Functional Analysis Of The Unannotated Genes In Mycobacterium Tuberculosis

Tuberculosis,which is caused by Mycobacterium tuberculosis(Mtb),is one of the main causes of human death.When host was infected with Mtb,infected cells will trigger a local inflammatory response that will attract immune cells into the site of infection to form the granulomas which is the pathological hallmarks of tuberculosis.The correct annotation of genes and their functional analysis are the prerequisites and foundations for efficient research of life science and medicine.In 1998,the complete genome of Mtb H37 Rv was sequenced,which is strongly supported the research for basic biological regulatory pathways in Mtb.However,due to the insufficiency of genome annotation technology,some genes of Mtb still not be identified,which restricts people’s in-depth and correct analysis of the biological process and pathogenic mechanism of Mtb.By the proteogenomics strategy,Xu’s group re-annotated the H37 Rv genome sequence and found 22 unannotated genes includingRv2742 and Rv2203 c.MSMEG＿2697 is the homologous gene of Rv2742 in M.smegmatis.In this study,we silenced the MSMEG＿2697 in M.smegmatis usingCRISPRi and ectopically expressed Rv2742 and MSMEG＿2697 in M.smegmatis.Results showed that knockdown of MSMEG＿2697 and overexpression of MSMEG＿2697 or Rv2742 had no significant effect on the growth,colony morphology,and drugsensitivity of M.smegmatis as well as its survival after infection of macrophage RAW264.7.Taken together,these results suggest that Rv2742 is not essential for bacterial growth.Sequence alignment of Rv2203 c revealed that there is no homologous gene in M.smegmatis,but Rv2203 c has a point mutation(G520A)in BCG(Rv2203cMT)compared to H37 Rv and H37Ra(Rv2203cWT).To further study the role of Rv2203 c,we ectopically expressed Rv2203 c in M.smegmatis.Results showed that,compared with the control strain,the growth rate of M.smegmatis overexpression of Rv2203 cMT was significantly decreased and the growth rate of M.smegmatis overexpression of Rv2203 cWT was even more decreased.In addition,we found that the morphology and surface structure of the colony of M.smegmatis overexpression of Rv2203 c is round,shiny and smooth,which is different with that of control strain.These results indicate that Rv2203 c may be associate with the cell wall of Mtb.Next,we tested the sensitivity for isoniazide and rifampicin of M.smegmatis overexpressingRv2203 c.Results showed that overexpression of Rv2203 c in M.smegmatis did not change the sensitivity to isoniazide and rifampicin.Finally,we infected RAW264.7 with M.smegmatis overexpressingRv2203 c and found that the overexpression of Rv2203 c impaired the survival of M.smegmatis in macrophages.In summary,we constructed knockdown models usingCRISPRi system and overexpression models to study the role of unannotated genes in M.smegmatis.The results of this study will unravel the function of unannotated gene which will provide a new target and strategy for effective therapy of tuberculosis.