Regulation Of Host Type Ⅰ Interferon Response By Mycobacterium Tuberculosis Rv3248c Protein Targeting CGAS-STING Signaling Pathway

Tuberculosis(TB)caused by Mycobacterium tuberculosis(MTB)infection is a chronic and consumptive disease that poses a serious threat to global public health.In recent decades,the emergence of multi-drug resistance(MDR)and extensively drug-resistant(XDR)tuberculosis has presented new challenge for the prevention and treatment of tuberculosis.Currently,traditional anti-tuberculosis drugs can no longer meet clinical demands,making the development of new anti-tuberculosis drugs a top priority.The identification of effective drug targets for screening is the main bottleneck in the development of new anti-tuberculosis drugs.The pathogenesis of MTB involves complex pathogen-host interaction.Following infection with MTB,a variety of effector proteins target host cells,thereby regulating the host immune response.Potential targets for anti-tuberculosis drugs include factors related to MTB immune evasion,latent infection-related pathogenic factors,and host factors.After entering the host cells,the DNA released by MTB can bind to cGAS,leading to activation of the cGASSTING pathway and regulation of the type Ⅰ IFN response.In addition,MTB c-di-AMP can mediate the activation of STING pathway in a way independent of cGAS.However,the specific mechanism by which MTB regulates the host cGAS-STING pathway remains poorly understood and the potential of targeting this pathway as a drug target for tuberculosis treatment requires further exploration.In this study,the effector proteins interacting with STING in MTB were screened by GST-pull down and mass spectrometry.Among the 10 candidate proteins screened,it was further confirmed that Rv3248c protein was involved in the regulation of type Ⅰ IFN response.Then the biological function of Rv3248c was identified,and the regulatory function of Rv3248c protein on host cGAS-STING signal pathway was discussed.These findings provide a new idea for the identification of new anti-tuberculosis drug targets.1.Screening of interaction effector proteins of host STING signal molecule targeting H37Ra of MTBIn this study,we first analyzed the functional domain of STING signal molecule in cCASSTING pathway by using protein structure prediction websites such as UniProt and constructed and expressed STING protein.Then,the effective proteins interacting with STING in MTB H37Ra were screened by GST-pull down and analyzed by mass spectrometry,and 10 candidate interaction proteins were obtained.After conducting literature search and functional analysis,some proteins were selected for the construction of over-expressed recombinant M.smegmatis.The results showed that 6 strains of recombinant M.smegmatis were successfully constructed.In order to explore whether the screened effector proteins are involved in the regulation of host type Ⅰ IFN response,the constructed recombinant bacteria were used to infect the J774-DualTM cell line containing IRF3-Luc reporter gene,and then the luciferase activity in the supernatant was determined.The result showed that compared with the control bacteria,the activity of luciferase in the supernatant of cells infected with recombinant bacteria rMS::pMV261-Rv3248c decreased significantly,indicating that Rv3248c may be involved in inhibiting the activation of host type Ⅰ IFN signal pathway.Next,the biological characteristics of recombinant bacteria rMS::pMV261-Rv5248c were determined,and the results of colony morphology observation showed that the colony surface of recombinant bacteria overexpressing Rv3248c was less wrinkled and smoother than that of control bacteria.The results of biofilm formation assay of recombinant bacteria showed that the biofilm formation of recombinant bacteria with Rv3248c overexpression was lower than that of control bacteria.indicating that Rv3248c inhibited the biofilm formation of mycobacteria.Through the H2O2 and SDS stress tests,the results showed that the stress resistance and survival ability of Rv3248c overexpression recombinant bacteria decreased.2.MTB Rv3248c regulates host STING-mediated type Ⅰ IFN responseThen,we focused on Rv3248c protein and successfully constructed both Rv3248c eukaiyotic expression plasmid and prokaryotic recombinant protein to explore its regulatory function on the host type Ⅰ IFN response.Firstly,qRT-PCR and ELISA were used to determine the transcriptional level and expression of IFN β in RAW264.7 cells infected with recombinant bacteria.The results showed that Rv3248c significantly down-regulated the transcriptional level and expression of IFN-β.At the same time,Western blot assay was used to determine the activation level of cGAS-STING pathway and its downstream JAK-STAT pathway after RAW264.7 cells infection.The results showed that the phosphorylation level of STING molecule and the expression level of TBK1 molecule in macrophages infected by Rv3248c recombinant bacteria were significantly down-regulated,indicating that Rv3248c is involved in inhibiting the activation of cGAS-STING signal pathway in host cells.In order to further confirm the effect of Rv3248c on the activation of type Ⅰ IFN signal pathway,Rv3248c eukaryotic expression plasmid and IRSE reporter plasmid were respectively co-transfected with STING,TBK1 and IRF3 eukaryotic expression plasmids,and the luciferase activity in the supernatant was determined.The results showed that Rv3248c could significantly inhibit the activation of type Ⅰ IFN pathway induced by TBK1.In order to explore whether Rv3248c directly regulates the host type Ⅰ IFN response,the recombinant Rv3248c protein was used to stimulate RAW264.7 cells.and then the activation level of cGAS-STING signal molecule in RAW264.7 cells was measured by Western blot assay.The results showed that Rv3248c recombinant protein could inhibit the phosphorylation level of STING molecule induced by 2’,3’-cGAMP in macrophages and down-regulate the expression level of TBK1 molecule induced by LPS.In order to explore the effect of Rv3248c on the intracellular survival of mycobacteria.RAW264.7 cells were infected with recombinant strain rMS::pMV261-Rv3248c and the intracellular mycobacteria were counted.The results showed that Rv3248c down-regulated the intracellular survival of mycobacteria.In order to further explore the regulatory function of Rv3248c on host type Ⅰ IFN response in vivo,mice were infected with recombinant strain rMS::pMV261-Rv3248c by intraperitoneal injection,and the transcription levels of cytokines in mouse tissues were detected by qRT-PCR.The results showed that the transcription levels of IFN-β,IL-6 and IL-1β in lung and spleen of mice infected with Rv3248c recombinant bacteria were significantly down-regulated.Furthermore,the colonization ability of recombinant Rv3248c bacteria in mouse was determined.The results showed that the overexpression of Rv3248c inhibited the survival of mycobacteria in vivo.In addition,the histopathological sections of mice were analyzed,and the results showed that Rv3248c reduced granulocyte infiltration and hepatocyte necrosis induced by M.smegmatis infection.In conclusion,our study shows that MTB Rv3248c inhibited the activation of type Ⅰ IFN signal pathway by down-regulating the phosphorylation of STING molecule and the expression of TBK1 molecule.In vivo and in vitro experiments revealed that Rv3248c significantly down-regulated the transcription levels of type Ⅰ IFN and inflammatory responserelated genes,and reduced the intracellular survival level of mycobacteria and the load in mice.These results suggest that Rv3248c has the potential to be a target of anti-tuberculosis drugs and tuberculosis vaccines.