The Effect Of MSMEG₆402 Deletion Mutant On Cell Wall Structure In Mycobacterium Smegmatis Mc² 155

The major components of the mycobacterial cell wall consist of mycolic acid, arabinogalactan （AG） and peptidoglycan, in which arabinogalactan is the most important structure.Ethambutol （EMB）, a first line anti- mycobacterial drug, has been reported to impact the integrity of cell wall through inhibiting polymerization of arabinan residue to inhibit the growth of bacteria. Through microarray analysis and bioinformatics study, we found that the synthesis of decaprenyl-phospho-arabinose （DPA）, the only arabinose donor for AG biosynthesis, might be affected by EMB treatment. Further more, Rv3807c was found to be associated with the synthesis of DPA. Rv3807c phosphase activity assay can not be carried out due to lacking of specific substrate, in order to indicate the relationship between Rv3807c and DPA biosynthesis, in this study, the biological function of Rv3807c will be explored in several respects.M smegmatis mc2155, a rapid growing and non-virulent species, was employed as a model of mycobacterial system in this study. Bioinformatics analysis also found that MSMEG₆402 in mc2155 is the corresponding orthologs to Rv3807c.In order to identify the contribution of Rv3807c （MSMEG₆402） to DPA biosynthesis and its role for EMB treatment, in this study, we constructed the MSMEG-₆402 mutant and over-expression strain respectively, and investigate the correspondent responses on the arabinan metabolism, cell growth, and cell morphology and gene expression. Based on the evidences of alterations of molecular expression and cell biological behavior, the biological function of Rv3807c will be predicted and the relationship of DPA synthesis and EMB treatment will be investigated further.Firstly,through SAM statistical analysis, the differentially expressed genes due to EMB treatment was identified by GSE1642 microarray data, which obtained from GEO database（http://www.ncbi.nlm.nih.gov/geo/）. Secondly, the overexpression of Rv3807c in mc2155 resulted in up-regulating the expressions of ispD and ispF for M.sm-ΔM-₆402 mutant strain.MSMEG₆402 was amplified from mc2155 genomic DNA and cloned into pMD18-T plasmid. After confirmation by DNA sequencing, MSMEG₆402 was disrupted by inserting the kan^R cassette from pUC4K. The DNA fragment of MSMEG₆402:kan^R was ligated to pPR27-xylE to generate a conditional replication plasmid pPR27- MSMEG₆402::kan^R （pXYI）.The Rv3807c was amplified from M. tuberculosis H37Rv genomic DNA and was cloned into pMD18-T. The positive recombinant plasmid was confirmed by sequencing and the Rv3807c was ligated to the NdeI and BamHI sites of pET23b-Phsp60. The DNA fragment containing Phsp60- Rv3807c was cloned to the XbaI and BamHI sites of pCG76 to yield a rescue plasmid pCG76-Phsp60-Rv3807c （pXYII）.The pXYI plasmid was electroporated to mc2155 competent cells and transformants were grown on LB agar plates containing kanamycin and gentamicin at 30°C. The S1 mutant （carrying both MSMEG₆402 and MSMEG₆402::kan^R copies ） grown at 42°C was selected by PCR.The rescue plasmid pXYII was electroporated into the S1 mutant. Transformants were grown on LB agar plates containing kanamycin and streptomycin at 30°C. The M. sm-ΔM₆402 mutant strain grown on LB agar plates containing 10% sucrose, kanamycin and streptomycin was selected by PCR and verified by DNA sequencing.Firstly, the growth of M. sm-ΔM₆402 and mc2155 strain was followed by monitoring the optical density （OD） at the absorption of 600 nm at 30°C and 42°C respectively. The wild type mc2155 and mc2155 with rescue plasmid were used as controls, and the OD600 was detected at the interval of 24 hours and the growth curves at both 30°C and 42°C were obtained. Secondly,the assay of EMB-sensitivity were decided by MTT for M.sm-ΔM₆402 and mc2155. Cell shapes for M.sm-ΔM₆402 and mc2155 were observed through JSM-6360LV scanning electron microscopy （SEM）.Ultra-thin sections from M.sm-ΔM₆402 and mc2155 were observed by JEM-2000EX TEM. On the other hand, the detection of the ratio of arabinose （Ala） and galactose （Gal） through HPLC with the CarboPac PA-1 anion-exchange chromatography and 0.1 M NaOH elution buffer. The standards of Ara and Gal （Biosharp）, polysaccharides samples from wild-type and mutant strains were separated and identified. The computation of the ratio of Ara and Gal was based on the hump time. Lastly, the detection of gene expression changes through RT-PCR. RNA samples from three biological replications were extracted and then reverse transcribed to cDNA with reverse transcription reagent kit. PCR reactions were completed and the ratio between the gene of interest and the internal reference （gap） was used for expression analysis. Through SAM statistical analysis, the differentially expressed genes due to EMB treatment was identified by GSE1642 microarray data, which was obtained from GEO database. After cluster and annotation, we found that the expression of the genes those were involved in DPA synthesis was dramatically changed. Rv3807c was found to be associated with DPA biosynthesis.IspF and IspD which are correlation genes for DPA biosynthesis were up-regulated for mc2155 strain with Rv3807c fusion protein overexpressed. The significant gene deletion was obtained by homologous recombination and then verified by PCR and Southern blotting. For the single homologous recombination, PCR and Southern blotting results showed that two of 40 clones were found to have an insert at the expected position. For the second homologous recombination, PCR results showed three of selected clones were amplified 2.2 kb PCR produces, including MSMEG₆402 and KanR sequence, which were proved further through DNA sequencing. M.sm-ΔM₆402 mutant strains were obtained.The growth curve of M. sm-ΔM₆402 mutant showed that M. sm-ΔM₆402 could grow at 42°C but the growth rate at 42°C was slower than that at 30°C. Since the rescue plasmid pXYII was unable to replicate at 42°C no more Rv3807c protein was produced to complement MSMEG₆402::kan^R. Therefore, the results indicated that MSMEG₆402 is non-essential for the growth of M. smegmatis, but the disruption of MSMEG₆402 caused the reduced growth rate of mc2155.MTT analysis for mc2155 and M.sm-ΔM₆402 was performed under different EMB treatmemnt concentration.MTT results showed the differences of inhibition rates between wide-type strain and M.sm-ΔM₆402 mutant strain. Inhibition rate was increased for M.sm-ΔM₆402 mutant.The changes on cell appearance were detected through SEM on M. sm-ΔM₆402 strain. Deformation and bulge on the side of mutant cells were observed, and some cells were cracked around the bulges after the gene was defected at a non-permissive temperature. As a contrast, Control group cells showed regular long rod-shape.Cytoplasm of mutant appeared to be detached from the cell wall and possessed a discrepancy density compared to wild cells through TEM analysis. Cell wall structure tended to be curled, and even tend to cause cellular autolysis. As a contrast, mc2155 cells wall showed regular patterns, instead the cells treated by EMB were damaged heavily.In this study, the ratios of Ara to Gal were respectively 2.1,1.7 and 1.1 for wild type mc2155, M. sm-ΔM₆402 strain and mc2155 cell treated by EMB. The ratio of Ara to Gal was reduced to 1.7:1 indicating inhibition of Ara chain biosynthesis possibly caused by M. sm-ΔM₆402 strain although the inhibition was not as strong as that by EMB.RT-PCR results showed metK was found to be down-regulated in mRNA level due to MSMEG₆402 deletion.ConclusionsIn short, MSMEG₆402 gene deletion may affect biosynthesis of mycobacterial cell wall, reduce content of Ara residues for AG, and change growth characteristic. However, we were still struggling on enzymatic evidence catalyzed by MSMEG₆402; further works are determination of its precise role.