The Role And Molecular Mechanism Of PPE36 In Mycobacteria-infected Macrophages

TB is an infectious disease caused by the bacillus Mycobacterium tuberculosis,which has existed for millennia and remains a major global health problem.It causes ill-health for approximately 10 million people each year and is one of the top ten causes of death worldwide.According to the 2017 WHO report,an estimated 10.4 million people fell ill with TB in 2016,3.0 million TB deaths worldwide.Between 5% and 15% of individuals infected with M.tuberculosis will progress(over months to a few years)to active TB disease.The successful establishment of MTB infection largely depends on its early interactions with host innate immune cells.Macrophages play a central role in mycobacterial pathogenesis,since they are the primary cellular niche for MTB during both early and chronic infection.Macrophages can eliminate MTB via multiple mechanisms,including the production of oxygen and nitrogen components and cytokines,phagosome acidification and the autophagy of intracellular MTB,among other processes.Although macrophages are known as professional killers for pathogens,MTB has adopted remarkable strategies to circumvent host defenses,building suitable conditions to survive and proliferate.Because MTB interferes with a number of cellular processes that are critical for intracellular survival of MTB,such as phagosome maturation,apoptosis and autophagy of infected macrophages.Many pathogenic bacteria have evolved sophisticated strategies,including the secretion of effector proteins into mammalian cells,to subvert signaling pathways of the innate immune system.Comprehension of the molecular events governing MTB survival within macrophages is fundamental for the improvement of vaccine-based and therapeutic strategies in order to help the host to better defend itself in the battle against the fierce invader MTB.PPE36,a protein encoded by the gene Rv2108 specific for the MTB complex,consists of 243 amino acids comprising the PPE domain of ~180 amino acids and a short C terminus of unique sequence.PPE36 were also found to be surface exposed,this antigen may be involved in the establishment of the host cellular immune responses against the M.tuberculosis,which led to the belief that they might play a role in the virulence of MTB and play an important immunological role either in diagnosis or in protection.In this study,we constructed overexpression-PPE36 in RAW264.7 cell lines,Mycobacterium smegmatis(MS_PPE36),deletion of PPE36 in BCG strains(BCG? PPE36)and complemented the PPE36 in BCG? PPE36 strains.We observed that PPE36 promoted the survival of mycobacteria in macrophages and in mycobacterium infected mice.Many studies reported that MTB interferes with a number of cellular processes that are critical for intracellular survival of MTB,such as phagosome maturation,apoptosis and autophagy of infected macrophages.In order to explore the mechanisms of mycobacterium survival,the amino acid sequence of PPE36 from the Mycobacterium bovis bacillus Calmette-Guerin(BCG)strain is identical to that of MTB PPE36.To explore the underlying mechanisms,we next investigated whether PPE36 modulates the expression of inflammatory cytokines in macrophages by analyzing the expression of mRNA encoding TNF-? and IL-1? in macrophages infected with BCG?PPE36 or PPE36-overexpressing.Expression of MTB PPE36 in M.smegmatis suppressed the cytokine expression at various time points.In contrast,deletion of PPE36 in BCG promoted the expression of TNF-?,IL-6 and IL1-? mRNA and decreased bacterial survival in RAW264.7 cells infected with BCG?PPE36.Thus,MTB PPE36 was able to promote survival of mycobacteria in macrophages by inhibiting cytokine production in host cells.As multiple virulence factors(such as ESAT-6 and CFP-10)of MTB are absent in the BCG strain,we challenged C57BL/6 mice intratracheally with wild-type BCG or BCG?PPE36,deletion of PPE36 in BCG increased levels of TNF-? mRNA,IL-6 mRNA and IL-1? mRNA in the lung of infected mice.Consistent with that,deletion of PPE36 in BCG resulted in a drop in the bacterial load in the lungs of mice.To explore the underlying mechanisms,we first investigated whether MTB PPE36 could modulate NF-?B and MAPKs signaling pathways.Transient expression of MTB PPE36 in human HEK293 T embryonic kidney cells suppressed the activation of NF-?B induced by TNF-?.MS_PPE36 also diminished phosphorylation of the inhibitory cytoplasmic NF-?B regulator I?B?,as well as phosphorylation P65 and IKK?/?.To investigate the effects of MTB PPE36 on modulating NF-?B activation in macrophages,we infected macrophage-like RAW264.7 cells with deletion of PPE36 in BCG.Deletion of PPE36 in BCG enhanced the phosphorylation of p65,I?B? and IKK?/? and ERK,p38.Together these results indicated the possibility that PPE36 from BCG could block the activation of immune system signaling dependent on the NF-?B and MAPKs pathways in macrophages infected with mycobacteria.To search for the targets of PPE36 in host cells,we first investigated whether MTB PPE36 could modulate the key factor in the NF-?B or MAPKs signaling pathways.MTB PPE36 suppressed activation of NF-?B mediated by MyD88,as MTB PPE36 inhibited the expression of MyD88,ubiquitination is important in regulating various eukaryotic cellular processes,including cell-cycle progression,vesicular trafficking and innate immune system responses.We investigated whether ubiquitin is required for this process,we demonstrated here that MTB PPE36 directly and specifically promoted the MyD88 ubiquitin and then inhibited the dephosphorylate IKK?/?,I?B,P65,ERK and p38,which led to the suppression of innate immunity.Taken together,our data showed that overexpression of MTB PPE36 in M.smegmatis diminished the abundance of TNF-?,IL-6 and IL-1? and promoted the survival of mycobacteria in macrophages.Deletion of PPE36 in BCG increased the production of TNF-? and IL-1? and reduced the survival of mycobacteria in macrophages and mice.Cytokines were increased in the complemented PPE36 strains.Here we found that PPE36 suppressed innate immunity dependent on pathways of the NF-?B and MAPKs pathway and inhbited the expression of MyD88 by exploiting host ubiquitin.Our findings revealed how pathogen subverts innate immunity by coopting host ubiquitin and suggesedt a potential tuberculosis treatment.