Functional Study Of WecA Gene In Mycobacteria

Tuberculosis (TB) is one of the most serious infective desease which threats the worldwide health. Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. TB has a certain cell wall structure, which is essential for its existence and reproduction. The mybacterial cell wall core consists of mycolic acids, arabinogalacan (AG) and peptidoglycan (PG). AG is attached to the PG via a linker disaccharide, the disaccharide linker plays so important role through the synthesis of mycobacterial cell wall that the integrality of cell wall will be destructed and the cell can not exist if the disaccharide linker destroyed.The synthesis of the disaccharide linker is depended on two enzymatic reactions. In the first reaction, C50-P is added to N-acetylglucosamine-P group to form Cso-P-P-GlcNAc by a transferase, then the product accepts a Rha group under the catalysis of WbbL and converts to the final product disaccharide linker D-N-acetylglucosamine-L-rhamnose. Obviously, the enzyme in the first reaction is the key point of disaccharide linker synthesis. But in mycobacteria, this enzyme is not found till now and the gene which encodes this enzyme is uncertain.In E. coli, the function of WecA is acting as a GlcNAc-1-P transferase to synthesis one of the most important components in LPS:O-PS. The BLAST analysis between WecA of M. tuberculosis and E. coli is showed that the two enzymes have 27% similarity. Based on this BLAST result, WecA of M. tuberculosis is probably has transferase enzyme activity as its homolog WecA of E. coli has. In M. tuberculosis, the function of WecA enzyme has not been clearly identified, but it may possibly have the same catalytic activity as a GlcNAc-1-P transferase and play a necessary role to help the synthesis of disaccharide linker.The objective of our study are:(1) To introduce firstly pMD18-SmwecA plasmid containing Mycobacterium smegmatis (Sm) wecA gene and pMD18-Tb wecA plasmid containing Mycobacterium tuberculosis (Tb) wecA gene into wecA gene defective strain E. coli MV501 to investigate if WecA in mycobacteria have the similar enzyme activity as E. coli WecA has. Mycobacterial WecA protein function will be detected by HPLC and LC/MS/MS. (2) To generate Mycobacterium smegmatis mc2155 wecA gene knockout strain by homologous recombination strategy and test the essentiality of wecA gene for mycobacterial growth, and then to observe the cellular morphological and structural changes when mc2155 wecA gene knockout strain lacked Tb WecA by scanning electron microscope (SEM) and transmission electron microscope (TEM). Inhibitory function of Tunicamycin at different concentrations to Tb WecA protein in mc2155 YJ-2 (wecA gene knocked out strain) will be tested. (3) To overexpress Tb WecA protein in three different E. coli strains and detect WecA enzyme activity.Followings are results we got in this study:1. LPS synthesis of E. coli wecA gene defective strain MV501 was complemented with Sm wecA and Tb wecA gene respectively.pMD18-Sm wecA (pYJ-1) and pMD18-Tb wecA (pYJ-2) was electroporated into wecA gene defective strain E. coli MV501, then LPS was extracted and electrophoresised. E. coli MV501 electroporated with pUC18 plasmid was used as a control. Only LPS from MV501 (pYJ-1) and MV501 (pYJ-2) cells showed a ladder like pattern characteristic of O side chain material.The membrane protein of MV501 (pYJ-1) and MV501 (pYJ-2) was extracted and incubated with substrates of C50-P and UDP-GlcNAc, after the reactions have completed, supernatant was separated and detected with HPLC and LC/LC/MS. The analyzing results of HPLC and LC/LC/MS suggested that the substrate of UDP-GlcNAc was decreased in above two reactions compared to the membrane from MV501 (pUC18).2. Mycobacterium smegmatis wecA gene knock out strain was constructed and essentiality of wecA gene was tested. The inhibitory activity of tunicamycin to Tb WecA enzyme function was detected. (1) Constrction of mc2155 YJ-2 mutants (wecA gene knockout strains)pUC4K was digested by BamHI to obtain the kanamycin resistance cassette (KanR). KanR was cloned to Sm wecA in pYJ-1 then subcloned to pPR27-xylE to construct pPR27-xlyE-Sm wecA::KanR (pYJ-4).Tb wecA gene was cloned to pET23b-Phsp60 to generate pET23b-Phsp60-Tb wecA (pYJ-5), and Phsp60-Tb wecA fragment was cloned to pCG76 yielding pCG76-Tb wecA (pYJ-6). The expression of Tb WecA was controlled by the promoter of heat shock protein from M. bovis BCG. The plasmid pCG76 carries the same temperature-sensitive mycobacterial replication origin as pPR27-xylE.pYJ-4 was electroporated into wild type Mycobacterium smegmatis mc2155 at 42℃, the single crossover event between Sm wecA-KanR in pYJ-4 and Sm wecA gene in the mc2155 genome occurred and resulted in mc2155 YJ-1 mutant, which carried both Sm wecA and Sm wecA::KanR. There were two homologous recombination pathways identified by Southern hybridization.Rescue plasmids pCG76-Tb wecA were electroporated into mc2155 YJ-1 and the transformed mc2155 YJ-1 cells were incubated at 30℃on LB agar containing 10% sucrose and appropriate antibiotics. Since sacB gene product (levansucrase) was lethal to mc2155 YJ-1 for the existence of sucrose, the double crossover event between Sm wecA::KanR and Sm wecA of mc2155 YJ-1 genome occurred, and the Sm wecA gene was replaced by Sm wecA::KanR in the presence of pCG76-Tb wecA, resulting in mc2155 YJ-2 mutant.(2) Growth of mc2155 YJ-2 mutantsThe growth curve of mc2155 YJ-2 was detected at 30℃and 42℃. mc2155 YJ-2 grew only at 30℃, but not at 42℃. The results confirmed that Tb wecA gene was essential for mycobacterial growth.In the following temperature shift experiment, mc2155 YJ-2 were grown at 30℃for 20 h to produce Tb WecA enzyme and then the cells were shifted to 42℃. Growth curves were measured in this Tb WecA lacking condition.(3) Morphological and structural changes of mc2155 YJ-2 mutantsIn temperature shift experiment, certain amount of mc2155 YJ-2 cells was acquired. The Scanning electron micrographs showed irregular surface with a crumpled appearance at 72 h and cell lysis appeared at 144 h, most cells began to swell, as contrasted with the control samples which were cultivated at 30℃.TEM analysis showed that mc2155 YJ-2 grown at 42℃for 144 h became bigger (in diameter) and exhibited pear shape, compared to mc2155 YJ-2 cells grown at 30℃. The vacuoles also were observed in mc2155 YJ-2 cells grown at 42℃for 144 h.(4) Inhibitory activity of tunicamycin to Tb WecA enzymeTunicamycin with different concentrations was added to mc2155 YJ-2 strain which had been cultured of 24 h under 30℃and absorbance was measured every 24 h until 120 h. From the growth curves we concluded that tunicamycin had inhibitory effects to Tb WecA enzyme.3. pET16b-Tb wecA (pJY-7) was constructed and expression of Tb WecA in different E.coli strains was investigated.pET16b-Tb wecA (pJY-7) was constructed and transformed into three E. coli expression strains BL21(DE3), C41(DE3) and ER2566, respectively. The membrane protein was extracted and analyzed by dot blot, SDS-PAGE and Western blot. The expressed Tb WecA protein was detected in three E. coli strains by dot blot. However, the expressed Tb WecA protein could not detected by SDS-PAGE and Western blot. The membrane protein from three strains was prepared and incubated with substrates of C50-P and UDP-GlcNAc. As soon as the reactions completed, the supernatant was separated and detected with HPLC. The analyzing results suggested that the substrate of UDP-GlcNAc was decreased from above reaction compared to the control reaction.Summary:1. LPS synthesis of E. coli wecA gene defective strain MV501 was complemented with Sm wecA and Tb wecA gene respectively.2. The membrane protein of MV501 (pYJ-1) and MV501 (pYJ-2) contain GlcNAc-1-P transferase activity. Therefore, Sm wecA and Tb wecA was the gene to encode GlcNAc-1-P transferase.3. Mycobacterium smegmatis mc2155 wecA gene knock out strain, YJ-2, was constructed to clarify the essentility of Tb wecA for mycobacterial growth.4. The growth curves of me2155 YJ-2 after temperature shift further proved that wecA is essential to mycobacterial growth.5. The cell morphorlogical and stuctural changes of mc2155 YJ-2 were observed by SEM and TEM when lacking Tb WecA protein. The SEM and TEM results showed that lacking WecA caused a serious of changes in cells and made cell death in the end.6. Tunicamycin with different concentrations could inhibit the growth of mc2155 YJ-2. Therefore, tunicamycin can affect the function of Tb WecA.7. mc2155 YJ-2 strain can be used a cell model to establish a fast method to screen lead compounds that are potential anti-tuberculosis drugs for further effective tuberculosis therapeutics.8. The expressed Tb WecA membrane protein in different E. coli strains was detected by dot blot. WecA assay was performed using Tb WecA membrane protein and GlcNAc-1-P transferase activity was detected by HPLC method.Further studies:1. To select suitable vector and expression strains and optimize the expression conditions to realize overexpression of Tb WecA membrane protein.2. To develop a simple and effective TLC method to determine WecA activity.3. To study proteomics of mc2155 YJ-2 by using two-dimensional electrophoresis. Select differently expressed protein dots of interests according to the results of 2-D electrophoresis. Peptide mass fingerprinting was obtained by mass spectrometry for identifying these differently expressed proteins. The genes of interest were obtained by molecular cloning in order to find out the relationship between wecA gene and other genes.4. To analyze the structural differences of cell wall between mc2155 YJ-2 and wild type mc2155 using HPLC.5. To establish a fast and accurate Tb WecA enzyme assay to screen lead compounds that are potential anti-tuberculosis drugs for further effective tuberculosis therapeutics. At the same time, these compounds may be further confirmed by the wecA gene knockout strain as a cell model.