Expression Of M. Smegmatis WecA Membrane Protein And Effect Of Weca Gene Knock-out On Other Protein Expression In M. Smegmatis

Mycobacterium tuberculosis is the pathogenic bacterium of tuberculosisOne-third of the global human population harbors M. tuberculosis in dormant form.For the past few years, it appears multi-drug and even extra-drug resistant strains inM.tuberculosis as using several drugs for long time. Therefore, it’s necessary toidentify some new drug targets of anti-tuberculosis.Mycobacterium smegmatis and M. tuberculosis belong to the Mycobacterium，and have the same structure of cell wall. Rapid growth and pathogenicity of M.smegmatis mc2155strain was used as an experimental model for the study ofmycobacterial cell wall. The mycobacterial cell wall has its unique structure, its core（major） structure consists of three parts, from the outside to the inside is the mycolicacid, arabinogalactan （AG） and peptidoglycan （PG） in turn. The arabinogalactan andpeptidoglycan is covalently linked by L-rhamnose-D-N-acetyl-glucosaminedisaccharide.L-rhamnose-D-N-acetyl glucosamine disaccharide molecule is an importantcomponent to keep the structure of mycobacterial cell wall stable, also a necessary forthe survival of Mycobacterium. The biosynthesis of the linker disaccharide consists oftwo steps catalyzed by two enzymes respectively： the firs step,N-acetylglucosamine-1-phosphate （GlcNAc-1-P） group of UDP-GlcNAc is added toC50-P （Decaprenyl phosphate） to form C50-P-P-GlcNAc by the catalyzed of WecA（N-acetylglucosamine-1-phosphate transferase）; the second step, rhamanopyranosyl ofdTDP-Rhamnose is added to C50-P-P-GlcNAc to form L-rhamnose-D-N-acetylglucosamine disaccharide by the catalyzed of WbbL enzyme （rhamanopyranosyltransferase）.WecA play an important role in the synthesis of disaccharide molecule. If we find out the genes that encode WecA enzyme, we could find the way to inhibitsynthesis of WecA enzyme, and further to inhibit the L-rhamnose-D-N-acetylglucosamine disaccharide molecule, at last to influence mycobacteria growth. Theprevious studies in our laboratory have shown that MSMEG₄947gene is the genewecA which coding M. smegmatis WecA protein, and proves that wecA is anessential gene for mycobacterial growth. In conclusion, WecA protein is likely to bean anti-mycobacterial potential target, especially for Mycobacterium tuberculosis.The objectives of this study:1. Amplifying Sm wecA from M. smegmatis mc2155genomic DNA throughPCR, and cloning Sm wecA to pMD18-T to generate pMD-Sm wecA cloning vector;2.ligating Sm wecA to pET29b to generate pET29b-Sm wecA. expression vector, andtransforming pET29b-Sm wecA to BL21（DE3）, CD41（DE3） and ER2566cellsrespectively for expression of WecA protein;3. The WecA enzyme activity wasdetected by high performance liquid chromatography （HPLC）;4. Usingtwo-dimensional electrophoresis technology to analyze the total protein of M.smegmatis cells for finding proteins that interacted with WecA enzyme.The results are followings:1. Sm wecA amplified from M. smegmatis mc2155genomic DNA throughPCR.Sm wecA was cloned to pMD18-T to generate pMD-Sm wecA cloning vectorAccording to the primer designed by Sm wecA （MSMEG4947） genenucleotide sequence, Sm wecA amplified from M. smegmatis mc2155genomic DNAthrough PCR.Sm wecA was cloned to pMD18-T to generate pMD-Sm wecA cloning vector.and was resequenced in Takara Biotechnology （DALIAN） CO.,LTD.2. Sm wecA ligated to pET29b to generate pET29b-Sm wecA expression vector,and pET29b-Sm wecA transformed to BL21（DE3）, CD41（DE3） and ER2566cellsrespectively for expression of WecA protein.pMD-Sm wecA was digested by NdeI and XhoI, and was ligated to NdeI andXhoI sites of pET29b to generate pET29b-Sm wecA.pET29b-Sm wecA were transformed into three different E. coli stainsBL21（DE3）, CD41（DE3） and ER2566, respectively. At37centi-degree, expression ofthe WecA proteins was induced by1mM IPTG. The supernatant, pellet and totalproteins were analyzed by Dot blot, SDS-PAGE and Western blot.At25centi-degree,1mM IPTG was used to induce expression of WecA protein for16hours in E. coli ER2566cells carrying plasmid pET29b-Sm wecA. WecAprotein was solublized by detergent DDM （n-Dodecyl-β-D-maltoside） formmembrane fraction of E. coli ER2566cells, and was analyzed by Western blot.3. The WecA enzyme activity was detected by high performance liquidchromatography （HPLC）Sm WecA was purified by histidine-Ni2+affinity chromatography （Ni-NTA）.A reaction system was built, substrates C50-P, UDP-GlcNAc and purified WecAprotein were incubated together for30minutes. Chloroform/methanol/ammoniamixture was utilized to extract the reaction product, and in aqueous phase of thereaction product, the consumption of UDP-GlcNAc was analyzed by HPLC.N-acetylglucosamine-1-phosphate transferase activity of WecA protein was proved bypeak area of UDP-GlcNAc reduced.4. Two-dimensional electrophoresis technology was utilized to analyze the totalprotein of M. smegmatis cells for finding out proteins interacted with WecA enzyme.In a small volum of culture, the growth curves of M. smegmatis mc2155YJ-2strain after temperature shift show the time for extraction of protein. After enlargingthe volum of culture, total protein of M. smegmatis mc2155YJ-2cells wasextracted.And total protein of M. smegmatis mc2155YJ-2cells without temperatureshift as a control.Two kinds of protein samples were treated with hydration buffer, isoelectricfocusing electrophoresis and SDS-PAGE electrophoresis were run in turn. Silverstaining kit was used to stain two pieces of gel, and different spots from two pieces ofgel were found out.Conclusions:1. Amplifying Sm wecA from M. smegmatis mc2155genomic DNA throughPCR. pET29b-Sm wecA was constructed. WecA membrane protein was expressed bypET29b-Sm wecA vector in E. coli ER2566.2. WecA protein of M. smegmatis was expressed and purified.Through themethod of HPLC activity of WecA protein was detected, and it is confirmed thatMSMEG₄947is wecA gene coding M. smegmatis WecA protein. The study laid afoundation for revealing the M. smegmatis WecA enzyme properties, and as M.tuberculosis WecA enzyme activity of important complement reflected thatMycobacterium WecA enzymes had GlcNAc-1-P transferase activity.3. Two-dimensional electrophoresis technology was utilized to analyze the total protein of M. smegmatis cells before and after knocking out the Sm wecA. And thisassay would explore a road to find proteins interacted with WecA enzyme.