involved in regulating the initiation of autophagy.mTOR plays an important role in regulating cell growth,development and metabolism,and is also an important protein that initiates autophagy.When mTOR activity is enhanced,it will promote the phosphorylation of the 757 site of the ULK-1 complex and inhibit the occurrence of downstream autophagy.Next,we explored whether Rv0790c affects the activity of mTOR and then affects the phosphorylation of downstream proteins ULK-1 and P70S6K,thereby inhibiting the onset of autophagy.By using Ms::Rv0790c to stimulate macrophages from different sources,it was found that the Rv0790c group could increase the phosphorylation of P70S6K(mTOR activity index)and enhance the phosphorylation expression level of ULK-1757 at the same time as the empty group.The combination of Rv0790c and mTOR activates the activity of mTOR and regulates the phosphorylation of ULK-1757 site,thereby inhibiting the occurrence of autophagy;in addition,we stimulate RAW264.7 cells through Ms::Rv0790c and Ms::Vector,through Confocal The test found that compared with the Ms::Vector group,the p62 protein in the Ms::Rv0790c group significantly reduced the formation of particulate aggregates,suggesting that the number of autophagosomes was reduced,thereby inhibiting the level of autophagy.In summary,this study innovatively found that Rv0790c interacts with the autophagy signaling pathway proteins mTOR and p62,inhibits macrophage autophagy,and promotes mycobacteria in macrophages by affecting mTOR activity and the formation of p62 autophagy receptors.Within survival.A comprehensive and systematic study of the function of Rv0790c in macrophages provides insights and ideas for understanding the interaction between Mtb and macrophages,and also provides a solid theoretical basis for the development of emerging targeted drugs for the treatment of tuberculosis.