Functional Studies Of Mycobacterial UDP-N-Acetylglucosamine Enolpyruvyle Transferase(MurA)

Mycobacterium tuberculosis is the pathogen of tuberculosis,which infected approximately 1/3 of the worldwide population.Due to the emergency of multi-drug resistant and even extra-drug resistant strains recently,the existing anti-tuberculosis drugs have been unable to meet the requirements of tuberculosis treatment and the spread control.Therefore,identifying targets for developing new anti-tuberculosis drugs has been a major task currently.M.tuberculosis has a unique cell wall which makes the bacteria to survive in the adverse environment.The core structure of the cell wall consists of peptidoglycan,arabinogalactan and mycolic acid.The peptidoglycan is the structural basis for M.tuberculosis to sustain the morphous and osmotic pressure.The peptidoglycan is a reticular molecule that is the connection of N-acetylglucosamine and N-acetylmuramate and cross-links with oligopeptide.UDP-N-acetylglucosamine(UDP-GlcNAc)and UDP-N-acetylmuramate(UDP-MurNAc)are the sugar donors of N-acetylglucosamine and N-acetylmuramate,respectively.The formation of UDP-MurNAc from UDP-GlcNAc is catalyzed by two enzymes and MurA,UDP-N-acetylglucosamine enolpyruvyle transferase catalyzes the first reaction.By the catalysis of the MurA enzyme,the enolpyruvyle group is transferred from phosphoenolpyruvate(PEP)to the 3'-OH of UDP-GlcNAc,resulting in UDP-N-acetylenolpyruvylglucosamine.The function of MurA enzyme is proved to be essential for M.tuberculosis.Furthermore,the metabolic pathway of N-acetylmuramate as well as MurA enzyme does not exist in mammalian cells.Therefore,MurA is an ideal target to develop anti-tuberculosis drugs.The MurA enzymes from E.coli,H.influenzae,etc.have been characterized and their cystal structures have been determined.Unfortunately,no research data in the kinetic characteristics or the 3D structure of M.tuberculosis MurA has been reported.The major issue is the low solubility of the MurA protein over-expressing in E.coli.In this study,we ultilized a cold-shock expression vector pCold? and induced the MurA protein expression at a low temperature to enhance the its solubility.We purified MurA protein and obtained the kinetic parameters of the enzyme.Due to the pathogenicity,infectivity and low metabolic rate of M.tuberculosis,it is not suitable to investigate the effect of MurA siliencing on M.tuberculosis directly.In this study,we ultilized the fast-growing Mycobacterium smegmatis,which had the same cell wall structure as M.tuberculosis but had no pathogenicity or infectivity,as a model mycobacterial strain in this study.We constructed a conditional murA knockdown strain by using the tetracycline-induced pMind expression vector through antisense RNA methodology and then determined the effect of down-regulation of MurA on mycobacteria.The objectives of this study:1.To obtain purified M.tuberculosis MurA protein;to establish the activity assay of MurA enzyme;to analyze the kinetic properties of MurA enzyme;to screen MurA inhibitors from the secondary metabolite library of marine actinomycetes.2.To construct conditional murA knockdown strain though antisense RNA technology;to investigate the effect of MurA down-regulation on the growth,morphous,structure,drug-sensitivity and proteomics of mycobacteria.3.To construct an acetamide-induced pVVAP expression system as a new research tool for functional analysis of MurA and other proteins.The results of this study:1.Soluble expression,kinetic analysis and inhibitors screening of M.tuberculosis MurA enzyme1.1 MurA enzyme was expressed in soluble formpCold-Mtb was transformed to E.coli BL21 strain.MurA protein was induced by 0.4 mM IPTG at 16? and then purified by Ni-NTA affinity chromatography.Small molecules those might affect MurA activity were removed by ultrafiltration.The results of SDS-PAGE and Western blot detection showed that the purified MurA protein with suitable concentration and purity was obtained.1.2 The activity assay of MurA enzyme was establishedThe UDP-N-acetylglucosamine enolpyruvyle transferase activity of purified MurA protein was detected by HPLC.The result showed that 60.1%decrease in substrate UDP-GlcNAc was observed in the MurA reaction,indicating that the purified MurA protein had the activity of UDP-N-acetylglucosamine enolpyruvyle transferase.In order to establish a simple and rapid enzymatic assay suitable for MurA kinetic analysis and high-throughput screening of inhibitors,a malachite green-based colorimetric assay was developed.The inorganic phosphate generation in the MurA reaction was detected by malachite green.The result of the colorimetric assay further confirmed the UDP-N-acetylglucosamine enolpyruvyle transferase activity of MurA protein.The specific activity of MurA enzyme was 0.305 ± 0.006 ?M min-1 ?g-1.1.3 Kinetic properties of MurA enzyme were measuredThe colorimetric assay was utilized to study the the kinetic properties of MurA enzyme.It was demonstrated that the initial velocity of MurA enzyme was limited to within 15 min and 10?g/ml MurA protein.The MurA had relative maximal activity at a temperature of 37? and reaction buffer of pH 7.5.The MurA activity was independent of Ca2+or Mg2+.The Km and Vmax values of MurA against substrate UDP-GlcNAc were 2.743±0.231 mM and 0.430 ± 0.027 ?M min-1 respectively,whereas the Km and Vmax values of MurA against substrate PEP were 0.199± 0.013 mM and 0.767 ±0.003 ?M min-1,respectively.1.4 Screening of MurA inhibitorsSixty components from the secondary metabolite library of marine actinomycetes were added into the MurA enzymatic reactions.The results of malachite green-based colorimetric assay showed that 6 components could obviously inhibit the enzyme activity of MurA.2.Construction of M.smegmatis murA antisense RNA expression system and the effect of MurA down-regulation on M.smegmatis2.1 M.smegmatis murA antisense RNA expression system was constructedThe DNA fragment from 5' end of M.smegmatis murA gene was inserted reversely to pMind vector and then transformed to M.smegmatis me2155 strain,yielding the conditional murA antisense RNA expression stain.The antisense RNA of murA was expressed under the induction of tetracycline.2.2 Polyclonal antibody against M.smegmatis MurA was preparedThe purified M.smegmatis MurA protein was injected into BalB/c mice.The antiserum was obtained and detected by Elisa and Western blot.The results showed that the polyclonal antibody has both high titer and specificity,suggesting that it could be used to detect the expression level of MurA protein in M.smegmatis murA knockdown strain.2.3 The optimal conditions of pMind system to induce murA antisense RNA was determinedTetracycline at multiple concentrations was added to induce the expression of murA antisense RNA,the inducing condition was optimized according to the growth of the bacteria.The result showed that with the induction of 20 ng/ml tetracycline for 36 h,the expression of MurA protein in M.smegmatis murA knockdown strain was effectively decreased,which caused the inhibition of bacterial growth.2.4 The effects of MurA down-regulation on the growth,morphous,structure,drug-sensitivity and proteomics of M.smegmatis were determinedThe morphous and structural alternations of M.smegmatis murA knockdown strain under the induction of 20 ng/ml tetracycline were observed by scanning electron microscope(SEM)and transmission electron microscope(TEM).The results showed that M.smegmatis with low-expression of MurA exhibited the swelled cellular shape,unintegrated cell wall,exudation of contents and the adhesion between cells.The minimal inhibitory concentration(MIC)of M.smegmatis murA knockdown strain against isoniazide(INH)and ethambutol(EMB)was measured.The results showed that the low-expression of MurA did not change the sensitivity of M.smegmatis against INH or EMB.The proteomics change of M.smegmatis murA knockdown strain was analyzed by 2-D electrophoresis.The results showed that the down-regulation of MurA caused multiple alternations of protein expression level in M.smegmatis.Through MOLDI-TOF-MS,5 differential protein spots,including 2 down-regulated proteins and 3 up-regulation proteins were selected for further identification.3.Construction of an acetamide-inducible expression vector pVVAP and application of pVVAP in mycobacterial conditional antisense RNA expression system3.1 The acetamide induced pVVAP expression vector was constructedThe acetemidase promoter from M.smegmatis mc2155 was amplified by PCR and cloned to the XbaI and NdeI sites of pVV16 vector,yielding the pVVAP vector.3.2 The effect of pVVAP expression system on inducing antisense RNA was confirmedM.smegmatis glmM gene,an essential gene,was used as a model to validate the effect of pVVAP.The DNA fragment from 5' end of glmM was inserted reversely to the pVVAP vector and then transformed to M.smegmatis mc2155 strain.The bacterial growth and the GlmM expression level in M.smegmatis carring pVVAP-As glmM plasmid were measured to evaluate the acetamide inducible expression system.The results showed that with the induction of 1.5%(w/v)acetamide for 72 h,the expression of GlmM protein in M.smegmatis glmM antisense RNA expression strain was effectively decreased,which caused the inhibition of bacterial growth.Therefore,the pVVAP constructed in this study was an excellent inducible vector to down regulate the target genes in mycobacteria.Conclusions:1.Soluble MurA protein was expressed in E.coli BL21 strain by using pCold?expression vector.The purified MurA was proved to possess the activity of UDP-N-acetylglucosamine enolpyruvyle transferase.2.Malachite green-based colorimetric assay of MurA enzyme was established and by using this assay,the kinetic properties were determined.Furthermore,MurA inhibitors were screened from the secondary metabolite library of marine actinomycetes.3.The down-regulation of MurA canse the slow-growth,the morphologic,structural and proteomic alternations of M.smegmatis.4.pVVAP,an acetamide inducible expression vector,was constructed for mycobacterial antisense expression,which would provide a new genetic tool for functional analysis of MurA or other proteins.Further studies:1.To screen MurA inhibitors from compound libraries by using colorimetric assay established in this study;to determine the type of inhibition as soon as inhibitiors of MurA are found;to clarify the mechanism of inhibitor through the crystal structure of MurA-inhibitor complex.2.To investigate the effect of MurA down-regulation on the susceptibilities of M.smegmatis against other anti-tuberculosis drugs,such as rifampicin,pyrazinamide,streptomycin,etc.This work will provide possibility on combination treatment of tuberculosis therapy.3.To validate the differential proteins by Real-time PCR technology;to study the protein-protein interaction network of MurA.This work will be helpful to discover new drug targets related to MurA protein.4.To apply the pVVAP expression system on MurA functional analysis,especially on studing the effect of MurA low-expression on mycobacterial biofilm formation.