Molecular Mechanism Of Mycobacterium Tuberculosis Rv0927c Regulating Host Innate Immunity And Cellular Activity

Tuberculosis(TB)is a chronic,zoonotic infectious disease caused by infection with the Mycobacterium tuberculosis complex.One-third of the world’s population is infected with Mycobacterium tuberculosis(M.tb)and approximately 5-10%of those infected will develop active TB.In the past 200 years,one billion people have died from TB.According to the World Health Organization(WHO),about 9.87 million people were infected with Mycobacterium tuberculosis in 2020.M.tb enters the lungs from the respiratory tract and is phagocytosed by alveolar macrophages.Macrophages infected with M.tb release inflammatory cytokines,chemokines and lipid mediators that work together to recruit other immune cells to the lungs and initiate the innate immune response.At the same time,dendritic cells migrate from the lungs to lymph nodes and they present antigens to T lymphocytes,triggering the adaptive immune response to control mycobacterial infection.Inflammatory signaling pathways,such as the NF-κB signaling pathway and MAPK signaling pathway,are involved in the initiation of host innate immune responses.Once activated by pathogens,these pathways facilitate the expression of inflammatory cytokines to destroy invasive pathogens.However,M.tb has evolved multiple strategies to disrupt these signaling pathways for better survive within macrophages.Macrophages infected with M.tb will switch its metabolism from primarily oxidative phosphorylation to glycolysis to rapidly gain energy to defend against invasive pathogens,which has been termed the Warburg effect.This effect is regulated by HIF-1α,which plays an important role in controlling intracellular bacterial infection and granuloma formation.In addition to macrophages,M.tb can also invade epithelial cells and is closely associated with lung tumorigenesis and development.However,the specific mechanisms by which M.tb proteins interact with host cells remain poorly understood.M.tb Rv0927c is distributed in the Mycobacterium tuberculosis complex as well as in the attenuated strain of Mycobacterium bovis BCG.Domain analysis showed that Rv0927c may belong to enoyl ACP reductase,which is related to fatty acid synthesis.In addition,it has been reported that compared with the wild M.tb strain,the Rv0927c transposition mutant strain induced more inflammatory cytokine secretion,suggesting that the Rv0927c protein may be involved in the regulation of host inflammatory responses.However,there are still no study about the effect of Rv0927c protein on the biological characteristics of mycobacterium and its immunoregulatory function during M tb infection.In this study,we explored the molecular mechanism of M.tb Rv0927c protein regulating host innate immunity and cell activity,which will provide a theoretical basis for the screening of new tuberculosis drug targets and has important significance for the prevention and treatment of tuberculosis.1.Biological function and immunological characterization of M.tb Rv0927c proteinRecombinant M.smegmatis rMS::pMV261-Rv0927c was constructed using the pMV261 shuttle plasmid and Rv0927c protein was expressed using the prokaryotic expression system.Rv0927c protein was purified and endotoxin removed.Growth curve measurements showed that the growth rates of the Rv0927c overexpression strain and the empty plasmid strain were consistent.The cytoplasmic and cell wall components of recombinant M.smegmatis were isolated by ultracentrifugation to determine the subcellular localization of Rv0927c.The results showed that Rv0927c was localized in the cytoplasm and cell wall.The protein’s localization is related to its function,we next investigated whether Rv0927c alters cell wall-related biological characteristics.Overexpression of Rv0927c made the colony and cell surfaces of recombinant M.smegmatis smoother,but had no significant effect on the formation of biofilms.In drug susceptibility experiments,overexpression of Rv0927c did not alter the drug susceptibility.In acid tolerance and SDS stress assays,there was no significant difference in the viability of Rv0927c overexpression strain and empty plasmid strain.RAW264.7 cells or BMDMs were infected with rMS::pMV261 and rMS::pMV261Rv0927c and cell supernatant was collected at different time points.The results showed that Rv0927c significantly inhibited the secretion of IL-6,TNF-α and IL-1β.Correspondingly,the secretion of inflammatory cytokines induced by M.smegmatis was inhibited,when cells were treated with Rv0927c protein.Then Mice were infected with recombinant M.smegmatis by intraperitoneal injection and the tissues were collected at different time points.The transcriptional levels of inflammatory cytokines in the tissues were detected by real-time PCR.The results showed that overexpression of Rv0927c in M.smegmatis significantly reduced the mRNA levels of IL-6,TNF-α and IL-1β in the lung and spleen of infected mice.These results suggest that Rv0927c is an immune regulator inhibiting the expression of inflammatory cytokine.2.The molecular mechanism of M.tb Rv0927c inhibiting host inflammatory responseTo investigate the effect of Rv0927c on inflammatory signaling pathways,RAW264.7 cells were infected with recombinant M.smegmatis or wild M.smegmatis together with the Rv0927c protein and the phosphorylation levels of p65,Erk,p3 8 and Jnk was detected.The results showed that Rv0927c significantly reduced the phosphorylation levels of p65 and p38,but did not affect the phosphorylation of Jnk and Erk.To further confirm the effect of Rv0927c on the NF-κB and MAPK signaling pathways,a dual fluorescent reporter experiment was performed,which showed that Rv0927c could significantly inhibit the activities of NF-κBLuc and AP-1-Luc with NF-κB predominant.The entry of p65 into the nucleus is a marker of the activation of the NF-κB signaling pathway.Rv0927c was expressed in HEK293-TLR4 cells and the localization of p65 was observed by laser confocal microscopy after LPS stimulation.The results showed that Rv0927c could inhibit the entry of p65 with a dosedependent relationship.Eukaryotic plasmid mutants covering the whole gene of Rv0927c was constructed and the effect of Rv0927c mutants on NF-κB-Luc activity was subsequently detected using a dual fluorescence reporter system.The results showed that the 30 amino acids at the N-terminal of Rv0927c protein are the key regions to exert anti-inflammatory function.These results suggest that Rv0927c inhibits the activation of NF-κB signaling pathway through the N-terminal 30 amino acids.To investigate the key signaling molecules that Rv0927c inhibits NF-κB signaling pathway-dependent,the analysis of the interaction between Rv0927c protein and TLRs was performed and the results showed that Rv0927c does not interact with TLR2 or TLR4.The effect of Rv0927c on IκBα and TAK1 phosphorylation was subsequently determined and the results showed that Rv0927c inhibited the phosphorylation of IκBα,but not the phosphorylation of TAK1.At the same time,co-immunoprecipitation results showed that Rv0927c had no effect on the formation of IRAK1-TRAF6 and TAK1-TRAF6 signalosome.These results suggest that Rv0927c inhibit the activation of NF-κB by targeting the phosphorylation of IκBα.Cells were pretreated with NF-κB and MAPK pathway inhibitors before infection and the results showed that blocking the p38 and NF-κB pathways with the inhibitors attenuated the ability of Rv0927c to inhibit the expression of IL-6 and IL-1β.The colonization ability of recombinant M.smegmatis in RAW264.7/BMDMs and mice was analyzed,which showed that macrophages or mice infected with rMS::pMV261-Rv0927c had significantly higher bacterial loads than rMS::pMV261-infected groups and induced more immune cell infiltration in mice tissues.These results suggest that Rv0927c promotes bacterial survival by downregulating host inflammatory responses.3.The molecular mechanism of M.tb Rv0927c regulating the activation of host HIF-la signaling pathwayTo explore the potential function of Rv0927c,transcriptome sequencing of RAW264.7 cells infected with recombinant M.smegmatis was performed.The results showed that host genes down-regulated by Rv0927c were enriched in the HIF-1α signaling pathway,which was consistent with the subsequent fluorescence quantitative results.The amount of HIF-1α protein in the nucleus represents the level of activation of the HIF-la signaling pathway.RAW264.7 cells were infected with recombinant M.smegmatis or RAW264.7-Lv5-Rv0927c cells were infected with the wild M.smegmatis.The results showed that Rv0927c reduced nuclear HIF1α protein.Silencing of HIF-1α expression in RAW264.7 cells using siRNA followed by intracellular colonization assays showed that silencing of HIF-1α disrupted the ability of Rv0927c to promote mycobacterial survival.To explore the effect of Rv0927c on the nuclear entry of HIF-1α,RAW264.7 cells were infected with recombinant M.smegmatis and the protein levels of HIF-la in the cytoplasm and nucleus were detected.The results showed that Rv0927c was able to inhibit the nuclear entry of HIF-1α.Inhibition of HIF-1α nuclear translocation was also observed under laser confocal microscopy.NF-κB signaling pathway is located upstream of the HIF-la signaling pathway and plays an important role in the regulation of the HIF-1α signaling pathway under normoxia.Pre-incubation with NF-κB inhibitor resulted in the inability of Rv0927c to inhibit HIF-la entry into the nucleus.COX-2 is a gene of NF-κB-dependent transcription.Infection of RAW264.7 cells with recombinant M.smegmatis decreased the transcript level and protein level of COX-2 and this effect was dependent on the NF-κB signaling pathway.Pre-incubation of RAW264.7 cells with COX-2 inhibitors followed by infection with recombinant M.smegmatis showed that the addition of COX-2 inhibitors resulted in a loss of the ability of Rv0927c to inhibit HIF-la entry into the nucleus.These results suggest that Rv0927c inhibits HIF-la entry into the nucleus via the NF-κB/COX-2 signaling pathway.Rv0927c was found to downregulate M.smegmatis-induced HIF-1α expression,but changes in HIF-1α transcriptional levels cannot support the decrease in protein levels,suggesting that Rv0927c may affect the stability of HIF-1α.RAW264.7 cells were infected with recombinant M smegmatis and protein synthesis inhibitor CHX or protein synthesis inhibitor CHX together with proteasome inhibitor MG 132 were added at the same time.The result showed that Rv0927c was able to degrade HIF-1α via the proteasome pathway.The expression level of VHL and the ubiquitination level of HIF-1α were also detected and the results showed that Rv0927c promoted the expression of VHL and the ubiquitination modification of HIF-1α.These results suggest that Rv0927c promotes the ubiquitination of HIF-1α by up-regulating the expression of VHL.thereby targeting HIF-1α for proteasomal degradation.After mycobacterial infection,host will tend to obtain energy through glycolysis to defend against pathogenic infection and HIF-1α is a transcriptional regulator of glycolysisrelated genes.RAW264.7 cells were infected with recombinant M smegmatis and the contents of extracellular lactate and intracellular glucose were detected.The results showed that the overexpression of Rv0927c result in the decrease of the contents of extracellular lactate and intracellular glucose and Rv0927c also reduced the cellular capacity for glucose uptake,indicating that the glycolytic capacity of the host was inhibited by Rv0927c.Finally,the ability of Rv0927c to promote mycobacterial survival was abolished,when the glycolysis inhibitor 2DG was added.These results suggest that Rv0927c regulate the intracellular survival of mycobacteria dependent on the glycolytic process.4.M.tb Rv0927c inhibits the proliferation of A549 and promotes its apoptosisFirstly,the Rv0927c eukaryotic plasmid was transfected into A549 cells and cell proliferation was examined using CCK-8,which showed that Rv0927c significantly inhibited the proliferation of A549 cells.The A549 cells stably expressing Rv0927c were obtained by virus infiltration and screening with puromycin.Then,the cells were inoculated on the backs of nude mice and the tumors were removed and measured after 45 days.The results showed that the tumor size and weight of the Rv0927c group were significantly smaller than those of the control group and there were fewer Ki-67 positive cells.These results suggest that Rv0927c can inhibit the proliferation of A549 cells in vitro and in vivo.The cells overexpressing Rv0927c were stained with Annexin V and PI,and the apoptosis was analyzed by flow cytometry.The results showed that the proportion of Annexin V+PI’ and Annexin V+PI+ cells in Rv0927c group was significantly higher than that in the control group,indicating that Rv0927c significantly upregulated the apoptosis of A549 cells.At the same time,the results of mitochondrial membrane potential detection showed that Rv0927c induced the decrease of mitochondrial membrane potential.The cleavage levels of apoptosis-related molecules were detected by Western blot and the results showed that Rv0927c promoted the cleavage of Capase-3,Capase-9 and PARP,while there was no significant effect on the cleavage of Caspase-8.These results suggest that Rv0927c promotes apoptosis through the endogenous pathway.The host-interacting proteins of Rv0927c were analyzed and eventually targeted to the TUFM gene.Silencing TUFM expression in A549 cells using siRNA promoted increased levels of apoptosis.Further,Rv0927c was overexpressed in TUFM-silenced cells and the apoptosis of the cells were examined.The results showed that the differences between the Rv0927c group and the control group disappeared.These results suggest that Rv0927c regulates the apoptosis of A549 cells by targeting TUFM.