The Overexpression Of MSMEG₆281 In M.smegmatis Mc²155 And Its Association With The Cell Morphological Characteristics

Tuberculosis (TB) caused by the infection of Mycobacterium tuber- culosis has become a global infectious disease. The emergence of mul- tidrug-resistant TB (MDR-TB) poses an important threat to TB control as MDR strains makes invalid of many anti-TB drugs. TB is recognized as one of the three most common infectious diseases in the world.Ethambutol (EMB) is a first-line anti-TB drug, which inhibits the growth of M.tuberculosis in vitro. It is reported that it inhibits the biosynthesis of arabinosyltransferase which is one of the key enzymes in the biosynthesis of arabinogalacan (AG). But some researches show that EMB might target multiple molecules. Rv3717, probably one of peptidoglycan hydrolases, is up regulated in H37Rv in response to the EMB treatment, which is regarded as the targeting molecule mined out by systemic bioinoformatic analysis for our research. In M.smegmatis mc²155 (mc²155), MSMEG₆281, is the homolog protein of Rv3717. The overexpression of MSMEG₆281 in M.smegmatis mc²155 and its association with the cell morphological characteristics are studied in current thesis.Objectives:1. To explore the morphological characteristics mc²155 cell with EMB treatment.2. To conform the positive regulation of EMB to MSMEG₆281 gene.3. To find the changes of the growth speed and the morphological chara- ctristics of mc²155 with overexpression of MSMEG₆281; and to spec- ulate its association with the polysaccharide of cell wall. Methods:1. The growth speed of mc²155 with/without EMB treatment was determined by the adsorption spectrophotometry method. The morphological chara- cteristics of mc²155 cells with/without EMB treatment were observed by scanning electron microscopy (SEM).2. The transcriptive expressions of MSMEG₆281 gene in mc²155 cells with/witout EMB treatment were analyzed by reverse transcription- polymerase chain reaction (RT-PCR).3. MSMEG₆281 was cloned in Nova Blue by cloning plasmid of pMD18-MSMEG₆281; and it was expressed in mc²155 by expressing plasmid of pVV16-MSMEG₆281. The expression of MSMEG₆281 in mc²155 was determined by SDS-PAGE and Western Blotting.4. The effect of the overexpression of MSMEG₆281 on the cell growth of mc²155 was analyzed by adsorption spectrophotometry method.5. The morphological characteristics of mc²155 following the overexpr- ession of MSMEG₆281 was studied by SEM.Results:1. M.smegmatis mc²155 was inhibited by EMB obviously. The M.smegm- atis mc²155 cells became shorter in response to the EMB treatment.2. The MSMEG₆281 was up regulated at mRNA level due to the stimu- lation of EMB.3. pMD18-MSMEG₆281 plasmid was successfully constructed, so was the expressing plasmid of pVV16-MSMEG₆281. SDS-PAGE and West- ern Blotting analysis indicated that the MSMEG₆281 was overpressed in mc²155.4. The growth of mc²155 was inhibited to some extent by the overex- pression of MSMEG₆281 relative to the wide type mc²155.5. The mc²155 cells with overxpression of MSMEG₆281 became longer in 48h compared with wild mc²155 cells, while they became shorter in 84h.Conclusions:1. EMB affects the shape of M.smegmatis mc²155 cells as well as inhibits the normal growth speed.2. MSMEG₆281 is up regulated at mRNA level following the EMB treatment. 3. The growth of mc²155 cells is inhibited by the overexpression of MSMEG₆281.4. The overexpression of MSMEG₆281 could change the morphological characeristics of mc²155 cell. MSMEG₆281 might be regarded as one of the autolysins for the cell.