Overexpression Of M. Tuberculosis GlmU In M. Smegmatis And Optimization Of GlmU Antisense RNA Expression System

Tuberculosis (TB) caused by infection of Mycobacterium tuberculosis is today amongst the worldwide health threats. The WHO estimates that one third of the world's population (two billion) are infected with M. tuberculosis. Tuberculosis kills someone every 20 seconds. The emergence of multi-drug and extra-drug resistant strains makes invalid of many anti- TB drugs. Thus, more efficient anti-TB drugs are desperately needed.The mycobacterial cell wall is required for the survival and growth of mycobacteria in the host, the core of the cell wall consists of peptidoglycan, arabinogalactan, and mycolic acid for connection. The mycolated arabinogalactan are covalently attached to peptidoglycan via the disaccharide linker (L-rhamnosyl-N-acetyl-glucosaminyl-phosphate) to build up the core structure of mycobacterial cell. UDP-N-acetyl-D-glucose- mine (UDP-GlcNAc) is an essential precursor of the rhamnose-GlcNAc. GlmU protein encoded by glmU gene is a bifunctional enzyme: Glucosa- mine-1-phosphate acetyl transferase and N-acetylglucosamine-1-phosphate uridylyltransferase, which catalyzes two sequential steps in UDP-GlcNAc biosynthesis. The research data from Zhang Wenli in our laboratory de- monstrate that glmU gene is essential for mycobacterial growth. Therefore, GlmU is a potential target to develop anti-tuberculosis drugs.The method to detecting the activity of GlmU has constructed in our laboratory. So we need to constructe an M. smegmatis strain to overexpress GlmU protein as a cell model to screen GlmU inhibitors. Otherwise, to investigate the effect of GlmU enzyme on the structure of the mycobacterial cell wall, we need to constructe a tetracycline inducible expression vector of glmU antisense RNA, pMind-Sm glmU(360bp)-AS to downregulate the expression of GlmU protein.The objectives of this study: (1) To overexpress Tb GlmU protein in M. smegmatis mc2155 and identify the overexpression Tb GlmU protein by Western blotting; (2) To optimize of antisense RNA expression system, mc2155/pMind-Sm glmU(360bp)-AS and detect GlmU protein by Western blotting; (3) To investigate morphological changes of M. smegmatis mc2155/ pMind-Sm glmU(360bp)-AS by scanning electromicroscope.Followings are results we have got in this study:1. Construction of pVV2-Tb glmU expression plasmid pET16b-Tb glmU was digested by Nde I and BamH I, then glmU gene was purified and ligated into the Nde I and BamH I sites of vector pVV2 to generate pVV2-Tb glmU. The N-terminus of GlmU protein was fused with histidine tag in pET16b plasmid.2. Expression and identification of Tb GlmU protein in M. smegmatis mc2155pVV2-Tb glmU was transformed into M. smegmatis mc2155 competent cells to express the GlmU protein. The mc2155 cells were broken by sonication, and the proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. The results showed that Tb GlmU protein in mc2155 was soluble expressed; and the overexpression Tb GlmU protein in M. smegmatis mc2155 was identified.3. Construction of pMind-Sm glmU(360bp)-AS plasmidpMD18-Sm glmU(360bp) was digested by Nde I and BamH I, then 360 bp fragment of glmU gene was purified and ligated into the Nde I and BamH I sites of a tetracycline inducible express vector pMind to generate pMind-Sm glmU(360bp)-AS.4. Expression of glmU antisense RNA induced by tetracycline and detection of GlmU protein in mc2155/ pMind-Sm glmU(360bp)-ASThe mc2155/ pMind-Sm glmU(360bp)-AS was induced by different concentration of tetracycline, and GlmU protein was detected by Western blotting with anti-GlmU polyclonal antibody. The results showed that 20ng/ml tetracycline has obviously inhibited the growth rate of mc2155/ pMind-Sm glmU(360bp)-AS, and, the GlmU of the mc2155 / pMind-Sm glmU(360bp)-AS induced by 20 ng/ml tetracycline had decreased obviously. So, glmU antisense RNA can downregulate the expression of GlmU protein.5. Morphological analysis of mc2155 / pMind-Sm glmU(360bp)-ASMorphology of mc2155/ pMind-Sm glmU(360bp)-AS induced by 20ng/ml tetracycline was analyzed by scanning electron microscope. The micrographs showed there is serious swell formed in the cells and some cells kinked each other. The results suggested that the reduction of GlmU has effect on the structure of cell wall, resulting in morphological changes of cells.Conclusions:1. We constructed a M. smegmatis mc2155 strain to overexpresse Tb GlmU protein. It will be as a cell model to screen the inhibitors of GlmU enzyme.2. We constructed a M. smegmatis mc2155 strain to express glmU antisense RNA and optimized expression condition of glmU antisense RNA. Lack of GlmU has profound negative effects on the cell mor- phology M. smegmatis mc2155.