Functional Analysis Of New Annotated Genes In Mycobacterium Tuberculosis

Tuberculosis（TB）is an ancient disease that caused by Mycobacterium tuberculosis（Mtb）.The pathogenic mechanism of Mtb is complex and still not clear.The complete genome of Mtb H37Rv was sequenced in 1998 and more than 4000 genes were predicted.Among them,some are related to the structure,growth and proliferation,metabolism of the bacterium,some are related to its pathogenesis,and most genes have been annotated,but there are a few are still unknown.Recently,Xu’s group in Beijing Proteome Research Center analyzed the genome sequence of Mtb and identified 22 unannotated genes.In this study,we functionally annotated four of those unannotated genes,Rv1118,Rv1156,Rv2706,and Rv3108c.We constructed the recombinant M.smegmatis strains that overexpressed those 4 above newly annotated genes of M.tuberculosis.Expressions of those four genes in recombinant M.smegmatis strains were verified by Western Blot,and results showed that all the genes except Rv1118 were expressed successfully in recombinant M.smegmatis strains.Then we analyzed the phenotypes of recombinant strains such as growth,colony morphology and biofilm,and found that there are no significant changes in the recombinant strains compared to the wild type strain or the parental strain.Furthermore,the growth of these recombinant strains was unchanged under stress condtions e.g.acidic condition（low p H）,0.05%SDS or 5 m M H₂O₂.These results suggested that Rv1156,Rv2706,and Rv3108c are not essential for the growth of Mtb under normal and stress conditions.Next we tested the sensitivity of these recombinant strains to several clinic drugs including methotrexate,streptomycin and rifampicin,results of drug sensitivity assays showed that the recombinant M.smegmatis strain overexpressing Rv3108c had a higher growth rate compared with wild type M.smegmatis strain,suggesting that Rv3108c maybe play an important role in Mtb resistance to rifampicin.Finally,we infected RAW264.7 with recombinant M.smegmatis strain overexpressing Rv3108c,the results of infection assays showed that the overexpression of Rv3108c had no effect on the survival of recombinant M.smegmatis strain in macrophages after infection,vice versa,the cell proliferation and the expression of intracellular inflammatory factors including IL-6,IL-1β,TGF-βand TNF-αwere not changed upon infection with the comparison of recombinant M.smegmatis strain ovrexpressing Rv3108c and wild parental or parental M.smegmatis strain.In summary,we found that Rv3108c could enhance the ability of Mtb resistance to rifampicin,suggesting that Rv3108c may play a pivotal role in drug resistance.Other genes may be not essential for the growth of Mtb,but whether their functions in survival or other properties during their infection of host cells still need to be elucidated.Overall,the results of this study will provide new clues for unravelling the mechanism of drug resistance of Mtb,and Rv3108c maybe a new target for clinical drug to treat tuberculosis,especially drug-resistant tuberculosis which is a great thread to human health in the world.