Study On The Correlation Of MurA Gene And Mycobacterial Growth

Mycobacterium tuberculosis is the pathogen that causes tuberculosis, infectious disease, which has become one of the most serious threaten for human health. Because of the emergence of durg resistant strains of bacteria, tuberculosis incidence is rising in recent years. Therefore, it is essential to find a new and effective drug target and develop new anti-tuberculosis drugs for fighting against the pathogen.The core structure of Mycobacterium tuberculosis cell wall consists of mycolic acids, arabinogalactan and peptidoglycan. Peptidoglycan is basic for sustaining the morphous and osmotic pressure of Mycobacterium tuberculosis. The basal structure of pepetidoglycan is a reticulate molecule that is the connection of N-acetylglucosamine and N-acetylmuramate in turn, then crosslink with oligopeptide. UDP-N- acetylglucosamine and UDP-N-acetylmuramate are sugar donors of N-acetylglucosamine and N-acetylmuramate respectively. The formation of UDP-N- acetylmuramate from UDP-N-acetylglucosamine is catalyzed by two enzymes (MurA and MurB) and MurA, UDP-N-acetylglucosamine enolpyruvyle transferase catalyzes the first reaction. Moreover, MurA and MurB have no homologues in mammalian cells. Therefore, MurA and MurB may be potential targets to develop anti-tuberculosis drugs.Our laboratory has identified Mycobacterium tuberculosis Rv1315 is murA gene to encode UDP-N-acetylglucosamine enolpyruvyle transferase. To confirm the essentiality of murA gene for mycobacteria growth, we need to construct a murA gene knock out strain by using M. smegmatis mc~2155 as an experimental model.The objectives of the study: (1) to construct mc~2155 murA TW-2 (murA gene knock out) strain by homologous DNA recombination method; (2) to confirm the essentiality of murA gene for mycobacteria growth by cultivating murA gene knockout strain at 30°C and 42°C respectively. The results in the study are following:1. Amplification and cloning of the Msm murA geneM.smegmatis murA gene (1257 bp) with its upstream region (463 bp) was amplified from mc~2155 genomic DNA by PCR. The PCR product of 1720 bp was purified and cloned into pMD18-T vector, yielding pMD18-Msm murA plasmid. The Msm murA gene was confirmed by DNA sequencing.2. Construction of conditional replication plasmid pPR27-Msm murA::kanRThe kanamycin resistant cassette from pUC4K was cloned into Msm murA gene in pMD18-Msm murA, generating pMD18-Msm murA::kanR. Msm murA::kanR was cloned into plasmid pPR27-xylE, resulting in a conditional replication plasmid pPR27-Msm murA::kanR which has the same feature with pPR27-xylE.3. Construction of rescue plasmid pCG76-Mtb murAThe Mtb murA gene from pET29b-Mtb murA (constructed in our lab) was cloned into pET23b-Phsp60, yielding pET23b-Phsp60-Mtb murA. The Phsp60-Mtb murA was cloned into the vector of pCG76 to generate the a rescue plasmid pCG76-Mtb murA. pCG76-Mtb murA has the same feature with pCG76. Expression of Mtb murA gene was controlled by the promoter of heat shock protein 60 to complement the function of Msm murA::kanR.4. Selection of M. smegmatis mc~2155 murA TW-1 strain by Southern blot and PCRpPR27-Msm murA::kanR was electroporated into M. smegmatis mc~2155 competent cell, the transformants is cultivated at 30°C and then at 42°C. Three mc~2155 murA TW-1 strains with the first homologous recombination (between pPR27-Msm murA::kanR plasmid and mc~2155 genome) were selected by Southern blotting and PCR.5. Selection of mc~2155 murA TW-2 (murA gene knockout) strain by Southern blot and PCRpCG76-Mtb murA was electroporated into mc~2155 murA TW-1 competent cells and the transformants were cultivated with 10% sucrose at 30°C. Seven mc~2155 murA TW-2 (murA gene knockout) strains with the second homologous recombination (within mc~2155 murA TW-1 genome) were selected by Southern blot and PCR. The expressed Mtb MurA from pCG76-Mtb murA was able to complement the function Msm MurA in murA gene knockout strain.6. Growth curve of murA gene knockout strainThe murA gene knockout strain was grown at 30°C and 42°C respectively. The optical density at 595 nm was monitored at the interval of 24 h. M. smegmatis mc~2155 was as a control. The results showed that murA gene knockout strain was able to grow at 30°C, but not at 42°C. M.smegmatis mc~2155 grew at both 30°C and 42°C. It demonstrates that murA gene is essential for mycobacterial growth.7. Growth curve of murA gene knockout strain after temperature shifting from 30°C to 42°CThe murA gene knockout strain was grown at 30°C and switched to 42°C. The murA gene knockout strain growing at 30°C was a control. The optical density at 595 nm was monitored at the interval of 24 h. The growth curves showed that the murA gene knockout strain grown at 30°C had the normal growth pattern, but the murA gene knockout strain grew slowly after switching from 30°C at 42°C since a certain amount of Mtb MurA produced in the murA gene knockout strain grown at 30°C complemented the deficient murA gene. It proved that murA gene is essential for mycobacterial growth.Conclusion:Insteading of pathogen M. tuberculosis, M.smegmatis mc~2155 which is non-pathogenic and has the same structure of cell wall is as experimental model in the study. The murA gene knockout strain is constructed by homologous DNA recombination. The results demonstrate that murA gene is essential for mycobacterial growth. It strongly supports that murA gene is a potential drug target.