Functional Studies On Peptidoglycan Deacetylase Of Mycobacterium Tuberculosis Rv1096 Protein And Its Mannosylation Analysis

Tuberculosis caused by Mcobacterium tuberculosis still is a worldwide infectious disease.Most of cases were in the chronic and latent state,which imply that M.tuberculosis has developed the sophisticated strategies to adapt and evade the host immune surveillances.As well as known,bacterial cell wall which composed of many biological macromolecules is the first barrier exposed to the host immune system.There is increasing evidence that bacterial cell wall components are associated with bacterial virulence even immune evasion.Many bacteria modulate and evade the immune defenses of their hosts through cell wall modification such as peptidoglycan deacetylation to gain the ability to survive in host immune cells.The peptidoglycan deacetylases from Streptococcus pneumonia,Listeria monocytogenes and Lactococcus lactis have been characterized.Additionally the peptidoglycan deacetylation plays an important role in the virulence.As we known,Mycobacterium tuberculosis in the chronic and dormant state is mainly survive and replicate in human alveolar macrophage.All these suggest that the bacteria can use various mechanisms to avoid alveolar macrophage phagosome maturation,blocking lysosomal degradation.However,the PG deacetylase of M.tuberculosis has not been identified yet.Bioinformatic analysis of M.tuberculosis Rv1096,a cell wall protein,revealed that it is homologous to the other known deactylases such as the N-acetyl glucosamine from S.pneumioae named PgdA,the N-acetyl glucosamine from L.monocytogenes named Lmo0415and the peptidoglycan deacetylase from L.lactis named XyD.Rv1096 protein is also a member of O-mannosylated proteins of M.tuberculosis and has three possible mannosylation sites.It is suggested that Rv1096 protein maybe involved in the recognition process with the specific surface receptors of the pulmonary alveolar macrophages.Three parts following were performed in our study.1.To determine the deacetylase activity of Rv 1096 protein.The Rv1096 gene from M.tuberculosis H37Rv genome was cloned and Rv1096 protein was expressed in E.coli.The enzyme assay of deacetylase was established.Rv1096 protein was incubated with M.smegmatis PG as substrate to determine its deacetylase activity.The kinetic parameters were also determined.2.To check the bacterial survival ability owing to Rv1096 protein.M.smegmatis with expression of Rv1096 protein(M.smegmatis/Rv1096)as a model bacterium was constructed.M.smegmatis/Rv1096 was treated with lysozyme and cultured with macrophage.3.To determine the mannosylation of Rv 1096 protein by using ConA-lectin and its mannosylation sites by making Rv1096 mutants using site-directed mutagenesis method.Followings results we got in this study:1.Rv1096 protein possessed metallo-dependent PG deacetylase activity.(1)Rv1096 protein was a novel PG deacetylase,which was able to deactylate PG but had no effect on chitin.(2)Rv1096 protein was a metallo-dependent PG deacetylase and its activity was the highest in the presence of Co2+.(3)The kinetic parameters of Rv1096 protein were determined.Km,0.910±0.007 mM;Vmax,0.514±0.038 ?Mmin-1;and Kcat=0.099±0.007(S-1).2.M.smegmatis/Rv1096 showed resistant to lysozyme and increased survival capacity in microphages.(1)Rv1096 protein expressed in M.had the same deacetylae activity as Rv1096 protein expressed in E.coli.(2)M.smegmatis/Rv1096 was resistant to lysozyme and still had the character of acid-fast.(3)The survival ability of M.smegmatis/Rv1096 was higher than wild yipe M.smegmatis in microphages.3.Rv1096 protein was an O-mannsylated protein which mannosylation site was at 265Thr residue.(1)M.smegmatis was able to carry out the mannosylation of Rv 1096 protein.(2)The O-mannosylated site of Rv1096 protein was at 265Thr residue.(3)The glycan structure of Rv1096 protein was analyzed by mass spectrometry.Conclusions:1.M.tuberculosis Rv1096 protein is a metallo-dependent PG deacetylase.2.M.smegmatis/Rv1096 had the lysozyme resistance and increased survival capacity in microphages owing to the deacetylation of cell wall PG caused by Rv1096 protein.3.Rv1096 protein was an O-mannosylated protein and its mannosylation site was at 265 Thr residue.Further studies:1.To investigate the catalytic mechanism on deacetylase of Rv 1096 protein.To determine the active sites of deacetylase and the specificity of its substrate.2.To deterimine whether Rv1096 protein is a virulent factor or not and which kind of receptor on macrophage interacted with Rv1096 protein.3.To determine the structure and the linkage of the glycan in Rv1096 protein.