Glycosylation Of Cell Wall Protein Rv0431 From Mycobacterium Tuberculosis And Its Modulation To The Immune Responses Of Macrophages

Tuberculosis(TB)caused by the intracellular pathogen Mycobacterium tuberculosis(M.tuberculosis)remained a global major problem.WHO reported in 2014,about 9.6 million cases of active tuberculosis and 1.5 million death occurred.There were one-third of the world''population infected with M.tuberculosis and most of them remained asymptomatic.The immune responses of host against tuberculosis were complicated.M.tuberculosis lived mainly within host cells,usually macrophages,which constituted the first line of host defense.Mycobacterial proteins,especially cell wall-associated proteins,interacted with macrophages of host to regulate their functions and cytokine production.Macrophage played a critical role in the innate immune response and it was able to directly destroy the intracellular invaders.In the macrophage,the fusion of phagosome containing pathogens with lysosome led to the acidification of phagolysosome,which resulted in the hydrolysis and release of reactive oxygen intermediates.In current years,the advances in cellular mycobacteriology showed that M.tuberculosis evaded the killing of macrophage using complex strategies,including the alternative modes of entry to the macrophage,prevention of phagosome-lysosome fusion.The cytokines which were secreted by macrophage and other immune cells were critical in the control of tuberculosis,and IL-12,IFN-ganda-TNF were established to be most importance of host resistance to M.tuberculosis.It was reported that mycobacterial proteins in cell wall interacted with macrophages and regulated the production of cytokines in host.For example,M.tuberculosis Rv3402c and glycoprotein Sod C both regulated host pro-inflammatory cytokine productionRv0431 was defined as a cell wall associated protein by proteomic analysis of M.tuberculosis and was reported to regulate the host immunity.The data showed that the M.tuberculosis Tn:Rv0431 induced much more IL-12p40,tumor necrosis factor(TNF)-aand IL-6 than wild type M.tuberculosis in vitro.Rath et al.reported that Rv0431regulated the quantity of membrane vesicles containing the lipoprotein Lpq H and Sod C which were ligands for toll-like receptor(TLR)2.Therefore,we investigated the function of Rv0431,a cell wall-associated protein in the M.tuberculosis H37Rv strain.Glycosylation was originally considered to be restricted to in eukaryotes,but was also an important modification in some bacteria,such as mycobaceria.The apa(alanine-and proline-rich antigen)and SodC in M.tuberculosis were firstly reported to be O-mannosylated.Recent studi es indicated that glycoproteins were associated with the interaction between bacteria and the host and evade the host immune response.Apa was able to recognize the immune molecules,surfactant protein A(SPA)and regulated T-cell dependent immune responses.Based on above research,three parts following were performed in our study:1.The Rv0431 protein was expressed in the fast-growing and nonpathogenic Mycobacterium smegmatis(M.smegmatis)and E.coli.Site-directed mutation and western blot was perfomed to detect whether Rv0431 was glycosylated,and to confirm the glycosylation site.The purified protein from M.smegmatis and E.coli was run on SDS-PAGE and blotted on a nitrocellulose membrane.The blot was then incubated with a monoclonal(anti)-polyhistidine His-1 antibody The blot was incubated for 2 h with alkaline-phosphatase conjugated anti-mouse-Ig G antibody.The Rv0431 protein bands were developed in 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium solution.Concanavalin A(ConA)affinity analysis and site-directed mutagenesis was performed to identify the Rv0431 protein as a mannosylated protein.We expressed Rv0431 protein in MS?5447 knockout strain((35)M5447)and further identify Rv0431from(35)M5447 strain were deglycosylated.2.Intracellular survival assay was performed to detect whether Rv0431 were able to enhance the survival of mycobacteria in macrophages.The level of IL-10,IL-12 and TNF-ain the supernant of infected macrophages were detected.Killing of M.smegmatis/vector,M.smegmatis/Rv0431,as well as M.smegmatis/Rv0431_m1-4 by RAW264.7 cells was measured by a colony-forming assay.RAW264.7cells were infected with M.smegmatis transformant.After 4 hours,the residual excellular bacteria in the culture were eliminated by washing three times with Dulbecco''s Modified Eagle Medium(DMEM).At 6,24 and 48 hours after infection,The cells were lysed by 0.025%(w/v)SDS.The lysate of 10?l was plated in duplicate on MB 7H11 agar plate.After incubation at 37°C for 3 days,colonies were counted.The pCG76-gfp plasmids were transformed into M.smegmatis/vector,M.smegmatis/Rv0431 and M.smegmatis/Rv0431_m3.RAW264.7 cells were incubated with transformants M.smegmatis for 24 hours.RAW264.7 cells were incubated in DMEM(Hyclone)containing Lyso-Tracker Red(25 n M)at 37°C for 30 minutes.The cells then were fixed with 2%paraformaldehyde at 37°C for 30 minutes.The imaging was performed by using a Lecia TCS SP8 inverted microscope.Images were collected with filters corresponding to 505-530 nm for green color,and 560-615 nm for red color.In the supernatant of culture infected with transformant mycobacteria,the level of IL-10,IL-12 and TNF-awere detected to explore the role of Rv0431 in modulation or evasion of immune responses.RAW264.7 cells were incubated with M.smegmatis/vector,M.smegmatis/Rv0431 and M.smegmatis/Rv0431_m1-4,respectively.After incubation for 6,24,and 48 h,the culture supernatants of RAW264.7 cells were collected and stored at-80°C for further detection.The IL-10,IL-12p40 anda-TNF level in the culture supernatants was detected by using a ELISA Kit(Abcam,UK)and the concentrations were calculated using a standard curve according to the instruction.3.Pull-down experiment was carried out to identify the receptor of Rv0431 on the macrophage.Rv0431 protein and lysate of macrophage were eluted and run SDS-PAGE.After that,silver staining was performed for visualizing the bands.Followings results we got in the study:1.The Rv0431 proteins expressed in E.coli and M.smegmatis were soluble and had a different molecular weight.Rv0431 was a mannosylated protein.(1)The Rv0431 protein from M.smegmatis had a higher molecular weight than that of the Rv0431 protein from E.coli.SDS-PAGE and Western blotting showed that molecular weight of Rv0431 protein from M.smegmatis and E.coli were about 25Kd and 18Kd respectively.So The Rv0431protein from M.smegmatis had a higher molecular weight(MW)than that of the Rv0431 protein from E.coli.(2)Rv0431 was bound by Con A lectin and was a mannosylated protein.Con A is a lectin specific for mannose and glucose and is capable of binding mannoprotein.The activity of peroxidase coupled with Con A was detected using HRP substrate and results showed that the Rv0431/M protein was bound by Con A,but the Rv0431 expressed in E.coli protein was not bound by Con A.The result suggested that the Rv0431 protein expressed in M.smegmatis was mannosylated.The glycosylation site of Rv0431 protein was predicted by the Net OGlyc 4.0 Server and the prediction results suggested that ⁵⁹Thr,⁶⁰Thr,⁶¹Thr,⁶²Thr were mostly glycosylated SDS-PAGE and Western blot showed that Rv0431_m3(⁶¹Thr mutation)had a lower molecular weight than that of Rv0431 protein and other three mutant proteins.The Con A affinity assay indicated that Rv0431_m3 did not bind to Con A lectin.Thus,the 61Thr was a glycosylation site and the Rv0431 protein was mannosylated.(3)Rv0431 expressed in(35)M5447 was deglycosylated.p Mind-Rv0431 plasmid was electroporated to?M5447 strain and M.smegmatis strain,respectively.Expressed Rv0431 protein was purified by Ni-NTA column.The results showed that Rv0431 protein expressed in M.smegmatis was mannosylated.However,the Rv0431 protein expressed in(35)M5447 lost Con A reactivity,which suggested the protein was deglycosylated.2.Rv0431 modulated the immune response of host as a virulence factor.(1)Rv0431 increased the survival of M.smegmatis in RAW264.7 cells.O-mannosylation contributed to the mycobacterial survival in macrophages.M.smegmatis/Rv0431 and M.smegmatis/Rv0431m3 showed significantly higher bacillary counts at 24h and 48h than M.smegmatis/vector.Moreover macrophages treated with M.smegmatis/Rv0431m3 showed decreased CFU compared to M.smegmatis/Rv0431 infected cells.Confocal microscopy showed that M.smegmatis/GFP/vector,but not M.smegmatis/GFP/Rv0431,fused with lysosomes.(2)Rv0431 increased the production of IL-10,and decreased the production of IL-12 and TNF-aof RAW 264.7 cells.O-mannosylation was involved in it.To further assess the effect of Rv0431 on macrophage,its ability to modulate production of IL-10,IL-12p40 anda-TNF was determined.As shown in Figure 6,there was no difference in IL-10,IL-12 anda-TNF production among the groups treated with M.smegmatis/vector,M.smegmatis/Rv0431 and M.smegmatis/Rv0431_m3 at 6 h after infection.However,after infection for 24 h and 36 h,macrophages treated with M.smegmatis/Rv0431 produced significantly higher level of IL-10 than M.smegmatis/vector.The level of IL-12 anda-TNF secreted by macrophage infected by M.smegmatis/Rv0431 were statistically decreased compared to M.smegmatis/vector treated cells.Furthermore macrophages stimulated by M.smegmatis/Rv0431_m3 secreted more IL-12,a-TNF and less IL-10 than M.smegmatis/Rv0431.There was no difference in level of IL-10,IL-12 and TNF-aamong the macrophages treated with M.smegmatis/Rv0431,M.smegmatis/Rv0431_m1,M.smegmatis/Rv0431_m2 and M.smegmatis/Rv0431_m4.3.Rv0431 probably recognized the mannose receptor(MR)on the macrophage.After silver staining,the bands were visualized and molecular weight was about 170KD,which was identical to MR.The bands were already excised off from the gel for further identification by mass spectrometry.Conclusions:1.Rv0431 protein in M.smegmatis was a mannosylated protein and ⁶¹Thr was mannosylation site.2.Rv0431 helped mycobacteria to enhance the intracellular survival and altered the pro-inflammatory cytokine response of macrophages.Rv0431 might be associated with virulence of M.tuberculosis and mannosylation seemed to be related to the virulence.Rv0431 contribute to the immune evasion of mycobacteria.3.Contribution of Rv0431 protein on immune evasion could be according to interaction of MR.Futher studies:1.To explore the structure and the linkage of the glycan in Rv0431 protein.2.To identify the receptor of Rv0431 and find out the signal pathway initiated by binding of ligand and receptor.To figure out whether there is the interaction between Rv0431 and its receptor and how to influence the fate of microorganism in macrophage.3.To investigate the role of Rv0431 in interaction of pathogen and host in vivo.