Effect Of Lacking D-glucose-1-phosphate Thymidylyltransferase On Mycobacterial Growth And Cell Morphology

Tuberculosis (TB) is today amongst the worldwide health threats. Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. 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 cell wall is necessary for mycobacterial viability. The mybacterial cell wall core consists of mycolic acids, arabinogalacan (AG), and peptidoglycan. AG is attached to the peptidoglycan via a linker disaccharide,α-L-rhamnosyl-(1→3)-α-D-N acetyl- glucosaminosyl- 1-posphate. L-rhamnose in the linker disaccharide is special for the bacteria and not available in human beings. So, it is strongly suggests that the linker disaccharide is an ideal and safe target for new anti-TB drugs.The L-rhamnosyl residue of linker disaccharide is provided with a sugar donor, dTDP-rhamnose. Four enzymes (RmlA-D) participate the dTDP-rhamnose biosynthesis. RmlA encoded by rmlA gene catalyzes the first reaction of the biosynthesis process: converting glucose-1-phosphate and dTTP to dTDP-glucose. The rmlA-D genes for the four enzymes are not located in a locus in the genome of M. tuberculosis H37Rv. The rmlA gene is isolated from any other rhamnosyl formation enzymes, the rmlB and rmlC genes are together in an operon, and rmlD gene is found in an operon with wbbL and manB.In our previous works, we studied and verified the essentiality of rmlB, rmlC and rmlD genes for mycobacterial growth. We also established M. tuberculosis RmlB-D enzyme assays to screen inhibitors for developingnew tuberculosis therapeutics.In this study, we generated Mycobacterium smegmatis mc2155 rmlA gene knockout strain by homologous recombination strategy and tested the essentiality of rmlA gene for mycobacterial growth. We then observed the cellular morphology changes when mc2155 rmlA gene knockout strain lacked RmlA by scanning electron microscope (SEM). We also studied the changes in protein profile when mc2155 rmlA gene knockout strain lacked RmlA by two-dimensional electrophoresis.Followings are results we got in this study:1. Construction of conditional replication plasmid pPR27-xylE-Sm rmlA::KanRAccording to the amino acid sequences of Tb RmlA gained from M. tuberculosis genome, BLAST search was run against the genome of M. smegmatis mc2155. Sm rmlA and its promoter sequence (about 500bp upstream of rmlA gene) was obtained. Sm rmA and its upstream sequence were amplified from mc2155 genomic DNA. The PCR products was purified and cloned into pMD18-T clone vector and sequenced to be right. pUC4K was digested by BamHI to obtain the kanamycin resistance cassette (KanR). KanR was then cloned to the StuI site of Sm rmlA in pMD18-Sm rmlA to create Sm rmlA::KanR mutated gene. The Sm rmlA::KanR fragment was then subcloned to pPR27-xylE to construct pPR27-xlyE-Sm rmlA::KanR. pPR27-xylE carries the temperature-sensitive mycobacterial replication origin and thus can replicate at 30°C but is lost at 42°C and it also harbors xylE gene and a counter-selectable marker sacB.2. Construction of rescue plasmids pCG76-Tb rmlA Tb rmlA gene was cloned to pET23b-Phsp60 to generate pET23b-Phsp60-Tb rmlA, and Phsp60-Tb rmlA fragment was cloned to pCG7 yielding pCG76-Tb rmlA. The expression of Tb RmlA 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.3. Screening of mc2155 M-1 mutantspPR27-xylE-Sm rmlA::KanR was electroporated to wild type mc2155 and transformed mc2155 cells were plated out on LB agar containing Kanat 42°C. Since the temperature-sensitive plasmid was able to replicate at 30°C but not at 42°C, the kanamycin resistant colonies that appear on LB agar containing Kan have necessarily integrated the KanR gene into their genome. Therefore, the single crossover event between Sm rmlA-KanR in pPR27-xylE-Sm rmlA::KanR and Sm rmlA gene in the mc~2155 genome occurred and Sm rmlA::KanR, sacB gene and xylE gene were integrated into the mc~2155 genome resulting in mc~2155 M-1 mutant, which carried both Sm rmlA and Sm rmlA::KanR. SmaI-digested genomic DNA from 7 yellow colonies grown at 42°C was analyzed by Southern hybridization of Sm rmlA probe and was proved to be mc~2155 M-1 strain, having the expected bands of 3.24 kb, 7.72 kb and 8.07 kb.4. Screening of mc~2155 M-2 mutants (rmlA gene knockout strains)Rescue plasmids pCG76-Tb rmlA were electroporated to mc~2155 M-1 and transformed mc~2155 M-1 cells were incubated at 30°C on LB agar containing 10% sucrose and appropriate antibiotics. Since sacB gene product (levansucrase) was lethal to mc~2155 M-1, the double crossover event between Sm rmlA::KanR and Sm rmlA of mc~2155 M-1 genome occurred, and the Sm rmlA gene was replaced by Sm rmlA::KanR in the presence of pCG76-Tb rmlA, resulting in mc~2155 M-2 mutant, that is Sm rmlA gene knock out strain. SmaI-digested genomic DNA from white colonies was analyzed by Southern hybridization, and all colonies were mc~2155 M-2 mutant (rmlA gene knockout strains).5. Growth of mc~2155 M-2 mutantsThe growth curve of mc~2155 M-2 was detected at 30°C and 42°C. mc~2155 M-2 grew only at 30°C, but not at 42°C. The results confirmed that Tb rmlA gene was essential for mycobacterial growth.In the following temperature shift experiment, mc~2155 M-2 were grown at 30°C for 20 h to produce Tb RmlA enzyme and then the cells were grown at 42°C. Growth curves were detected in this Tb RmlA lacking condition. The results showed that mc~2155 M-2 grew for about 1-2 days before its dying.6. Morphology of mc~2155 M-2 mutantsIn temperature shift experiment, certain amount of mc~2155 M-2 cells was acquired. The Scanning electron micrographs showed many seriouscollapse and reductus formed the third day and cell lysis appeared the sixth day, as contrasted with the control samples which were cultivated at 30°C.7. Effect of lacking RmlA on protein profile of mc2155 M-2 mutantsAfter temperature shift, mc2155 M-2 cells growing at 42°C for five days were collected and cytoplasmic protein were obtained. Changes in protein profile were tested by two-dimensional electrophoresis. The results showed that there were some changes in protein profile. The numbers of protein spots were less than those of control samples (mc2155 carrying pCG76). Because lacking RmlA may decrease many proteins relevant to cell wall metabolism, these protein spots need further confirmation by mass spectrometry.Summary:M. smegmatis mc2155 was used as model to clarify the essentility of Tb rmlA for the growth of mycobacterium tuberculosis. Conditional replication plasmid pPR27-xylE-Sm rmlA::KanR was constructed and electroporated to mc2155. The mc2155 M-1 mutants were screened by Southern blot analysis. Rescue plasmid pCG76-Tb rmlA was constructed and electroporated to mc2155 M-1 mutant, whose genomic Sm rmlA::KanR and Sm rmlA underwent second homologous recombination under selecting pressure and Sm rmlA was deleted. The mc2155 M-2 mutants (rmlA gene knockout strains) were screened by Southern blot analysis. We tested the growth of mc2155 M-2 at 30°C and 42°C. The result indicates rmlA gene is essential for mycobacterial growth. We tested the cell morphorlogical change by SEM when lacking RmlA protein. The SEM result shows that lacking RmlA causes cell lysis. We also checked proteomic profile of mc2155 M-2 when lacking RmlA. The result shows that the protein profile of mc2155 M-2 was changed compared to mc2155 cells. Therefore, Tb rmlA gene involved in dTDP-rhamnose synthesis in M. tuberculosis is a valid target for developing new anti-tuberculosis drugs.Further studies:1. To repeat and optimize the conditions for two-dimensional electrophoresis. Select differently expressed protein dots of interests according to the results of 2-D electrophoresis. Peptide mass fingerprinting were obtained by mass spectrometry for identifying these differentlyexpressed proteins. Genes of significance were obtained by molecular cloning in order to find out the relationship between rmlA gene and other genes.2. To analyze the structural differences of cell wall glycan (arabinogalacan) between mc2155 M-2 and wild type mc2155 using HPLC. To detect the differences of cell wall sugar composition of cell wall between mc2155 M-2 and wild type mc2155 by using GC.3. To establish a fast and accurate M. tuberculosis RmlA enzyme assay to screen and optimize lead compounds that are inhibitors of RmlA for further developing new tuberculosis therapeutics. At the same time, these compounds may be further confirmed by the rmlA gene knockout strain as a cell model.