Identification Of M.Smegmatis Glyoxalase Ⅱ And The Effects Of Its Knockdown On M.Smegmatis And Macrophage Cells

1.BackgroundTuberculosis（TB）is one of the most lethal diseases causing millions of deaths each year across the globe.Multiple drug-resistant（MDR）,extensive drug-resistant（XDR）,and totally drug-resistant（TDR）MTB strains have made the treatment extremely difficult.To overcome this hurdle,the development of new drug targets and an effective treatment strategy are desperately needed.This can be achieved by deciphering the role of essential genes which are involved in cell survival.One such enzyme is glyoxalase Ⅱ.The glyoxalase system（glyoxalase Ⅰ and glyoxalase Ⅱ）has a pivotal role in cellular survival and detoxification by converting methylglyoxal（MG）into lactate.Otherwise,the increased concentration of MG then modifies DNA,proteins,and lipids,resulting in abnormalities and cell death.Interestingly,there has been no prominent work so far on mycobacterial glyoxalases even though several mycobacterial genes have been identified as possible glyoxalases including MSMEG＿2975.Hence,MSMEG＿2975 possible glyoxalase Ⅱ has been selected to identify its enzymatic activity and decipher its role in bacterial physiology in this study.2.ObjectivesTo prove the predicted functional activity of MSMEG＿2975（putative glyoxalaseⅡ）experimentally along with enzyme kinetic analysis.Followed by the construction of an inducible knockdown strain of Mycobacterium smegmatis MSMEG＿2975 based on anRNA silencing mechanism.To study the effect of MSMEG＿2975 gene knockdown on bacterial growth,metabolism,survival under stress,antibiotic susceptibility,biofilm formation,and transcriptome.To investigate the effect of MSMEG＿2975 gene knockdown on the survival of M.smegmatis in macrophage cells and production of cytokines in macrophage cells.3.Methods and Results1.Cloning and expression of Mycobacterium smegmatis MSMEG＿2975 and identification of glyoxalase Ⅱ activityThe protein encoded by MSMEG＿2975 gene was predicted as glyoxalase Ⅱ through homology model.MSMEG＿2975 gene wasamplified by polymerase chain reaction（PCR）using the genomic DNA of Mycobacterium smegmatis mc2 155 followed by ligation into the cloning vector（pJET1.2/blunt）for high copy number amplification in cloning strain E.coli NovaBlue.For overexpression,theMSMEG＿2975cloned fragment was restriction enzyme digested and ligated into the expression vector pET-16b and inducible overexpression was achieved by using E.coli BL21（DE3）pLysS transformed with pET16b-GLYII.This expression strain contains an additional plasmid pLysS which prevents fromleaky expression of the recombinant proteins.The homology model of glyoxalase Ⅱ（MSMEG＿2975）was built with SWISS-MODEL,which provides structural details in the absence of experimental data and valuable information of its molecular function.This also facilitates in setting up enzyme assay of glyoxalase Ⅱ.The enzymatic activity of the purified MSMEG＿2975 protein by Ni-NTA resin,was performed by using S-d-lactoylglutathione（SLG）as the substrate.MSMEG＿2975 protein possessed the predicted glyoxalase Ⅱ activity as the formation of 2-nitro-5-thiobenzoate（TNB,a yellow-colored product）detected at 405 nm was increased with time.The Km and Vmax of Gly Ⅱ against SLG were 53.89 μM and 2.69 μM/min,respectively.2.Construction of inducible MSMEG＿2975 gene knockdown strain M.smegmatis KD using pMIND vector with RNA silencing based strategyThe first 420 bp DNA sequence from MSMEG＿2975genewas PCR amplifiedusing genomic DNA of M.smegmatis mc2155 and ligated into the NdeI and SpeI sites of pMIND plasmid in reverse orientation to generate pMIND-MSMEG＿2975-AS420.This resulted in the generation of antisense mRNA sequence upon induction with tetracycline.This cloned fragment was then confirmed by sequencing.This pMIND-MSMEG＿2975-AS420 plasmid was then transformed into electrocompetent M.smegmatis mc2155 cells prepared by using Electroporator 2510（Eppendorf）to construct the MSMEG＿2975conditional gene knockdown strain M smegmatis KD under tetracycline induction.3.The effects ofMSMEG＿2975 gene knockdown on bacterial growth,metabolism,survival under stress,antibiotic susceptibility,biofilm formation,and transcriptome The growth of M.smegmatis KD in the presence of 30 ng/ml tetracycline declined significantly compared to the growth of wild typ.M smegmatis mc2155 in the absence（0 ng/ml tetracycline）,in the presence of 30 ng/ml tetracycline,and compared to the growth of M.smegmatis KD strain with 0 ng/ml tetracycline.The growth of M.smegmatis KD showed a significant decline due to metabolic defects in minimal medium supplemented with or without glycerol compared to the growth of M.smegmatis KD cultured without tetracycline.The growth of M smegmatis KD in minimal medium supplemented with 1%ethanol showed a severe decline compared to the growth of M smegmatis KD cultured without inducer.Reduced survival of M smegmatis was observed under NaNO2,sodium dodecyl sulphate（SDS）,and lysozyme stresses.While the survival of M.smegmatis KD cultured without tetracycline was much better than the test strain.Upon induction of M smegmatis KD with 30 ng/ml tetracycline,the minimal inhibitory concentrations（MIC）for two antibiotics（isoniazid and rifampicin）were reduced.While for ethambutol there was no difference found in the MICs of the test and control strains.The results from the biofilm formation experiment showed that M.smegmatis KD induced with 30ng/ml tetracycline formed less biofilm than uninduced M.smegmatis KD.Transcriptomic data indicates that inducible knockdown of glyoxalase Ⅱ resulted in transcriptional dysregulation of more than five hundred genes significantly.Among these 519 genes,303 genes were down-regulated,while 216 genes were found to be upregulated.These dysregulated genes are predicted to play vital roles in essential metabolic pathways and processes including protein/peptide biosynthesis and lipid biosynthesis along with various transcription regulators that have been significantly altered and affected.4.The effects of MSMEG＿2975 gene knockdown on survival of M.smegmatis in macrophage cells and expression of cytokines in macrophage cellsThe macrophage cell line RAW264.7 was infected by M.smegmatis KD strain.The percent survival of M.smegmatis KD was significantly decreased in RAW264.7 at 24 h and 48 h after infection due to inducible knockdown of MSMEG＿2975.These results implicated the importance of glyoxalase Ⅱ for the intracellular survival of M.smegmatis within macrophages during the infection.The inducible knockdown of MSMEG＿2975 caused increase in expression of pro-inflammatory cytokines IL-1β,IL-6,and TNF-α inM.smegmatis KD infected macrophages after 24 h and 48 h of infection.However,the expression of anti-inflammatory IL-10 was decreased in M.smegmatis KD infected macrophages after 24 h and 48 h of infection compared to the expression of these cytokines in macrophages infected with M.smegmatis KD in the absence of inducer.4.ConclusionBased on the results obtained,this work has indicated the role of（glyoxalase II）in M.smegmatis as a crucial enzyme for detoxification and adaptation.Since,its knockdown hampered the bacterial growth,caused metabolic defects,reduced survival under stress,decreased the MICs of two first-line TB antibiotics,weakened the ability of biofilm formation,and caused dysregulation of hundreds of genes.The transcriptomic data also exhibited various new gene targets for further studies,involved in several metabolic pathways.This might be a stepping stone for establishing effective treatment strategies against mycobacterial pathogens.MSMEG＿2975 gene knockdown affected the survival of M.smegmatis in macrophage cells and expression of cytokines in macrophage cells.Hence,the development of mycobacterial glyoxalase Ⅱ as a new potent drug target is expected.Because,MSMEG＿2975 has more than 70%sequence similarity with Rv2581c（Mycobacterium tuberculosis）,Mb2612c（Mycobacterium bovis）,ML0493（Mycobacterium leprae）,MMAR₂126（Mycobacterium marinum）,and have similar predicted enzyme activity as glyoxalase Ⅱ.