Active GlmU Is Essential For Growth Of Mycobacteria And Its Lack Causes Dramatic Morphological Changes And Cell Lysis

Tuberculosis (TB) is caused by the pathogen of Mycobacterium tuberculosis (M. tuberculosis). It still remains a major threat to world health because of the emergence of drug resistant strains of bacteria and the co-infection with HIV in recent years. Therefore, it is very important to find a new and effective chemotherapeutics to fight against this causative pathogen.The structure of cell wall is basic for surviving and unique in mycobacteria. Its core consists of two layers. The highly impermeable outer layer is composed of mycolic acids. The inner layer consists of peptidoglycan. These two layers are covalently tethered via the connecting polysaccharide arabinogalactan, which is attached to the peptidoglycan through a disaccharide linker, a-L-rhamnosyl-(1,3)-a-D-GlcNAc-(phosphate). Thus, GlcNAc is an essential component both peptidoglycan (as in most bacteria) and the rhamnose-GlcNAc linker of cell wall in Mycobacteria.The activated nucleotide sugar of GlcNAc is UDP-GlcNAc, is biosynthesized by the bifunctional enzyme, GlmU (the gene coding it is named Rv1018). The GlmU has two kind of activity that include glucosamine-1-phosphate acetyl transferase, which catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) using acetyl Co-A as the acetyl donor, and N-acetylglucosamine-1-phosphate uridyltransferase, which catalyzes the formation of UDP-N-acetyl glucosamine (UDP-GlcNAc) from GlcNAc-1-P and UTP. The two active sites reside on the C and N terminal regions of the protein respectively.In this study, the strain of M. smegmatis mc~2155, which has advantage of fast growing, non-pathogen, high homology and the same cell wall structure as the M. Tb, was used herein instead of M. tuberculosis. The glmU gene was tried to be mutated;the strategy used here is Gene Knock-out according to the homologous recombination while the glmU gene is disrupted by inserting a marked gene inside it. At last, the glmU gene was tested whether it is essencial for bacilli's growth. Furthermore, the morphology for the strain of glmU mutation was observed by microscope including the scanning electric microscope (SEM) and optical electric microscope; and the chemical analysis were done for the sugar component and linkage in the cell wall by Gas Chromatography (GC) and HPLC Dionex ion-exchange columns.The results as following were achieved in this study.1. The gene clone of the Sm glmUThe amino acid sequence of M. tuberculosis H37Rv GlmU protein was acquired from TubercuList World-Wide Web Server (http://genolist.pasteur.fr/TubercuList/). The sequence was blasted against M. smegmatis mc~2155 genome, and the homologue of M. tuberculosis GlmU was found in mc~2155 genome. M. smegmatis glmU gene (1449 bp) with its upstream region (491 bp) was amplified from mc~2155 genomic DNA by using LA Taq DNA polymerase, and the primers which were designed by the sequence from upstream and downstream. The PCR product of 1940 bp was purified and cloned into pMD18-T vector to generate a plasmid pMD18-Sm glmU. Sequences of double-stranded pMD18-Sm glmU were obtained by BcaBest Sequencing primer and M13 primer in TaKaRa Biotechnology (Dalian) Company (Dalian, China). The plasmid of pMD18-Sm glmU, which sequcence matched completely with the glmU of M. smegmatis mc~2155 genomic DNA, was accheived.2. Construction of conditional replication plasmid pPR27-xylE-Sm glmU::KanR The kanamycin resistant cassette from pUC4K was cloned into Sm glmU gene in pMD18-Sm glmU, yielding pMD-Sm glmU:: kanR. After pMD-Sm glmU::kanR was digested and purified, Sm glmU::kanR fragment was put into the vector of pPR27-xylE, resulting in a conditional replication plasmid pPR27-xylE-Sm glmU::KanR, which was used to achieve the first single crossover event at the glmU locus of M. smegmatis mc~2155 genome. The parent plasmid pPR27 is a shuttle vector containing the replication origins for both E. coli and mycobacteria. Specially, the replication origin for mycobacteria has mutations sensitive to temperature. Therefore, it can replicate at 30°C but is efficiently lost at 42°C. The counter-selectable marker sacB gene is used for selection of the double crossover event in the presence of sucrose. The xylE gene in pPR27-xylE-Sm glmU::KanR encodes catechol 2, 3,-dioxygenase which converts catechol into a compound with a bright yellow color. Thus, the xylE gene is in use for screening strains for both single and double crossover event.3. Constructions of the rescue plasmid pCG76-Phsp60-Tb glmURescue plasmid pCG76-Phsp60-Tb glmU was constructed for complementation of Sm glmU::kanR in M. smegmatis mc~2155 glmU gene knock out strains with the second homologous recombination. Tb glmU gene was transcribed from the promoter of heat shock protein 60 (Phsp60) from M. bovis BCG. The parent plasmid pCG76 has the same temperature-sensitive mycobacterial replication origin as pPR27 and thus can replicate at the permissive temperature of 30°C, but can not at 42°C.4. Screening of M. smegmatis mc~2155 glmU SCO-1 strainspPR27-xylE-Sm glmU::KanR was electroporated into mc~2155, the transformants was put at 42°C to force occuring the first homologous recombination between Sm glmU::kanR of the pPR27-xylE-Sm glmU::KanR according to the characteristic of temperature-sensitive mycobacterial replication origin. SmaI-digested genomic DNA from 14 yellow colonies occuring the first single crossover event (SCO-1) was analyzed by Southern hybridization of Sm glmU probe labeled by Digoxin (DIG-Sm glmU). Southern blot results revealed that 9 colonies came from the first homologous recombination and 5 colonies were able to grow on Kan plates due to illegitimate recombination.5. Screening of M. smegmatis mc~2155 glmU KOT (glmU gene knockout) strainsThe rescue plasmid of pCG76-Tb glmU was electroporated to M. smegmatis mc~2155 glmU SCO-1 competent cells. Under selection of 10 % sucrose and expression of Tb glmU in M. smegmatis mc~2155 glmU SCO-1, the genR, sacB, xylE and Sm glmU genes will be deleted from the genome of M. smegmatis mc~2155 glmU SCO-1 when the second single crossover event (SCO-2) occurs, resulting in generation of M. smegmatis mc~2155 glmU KOT, that is, Sm glmU gene is knocked out. The genomic DNA from 6 white colonies was hybridized by DIG-Sm glmU. The Southern blot result showed that all of the 6 colonies selected were from the strain of Sm glmU gene knock out.6. Essentiality of glmU gene for mycobacterial growthTo confirm whether GlmU is essential for mycobacterial growth or not, growth curves of the M. smegmatis mc~2155 glmU KOT strain at both 30°C and 42°C were obtained. The results clearly showed that M. smegmatis mc~2155 glmU KOT grew only at 30°C, but didn't grow at 42°C at which pCG76-Phsp60-Tb glmU was unable to replicate. As contrast, wild type mc~2155 and wild type mc~2155 containing pCG76-Phsp60-Tb glmU grew at both 30°C and 42°C. Therefore, the results confirmed that Tb glmU gene was essential for mycobacterial growth.7. Time course of the growth of the M. smegmatis mc~2155 glmU KOT after shifting to the non-permissive temperature.The M. smegmatis mc~2155 glmU KOT cells were grown at 30°C to 0.09 of OD600, and then the cultures were switched to an incubator at 42°C. The M. smegmatis mc~2155 wild type was used as a control. The growth curve revealed that there was no any effect on M. smegmatis mc~2155 wild type after the temperature changed; but for the M. smegmatis mc~2155 glmU KOT cells, the OD600 began to decrease after reaching to the highest within 24 hours with the temperature shifting. At the same time, to the M. smegmatis mc~2155 glmU KOT cells, the CFU figure, which had same tendency as the growth curve, was obtained. Therefore, a further proof had been given to conform that Tb glmU gene was essential for mycobacterial growth through the result of growth curve and CFU duo to the time course of the growth. Furthermore, the result of CFU also showed that the decrease of OD600 is from the death of cells.8. Morphological changes of the M. smegmatis mc~2155 glmU KOT strain after treatment at the non-permissive temperature.Since UDP-GlcNAc is an important precursor for peptidoglycan of mycobacterial cell wall, we expected a change in morphology and possibly lysis of the bacteria. Thus the morphology of M. smegmatis mc~2155 glmU KOT cells was determined by Scanning Electric Microscope (SEM) and optical microscope. The both results showed that the M. smegmatis mc~2155 glmU KOT cells grown at 30°C exhibited a rod-shaped structure typical of wild type M. smegmatis mc~2155, whereas M. smegmatis mc~2155 glmU KOT bacteria grown at 42°C became dramatically shorter and rounder over time. Furthermore, the SEM result give a clear view that some of the cells grown at 42°C had an evidence of destroyed cell wall and even lysis. Those results suggested that lack of GlmU activity will result in profound negative effects on the bacterial cell morphology.9. The chemical analysis of sugar component by Gas Chromatography (GC) and HPLC Dionex ion-exchange columns (Dionex)The mAGP was extracted and applied to run GC colomn for the M. smegmatis mc~2155 glmU KOT cells with temperature shifting from 30°C to 42°C. The mc~2155 glmU KOT cells grown at 30°C were used as a control. The result showed that the amount of arabinose increased greatly when the active GlmU was lost, but the amount of galactose kept unchangeable.The soluble AG was prepared after removing peptidoglycan and mycolic acid by sodium hydroxide and was run Dionex after being digested by arabinase from Cellumonas gelida. The result revealed that Ara2 (di-arabinoses) and Ara6 (hexo-arabinoses) were main peaks for the mc~2155 glmU KOT cells, which were the same asthose in mc~2155 wild type. However, for the mc~2155 glmU KOT cells, the peak of Ara4 is much bigger than that of wild type mc~2155. Therefore, the result showed that the LAM-like AG existed in mAGP of the mc~2155 glmU KOT cells. Furthermore, since the peak area of Ara4 and Ara6 was increased in mc~2155 glmU KOT cells compared to control, it can be learned that there is more terminal branches of arabinan when the GlmU is lost.ConclusionsIn this study, essentiality of Tb glmU gene for mycobacterial growth was tested in the strain of M. smegmatis mc~2155.Firstly, the condition replication plasmid carring the Sm glmU::KanR was constructed and transformed into M. smegmatis mc~2155, the single crossover event (mc~2155 glmU SCO-1) was identified by Southern Blot through DIG-Sm glmU;Secondly, the rescue plasmid with Tb glmU was constructed and put into the M. smegmatis mc~2155 glmU SCO-1 strains. After the second homologous recombination occured, the knockout strain (mc~2155 glmU KOT) was achieved and identified by Southern blot.Thirdly, the growth curve was got at 30°C and 42°C respectively to the M. smegmatis mc~2155 glmU KOT strain, the result showed that GlmU is essential for mycobacterial growth. Furthermore, the CFU and growth curve after temperature shift from 30°C to 42°C were obtained, and gave a further proof of the growth essentiality in Mycobacteia.Fourthly, the morphologic phenotype was observed by SEM and optical microscope. The cells would become dramatically shorter and rounder over time when the active GlmU is not enough for cells to grow; without active GlmU, the cells became swollen and even lysed because the destroyed structure happened on the cell wall.Fifthly, both GC and Dionex results show that there is much more arabinose branches in the mAGP of mc~2155 glmU KOT cells when the active GlmU is lost. Therefore, we hypothesize that the EmbA and EmbB, arabinosyltransferases for the formation of terminal arabinan branch, would be affected by lacking GlmU, resulting in the change of arabinose in mc~2155 glmU KOT cells.The essentiality of GlmU and the effect of the loss of its activity on mycobacterial cells strongly support its development as a target for new TB drugs.