Mycobacterium Tuberculosis(Mtb) Protein EspR (Rv3849) Promotes Mtb Survival By Inhibiting The Expression Of Inflammatory Mediators And Cell Apoptosis In Macrophage

Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis,one of the three most infectious diseases that threaten humans.About 2million people die each year from tuberculosis.Macrophages are the first line of defense against Mtb as well asthe main host of Mycobacterium tuberculosis.Macrophages kill Mtb by phagocytosis,secretion of a variety of inflammatory cytokines and other mediators,and production of ROI,RNI and antibacterial peptides.In the mean time,macrophage autophagy and apoptosis also play important roles in clearing Mtb in vivo.Mycobacterium tuberculosis,as intracellular bacteria,also developed multiple ways to resistant macrophage killing,such as preventing fusion of phagosome and lysosome;and inhibiting host cell autophagy and apoptosis.The evasion strategies are mainly realized by Mycobacterium tuberculosis expressed virulent factors.EspR(Rv3849)is a14.7 kDa Mtb protein which promotes the survival of Mycobacterium tuberculosis in macrophages.Raghavan et.al.reported that EspR binds to the promoter of Rv3616c-Rv3614c(EspACD)and activates its transcription,whose products activates the ESX-1 secretion system.Interesting,EspR itself is also secreted out of Mtb by ESX-1 system,as a negative feedback control of the secretion system.This is not a usual stragety for bacteria to regulate its protein level,so we suspect EspR might have additional functions in host cells.So far there is no report about the functions of EspR in macrophage.If it does exert an inhibitory role on the immunological functions of macrophage,it might provide a new target for anti-Mtb drug development.Therefore,this paper aims to study the functions of EspR in the host cells.In this study,we constructed eukaryotic expression plasmid pMSCV-eGFP-EspR,packed it into retrovirus,infected RAW264.7 cells and developed a stable macrophagy cell line expressing EspR(RAW264.7-EspR).To investigate the effect of EspR onmacrophage immune function,we used BCG to stimulate RA264.7-EspR and the control cell line RAW264.7-Vector,and detected the expression of inflammatory mediators using real-time PCR,ELISA,and Western blot.It was found that EspR could inhibit the expression of IL-1?,IL-6,and iNOS in macrophages.To further explore the mechanism of this inhibition,we examined the activation of NF-?B and MAPK signaling pathways in BCG stimulated RAW264.7-EspR and RAW264.7-Vector,finding that EspR could inhibit the phosphorylation of p65,IKK,Erk1 / 2,SAPK/JNK and p38 in NF-?B and MAPK signaling pathways.We continued to examine the upstream-related molecules of NF-?B and MAPK signaling pathways,and found that EspR was able to impair the phosphorylation of IRAK1,through which it inhibit TLR activation.We also used Annexin-V/7-AAD and Western blotting to detect the effect of EspR on macrophage apoptosis,and found that EspR can inhibit BCG induced exogenous apoptosis of macrophage apoptosis,with its mechanism still under investigation.The above results suggest that EspR in macrophages can inhibit BCG induced activation of TLR signaling pathway,resulting in inhibition of expression of proinflammatory mediators IL-1?,IL-6,and iNOS.EspR can also inhibit BCG induced macrophage apoptosis.These functions of EspR hampered the bactericidal ability of macrophage and improved Mtb survival.