Biological Role Of Mycobacteria Protein O-mannosyltransferase MSMEG＿5447 And Glycoprotein Rv1096

BackgroundTuberculosis(TB)caused by Mycobacterium tuberculosis(Mtb)infection,is an infectious disease that threatens global public health and is the number one killer of a single infectious disease higher than AIDS.According to WHO report,in 2019,10 million people worldwide were diagnosed with TB and 30 countries,including China,India and South Africa are classified as high burden countries with tuberculosis,accounting for 87% of the globle TB incidence.Although the treatment effect of tuberculosis has been greatly improved,the treatment of MDR/RR-TB and TB/HIV co-infection is still grim.After Mycobacterium tuberculosis infection,it is engulfed by pulmonary macrophages and dendritic cells,the adaptive immune response is activated,and finally granuloma tissue is formed.Recently,O-mannosylation has been reported in some cell wall and secreted proteins of Mtb and is closely associated with Sec-translocation pathway.In the functional study of protein O-mannosylation,it not only associated with proteolysis and localization of proteins,but also was involved in the interaction of mycobacteria and host.The initial step of protein O-mannosylation of Mtb is catalyzed by protein O-mannosyltransferase(PMT).It can catalyze the first mannosyl group to serine or threonine residues of the protein and then extend the sugar chains by mmannosyltransferases(MT)and other glycotransferase.Rv1002 c gene encodes PMT in Mtb and its homologous gene in Mycobacterium smegmatis(Msm)is MSMEG＿5447.Both of them are key enzymes in the process of protein O-mannosylation.Previous studies mainly focused on the O-mannosylation of a specific protein,but few studies focused on the effect of global glycosylation on the biological characteristics in mycobacterium and its biological role in infection.In addition,our previous study found that Rv1096,the cell wall protein of Mtb,is an o-mannosylated protein with peptidoglycan deacetylase activity,which can resist the killing of host lysozyme by catalyzing the deacetylation of peptidoglycan of Mtb.However,the effect of O-mannosylation of Rv1096 protein on Mycobacterium infection is still unclear.Therefore,in this study,we used Msm as a model to investigate the biological characteristics of Mycobacterium and the biological role of O-mannosylation of proteins controlled by protein O-mannosyltransferase(PMT),and preliminarily explored the biological role of O-mannosylation of glycoprotein Rv1096.The results will help us to understand the biological function of protein O-mannosylation as a whole and provide theoretical basis for the development of more effective vaccines and drugs of anti-tuberculosis.Methods1.Effect of MSMEG＿5447 gene deficiency on biological characteristics of Msm△M5447 strain of protein O-mannosyltransferase MSMEG＿5447 gene deletion has been constructed in our laboratory.In order to analyze the function of MSMEG＿5447more comprehensively,we constructed a compensatory strain Comp which can compensate the function of MSMEG＿5447 gene in△M5447 strain.The effect of MSMEG＿5447 gene deficiency on the growth rate of Msm was determined by a microplate reader and the morphology of Msm colonies was observed by microscope.The subcellular components of Msm were separated by ultracentrifugation,and the O-mannosylation level of Msm proteins was detected by Con A lectin blot.The susceptibility of Msm to lysozyme was measured by resazurin colorimetric assay.The Msm were cultured in acidic medium and their tolerance to acidic environment was examined.The cell wall permeability of Msm was observed by measuring absorption rate of ethidium bromide.Biofilm formation of Msm was observed by culturing Msm in M63 and LB medium respectively.The glycolipids containing mannan,LAM(lipoarabinomannan)and its synthetic precursor LM(lipomannan),were prepared and detected by PAS staining.The changes of Msm proteome were analyzed by two-dimensional electrophoresis combined with mass spectrometry.2.The effect and mechanism of MSMEG＿5447 gene deficiency on Msm during macrophage infectionBased on the experiment of macrophage infection,the invasive ability and viability of bacteria in macrophages were observed by counting colony forming units(CFU)and fluorescent dye method.The levels of cytokines in macrophages were detected by q PCR and ELISA respectively.The activation of transcription factor NF-κB was detected by q PCR,Western blot and immunofluorescence.The co-localization of phagosome and lysosomes in infected macrophage was detected by Lyso Tracker Red and LAMP-1immunofluorescence assay,and the expression of lysosome-related hydrolase CTSD was detected by Western blot.The transcriptome changes of infected cells were detected by RNA sequencing technology to analyze the effects of Wt and△M5447 on macrophages.3.The role of O-mannosylation of Mtb Rv1096 during infectionBased on MS＿Rv1096 strain,Msm overexpressing Rv1096 and MS＿Rv1096am strain,Msm overexpressing Rv1096 am with 3 mutations at glycosylation sites(T265,S266,S267),the O-mannosylation of Rv1096 and O-mannosylation level of Rv1096 am were detected by Con A lectin blot.The subcellular fractions of MS＿Rv1096 were prepared by ultracentrifugation and subcellular localization of Rv1096 was detected.The localization of Rv1096 and Rv1096 am on cell surface was determined by trypsin digestion.After infecting macrophages,the viability of MS＿Rv1096 and MS＿Rv1096am within macrophages was tested by plate counting CFU and fluorescent dye method.The co-localization of lysosomes and phagosome containing Msm in macrophages was detected by Lyso Tracker Red dye.Results1.The MSMEG＿5447 gene deficiency affected the biological characteristics of MsmProtein O-mannosyltransferase MSMEG＿5447 complementary strain Comp was successfully constructed.The deletion of MSMEG＿5447 gene interferred the growth of Msm in 7H9 liquid medium,and also made the colony grown on 7H11 solid plate smaller and irregular structure but it grew normally in LBT liquid medium.Therefore,in the following experiment,the strains related to Msm were cultured in LBT medium.Lectin blot showed that the deletion of MSMEG＿5447 gene reduced the O-mannosylation of proteins in whole cell lysate(WCL),cell wall(CW),cell membrane(CM)of Msm.When analyzing the effect of MSMEG＿5447 gene on the resistance of Msm to pressure environment,we found that the lacking MSMEG＿5447gene reduced the resistance of Msm to lysozyme and acid environment,increased the cell wall permeability and reduced the formation of biofilm.Additionally,the lacking MSMEG＿5447 gene increased the synthesis of LAM in Msm.Proteomic analysis showed that the deficiency of MSMEG＿5447 gene affected expression of ornithine transaminase,glyceraldehyde phosphate dehydrogenase and glyceraldehyde phosphate isomerase.These proteins are mainly involved in the process of glycolysis,fructose-mannose metabolism and redox process of Msm.2.The MSMEG＿5447 gene deficiency affected survival of△M5447 strain in macrophagesIn this part,we first established the model of THP-1 cells infected with Msm,and analyzed the survival of WT,△M5447 and Comp strains at 0 h and 24 h after infection.We found that the number of△M5447 strain entering macrophages was not changed,but the number of viable Msm in macrophages decreased after 24 hours infection.Fluorescent dye assay also showed that the fluorescence intensity of△M5447-GFP in macrophages decreased significantly after 24 h infection.q PCR and ELISA results showed that the expression of TNF-α and IL-6 in macrophages infected by△M5447strain decreased significantly.The activation of p65 subunit of NF-κB in macrophages infected by△M5447 strain decreased indicating that the loss of O-mannosylation of protein attenuated the inflammatory response of host cells infected with Msm.We also found that the co-localization of phagosome containing△M5447 strain with lysosomes in macrophages significantly increased,and the expression of CTSD protein increased suggesting that the inhibition of phagosome lysosome fusion may be the potential molecular mechanism of O-mannosylation modification of proteins.RNA sequencing results showed that the differentially expressed genes in macrophages infected by△M5447 strain were significantly enriched in inflammatory signaling pathways.3.O-mannosylation of Mtb Rv1096 played a role during infectionIn this part,we focus on the cell wall glycoprotein Rv1096 of Mtb to explore the effect of O-mannosylation of Rv1096 on its function.The results of Con A lectin blot showed that the glycosylation sites of Rv1096 were T265,S266 and S267,and the glycosylation was significantly decreased in T265 mutant alone,thus we confirmed the recombinant Msm with overexpression of Rv1096 without O-mannosylation(MS＿Rv1096am).Further experiments showed that the O-mannosylation of Rv1096 protein did not affect the growth rate of Msm and the localization of Rv1096 protein in cell wall.The results by Colony forming units(CFU)counting and fluorescent staining showed that the viability of MS＿Rv1096am strain in RAW264.7 cells was decreased compared with MS＿Rv1096.The results by Lysotracker Red staining showed that deletion of O-mannosylation of Rv1096 increased fusion of phagosome and lysosome in macrophages infected with MS＿Rv1096am.Conclusions1.The deletion of MSMEG＿5447 gene not only attenuated the growth of Msm and reduced the formation of biofilm,but also impaired the tolerance to lysozyme and acid environment,increased the permeability of cell wall.It also affected the expression of glycometabolism related proteins.2.The deletion of MSMEG＿5447 gene reduced the survival of Msm in macrophages,attenuated the inflammatory response of Msm infected cells,and promoted the fusion of phagosome and lysosome.3.O-mannosylation of Rv1096 protein inhibited phagosome lysosome fusion and promoted MS＿Rv1096 survival in macrophages.