Mycobacterium Tuberculosis MmsA (Rv0753c) Negatively Regulates Type Ⅰ IFN Responses By Targeting Sting And Its Mechanism

TB(Tuberculosis),caused by infection with Mycobacterium tuberculosis(Mtb),is a potentially fatal chronic infectious disease.It was reported in WHO’s “Global Tuberculosis Report 2017” that an estimated 10.4 million people fell ill with TB in 2016,0.4 million of whom have HIV co-infection.Type Ⅰ IFN is a key biomarker of active tuberculosis,distinguishing from latent tuberculosis infection,and is related to the severity of the disease.Previous researches had shown that Type Ⅰ IFN production mainly depends on the STING(Stimulator of Interferon Genes)signaling pathways during Mycobacterium tuberculosis infection.STING is an important adaptor molecule in innate immunity,and its role as a link between DNA sensing and cell activation and as the sensor of cyclic dinucleotides(CDNs),consequently activating Type Ⅰ IFN responses.Mycobacterium tuberculosis DNA recognized by DNA sensor cGAS as well as c-di-AMP derived from Mycobacterium tuberculosis both activate STING-TBK1-IRF3 signal axis.Researches on the immunoregulation effect of STING have been paid wide attention recently.Multiple host proteins interacting with STING could modulate Type Ⅰ IFN responses.Some of them play a role of anti-pathogen immune defense(positive regulation),while others contribute to avoiding overeactive immune response to maintain immune homeostasis(negative regulation).In addition,a series of proteins from various viruses such as HCV-NS4 B,KSHV-v IRF1,HCMV-UL82 bind STING directly to evade immune system.Although it is very clear that Type Ⅰ IFN acts as a protective role in virus infection,its effect on the outcomes of Mycobacterium tuberculosis infection is still in debate.Based on the above research background,we proposed the scientific hypothesis that there are host or pathogen proteins targeting STING to regulate Type Ⅰ IFN responses.In the present study,we screened the proteins interacting with STING during Mycobacterium tuberculosis infection firstly.The cell lysis from RAW264.7 cells infected with Mycobacterium tuberculosis virulent strain H37 Rv were immunoprecipitated with anti-STING.Mass spectrum analysis showed the candidate proteins interacting with STING include Sox5,SFPQ,IFIT3 b,Bhmt and Ebf2 from host as well as mmsA(Rv0753c)from Mtb.We placed emphasis on IFIT3 b and mmsA when analyzing related literatures and documents.Preliminary research by exogenous co-immunoprecipitation had shown that mmsA,rather than IFIT3 b,interacts with STING.MmsA,namely methylmalmonate semialdehyde dehydrogenase,is a culture filtrate protein of Mycobacterium tuberculosis.Not many studies have focused on mmsA.It may contribute to distinguish latent tuberculosis infection(LTBI)from active tuberculosis.It is also reported that mmsA induces DC activation and promotes Th1 cell-type immune responses.However,its function related to the regulation of type I IFN has not been reported.We successfully constructed the prokaryotic,eukaryotic and mycobacterium-shuttle expression plasmids of mmsA,stably cell line RAW264.7 expressing mmsA as well as recombination Mycobacterium smegmatis overexpressing mmsA.The polyclonal antibody against mmsA has also been successfully prepared.The results indicated the mmsA interacted with STING by exogenous and endogenous coimmunoprecipitation.Meanwhile,the immunofluorescence with confocal assays showed that the co-localization of mmsA and STING were verified not only by exogenous transfection,but also by infection with Mtb.Furthermore,we successfully constructed the truncated plasmids of mmsA and STING.Results from domain mapping analysis suggested that the N-terminal of mmsA(1-251 aa)was essential for mmsA-STING interaction,whereas the N-terminal transmembrane domain of STING(1-190 aa)was responsible for its interaction with mmsA.In addition,the enzyme activity site of mmsA may partly impact the interaction with STING.To determine the effect of mmsA on the Type Ⅰ IFN responses,we found that mmsA suppressed the promoter activity of IFN-β by dual luciferase reporter gene assay.As expected,mmsA decreased the mRNA level of IFN-β and CXCL10 in macrophages by RT-qPCR.The immunoblotting analysis showed mmsA reduced the phosphorylation of TBK1 and IRF3,as well as the dimerization of IRF3.In addition,we also found mmsA disrupted the interaction of STING and TBK1,and suppressed the dimerization of STING.These results demonstrated that mmsA down-regulated TBK1-IRF3 signal.Colony forming unit assay suggested that mmsA reduced the intracellular survival of mycobacteria.Next we explored the regulatory mechanisms.Our results showed that mmsA induced the degradation of STING both in transient transfection into 293 T cells and stable transfection into RAW264.7 cells.Further findings indicated that mmsA mediated the autophagy,rather than the proteasome dependent degradation of STING and facilitated the association between STING and the autophagic cargo receptor p62.The results demonstrated mmsA promoted p62-mediated selective autophagic degradation of STING.Our study identified Mycobacterium tuberculosis mmsA(Rv0753c)as a negative regulator targeting innate immune adaptor molecule STING to modulate Type Ⅰ IFN responses,and explored potential regulatory mechanisms.We discussed the molecular regulation mechansims of STING signaling pathways during macrophages infected with Mtb in terms of protein-protein interaction,which is helpful for getting a deeper understanding the pathogenesis of LTBI.It provides new clues into the immunodiagnosis of LTBI as well as the risk prediction of the progression to active tuberculosis disease from LTBI.