Molecular Mechanisms Of Mycobacterial Adaptation To Isoniazid And Hypoxic Stresses

Mycobacteria are special species of actinomycetes,including Mycobacterium tuberculosis,the causative agent of tuberculosis,and nonpathogenic Mycobacterium smegmatis.Bacteria are inevitably exposed to various environmental stresses during their survival.Antibacterial drugs and hypoxia are two environmental stresses that M.tuberculosis has to cope with when invading hosts.The molecular mechanisms and signaling pathways utilized by mycobacteria for adapting to these stresses remain unclear.In this study,we tried to pursue the underlying molecular mechanisms that mycobacteria adapt to isoniazid and hypoxic stresses with model mycobacterial strains.The results are mainly as follows:(1)A signaling pathway of biofilm formation regulated by three transcription factors was found to explain the new mechanism that Mycobacterium smegmatis protect itself against isoniazid(INH)stress.Ms0179 gene,encoding a GntR-family transcriptional factor,was found to promote the INH resistance in M.smegmatis,which was confirmed in this study.The expression of Ms0179 was also found to be significantly induced by INH.Then,its adjacent Ms0180 was identified to be positively regulated by Ms0179,and overexpression of both genes can result in up-regulating of mmt(mannitol metabolism and transportation)operon(encoded by Ms5571-Ms5576 genes)respectively.Furthermore,Ms0180 protein was confirmed to directly bind to the upstream regulatory region of mmt operons and significantly promote the formation of mycobacterial biofilm as well as its INH resistance,which were also drive when overexpressing mmt operon genes in M.smegmatis.As mycobacterial biofilm enhances its drug resistance,it indicated that M.smegmatis coping with isoniazid stress from Ms0179 was due to the formation of bacterial biofilm,and the process was jointed by Ms0180 regulating mmt operon.Moreover,Ms5575,a gene in mmt operon and encoding a negative regulator,was found to inhibit the expression of this operon.But another intrinsic Ms5576 can encode a mannitol-2-dehydrogenase and metabolize D-mannose to D-mannitol,which then deregulated Ms5575 on mmt operon.These results indicated that mmt operon has the function of self-regulation.Therefore,this study clarified a regulatory mechanism of the process that M.smegmatis resist to INH stress through its biofilm formation.(2)A new mechanism of improving viability under hypoxic stress through induction of rTCA cycle related enzymes was discovered in mycobacterium.First,the expression of citE,korA and korB,the key genes involved in rTCA cycle were determined under hypoxic stress in M.bovis BCG and M.tuberculosis H37 Ra,and these genes were found significantly up-regulated.Subsequent study confirmed that CitE from M.tuberculosis(MtbCitE)is a citrate lyase,and several amino acid residues,such as Glu36,Asp37,Arg64,Glu112 and Asp138,played important roles in its lytic activity.The result that ATP significantly inhibit the activity of MtbCitE was found by analyzing the co-crystallization pattern of MtbCitE and ATP and combining with experimental studies,indicating that bacterial energy metabolism was coupling with the regulation of MtbCitE function.Furthermore,CitE was found to have effect on BCG survival under hypoxia by comparing hypoxic survival of citE knockout,over-expression strain and wild-type strain.Bacterial invasion assays also revealed that citE was significantly beneficial to intracellular survival of BCG by showing that the survival rate of citE knockout strain was lower than wild type strain.Thus,the citrate lyase CitE from M.tuberculosis was believed to play an important role in hypoxia adaption and host invasion.These findings further enhanced our understanding of the molecular mechanism of mycobacterial adaptation to hypoxic stress,and it may provide potential targets for developing new anti-tuberculosis treatments.