Construction Of Mycobacterium Tuberculosis De-mannosylated Rv1096 Mutant And Its Role In Infection

Tuberculosis is a chronic infectious disease seriously endangering human health around the world.Mycobacterium tuberculosis,a pathogen to cause tuberculosis,is able to evade the host immune system clear that is the key to treatment of tuberculosis.While the complex structure of cell wall of mycobacteria plays a crucial role in this infection and immune escape.As a pathogen causing TB,M.tuberculosis can not only infect lungs,but also invade the nervous system,immune systems,bones,skin,joints,and important parts of brain.Therefore,the studieson the biological process of M.tuberculosis infecting host cells,interaction between M.tuberculosis and macrophages and the immune response of host toM.tuberculosis may find new drug targets,which play a vital role in the fight against tuberculosis.O-mannose-based glycosylationhas been considered to be a protein-modifying type only in eukaryotic organisms.Recent studies have shown that O-mannose-based proteins are also present in actinomycetes including M.tuberculosis.In our preliminary studies,we identified Rv1096 protein O-mannosylation site is ²⁶⁵Thr by using prediction online tools of glycosylation and site directed mutagenesis method.In addition to O-mannosylation of threonine residue,Smith,et al.found that Rv1096protein ²⁶⁶Ser and ²⁶⁷Ser also are the sites for O-mannosylation.In this study,to validate all three sites(²⁶⁵Thr,²⁶⁶Ser,²⁶⁷Ser)for O-mannosylation,Rv1096am carrying three mutantion sites was cloned and expressed in M.smegmatis and identified.To understand the effect of de-mannosylated Rv1096,after M.smegmatis/Rv1096_am was incubated with mouse RAW246.7 macrophage,M.smegmatis survival rates in the macrophage and cytokines from the macrophage weredetected.?.Expression and identification of de-mannosylated Rv1096 mutant,Rv1096_amConstructed pVV2-Rv1096_amexpression plasmid and electroporatedit toM.smegmatis mc²155 for expressionofRv1096_amprotein.After purification,identified Rv1096_amprotein by ConA lectin.?.Effect of de-mannosylated Rv1096 mutant on macrophagesObserved the survival of M.smegmatis in RAW246.7 macrophage and detected the level of cytokines in medium after mouse macrophageRAW246.7 was incubated with M.smegmatis/Rv1096_am,M.smegmatis/Rv1096and M.smegmatis/vector respectively.The results of this study:?.Rv1096_am protein lost the ²⁶⁵Thr,²⁶⁶Ser,²⁶⁷Ser sites for O-mannosylationThe Rv1096_am gene with mutations at ²⁶⁵Thr,²⁶⁶Ser,²⁶⁷Ser was amplied by using Rv1096_m2 as a template.Expression plasmid pVV2-Rv1096_am was constructed and electroporated to Mycobacterium smegmatis mc²155,generating M.smegmatis/Rv1096_amm strain.The Rv1096_am protein was expressed and Rv1096am protein was not detected by ConA lectin.The results indicated that Rv1096_am protein lost the ²⁶⁵Thr,²⁶⁶Ser,²⁶⁷Ser sites for O-mannosylation.?.De-mannosylation of Rv1096 had influence on survival of M.smegmatis within macrophage and cytokine production by macrophageMouse macrophageRAW246.7 was incubated with M.smegmatis/Rv1096_am,M.smegmatis/Rv1096 and M.smegmatis/vector respectively.The viability of M.smegmatis/Rv1096_am strain in macrophages was reduced by detecting CFU.It indicated that the O-mannosylation of Rv1096 protein had effect on the survival of M.smegmatisin macrophages.The macrophage infected by M.smegmatis/Rv1096_amproduced high-level of IFN-?and IL-6.However,the TNF-?level was unchanged.The result demonstrated thatde-mannosylation of Rv1096 can promote the production of cytokines,strengthen the anti infection and inflammation of the body.Conclusion:1.pVV2-Rv1096_am expression plasmid was constructed successfully.The expressed Rv1096_am protein was purified and de-mannosylation of Rv1096 was confirmed by ConA lectin detection.2.De-mannosylation of Rv1096 protein reduced the viability of M.smegmatis in macrophage,therefore,the O-mannosylation of Rv1096 protein has a certain influence on the survival of M.smegmatis in macrophages.It can be inferred that the Rv1096 protein may be a virulence factor and its virulence was weaken to some extent after O-mannosyl modification was removed.Future research:1.To analyze the structure of sugar chain on Rv1096 proteinand explore the function of mannosylation of Rv1096 with its connection manner of sugar chain.2.To further study the role of Rv1096 O-mannosylation in the process of infection.To identify the receptor of host cell to Rv1096 and relatedsignal transduction pathwaysand investigatethe effect of de-mannosylation on the transcription level of host cells.