The Functional Study Of Mycobacterium Tuberculosis Rv1096 With Infection Of Macrophage

Tuberculosis(TB)is a chronic respiratory disease that has afflicted humans for years.So far,hundreds of millions of people have been dead from TB.Mycobacterium tuberculosis(MTB)is a kind of aerobic,slow growing bacillus with positive rich lipid acid staining.MTB has special cell wall components: mycolic acid,arabinogalactan,peptidoglycan(PGN).PGN is a special network of biomolecule.It has N-acetyl-glucosamine and N-acetyl muramic acid which are connected by ?-1,4 glycosidic bond.The stable structure of PGN keeps TB away from host immune surveillance and clearing system.It has been reported that PGN deacetylase contributes to lysozyme resistance in L.monocytogenes.However,the function of Rv1096 speculated as a PGN deacetylase in MTB geonome has not been studied during infection.To explore the mechanism how MTB virulent factor Rv1096 effects on MTB survival in macrophage,Rv1096 over expression plasmid p MV261-Rv1096 was constructed using shuttle vector p MV261.The p MV261-Rv1096 was transfected into M.smegmatis mc2155 by electroporation and the p MV261-Rv1096 contained mc2155 was named MS_Rv1096.RAW264.7 and BMDM cells were infected with MS_Rv1096 and the empty vector control MS_Vec,and the bacterial survival rate was determined in macrophages.The clony formation unit result showed that Rv1096 can promote the survival of M.smegmatis in macrophages.The NF-?B and MAPK signaling pathway related inflammatory cytokines were detected,after MS_Rv1096 infection with macrophages.Rv1096 overexpression in M.smegmatis inhibit release of TNF-?,IL-6,IL-1? in macrophages.Bacterial load of lung,liver,spleen was also measured in C57BL/6 mice infected with MS_Rv1096.and inflammation of mice model showed that the bacterial loads of MS_Rv1096 from mice lung,liver,spleen were higher than those of mice infected with MS_Vec.Further detection of the inflammatory response in lung and serum showed that MS_Rv1096 infection induced decreased inflammatory response.It has been known that L.monocytogenes PGN deacetylated by Pgd A can resistant to lysozyme digestion.The resistance of MS_Rv1096 to lysozyme was evaluated because Rv1096 is assumed to be a PGN deacetylase.We cultivated MS_Vec and MS_Rv1096 with different concentration of lysozyme,and recording 600 nm absorbance of MS_Vec and MS_Rv1096 at different time.To detect the enzyme activity of Rv1096,we analyzed PGN from MS_Vec and MS_Rv1096 by RT-IR.As expected,the MS_Vec showed more sensitivity to lysozyme than MS_Rv1096.It is known that PGN can be sensed by TLR2.We found that Rv1096 may deacetylate PGN to avoid stimulating TLR2,resulting in reduced inflammatory response and increased bacterial load.