Targeted suppression of MEP pathway genes DXS, IspD and IspF to explore the mycobacterial metabolism and survival

Rani, N and Surolia, A (2024) Targeted suppression of MEP pathway genes DXS, IspD and IspF to explore the mycobacterial metabolism and survival. In: International Journal of Biological Macromolecules, 272 .

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Abstract

Due to the uniqueness and essentiality of MEP pathway for the synthesis of crucial metabolites- isoprenoids, hopanoids, menaquinone etc. in mycobacterium, enzymes of this pathway are considered promising anti-tubercular drug targets. In the present study we seek to understand the consequences of downregulation of three of the essential genes- DXS, IspD, and IspF of MEP pathway using CRISPRi approach combined with transcriptomics in Mycobacterium smegmatis. Conditional knock down of either DXS or IspD or IspF gene showed strong bactericidal effect and a profound change in colony morphology. Impaired MEP pathway due to downregulation of these genes increased the susceptibility to frontline anti-tubercular drugs. Further, reduced EtBr accumulation in all the knock down strains in the presence and absence of efflux inhibitor indicated altered cell wall topology. Subsequently, transcriptional analysis validated by qRT-PCR of +154DXS, +128IspD, +104IspF strains showed that modifying the expression of these MEP pathway enzymes affects the regulation of mycobacterial core components. Among the DEGs, expression of small and large ribosomal binding proteins (rpsL, rpsJ, rplN, rplX, rplM, rplS, etc), essential protein translocases (secE, secY and infA, infC), transcriptional regulator (CarD and SigB) and metabolic enzymes (acpP, hydA, ald and fabD) were significantly depleted causing the bactericidal effect. However, mycobacteria survived under these damaging conditions by upregulating mostly the genes needed for the repair of DNA damage (DNA polymerase IV, dinB), synthesis of essential metabolites (serB, LeuA, atpD) and those strengthening the cell wall integrity (otsA, murA, D-alanyl-D-alanine dipeptidase etc.). © 2024 Elsevier B.V.

Item Type:	Journal Article
Publication:	International Journal of Biological Macromolecules
Publisher:	Elsevier B.V.
Additional Information:	The copyright for this article belongs to Elsevier B.V.
Keywords:	Amino acids; Bacteria; Enzymes; Metabolism; Metabolites; Polymerase chain reaction, Anti tuberculars; Bactericidal effects; Cell walls; Down-regulation; Knock downs; Methylerythritol phosphate; Methylerythritol phosphate pathways; Mycobacterial; Mycobacterium smegmatis; Pathway genes, Genes, 1 deoxy d xylulose 5 phosphate synthase; 2 c methyl d erythritol 4 phosphate cytidylyltransferase; 2c methyl d erythritol 2 4 cyclodiphosphate synthase; amikacin; beta lactamase; DNA polymerase; hopanoid; isoprenoid; kanamycin; menaquinone; methylerythritol phosphate; moxifloxacin; protein; protein translocase; pyrazinamide; reactive oxygen metabolite; unclassified drug, amino acid metabolism; Article; bacterial growth; bacterial strain; bactericidal activity; bioinformatics; controlled study; differential expression analysis; DNA damage; down regulation; enzyme activity; fatty acid synthesis; fluorescence intensity; gene ontology; metabolism; Methylerythritol phosphate pathway; Mycobacterium; Mycobacterium smegmatis; Mycobacterium tuberculosis; nonhuman; oxidative phosphorylation; oxidative stress; plant growth; real time polymerase chain reaction; real time reverse transcription polymerase chain reaction; RNA isolation; signal transduction; transcriptomics
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	17 Aug 2024 12:15
Last Modified:	17 Aug 2024 12:15
URI:	http://eprints.iisc.ac.in/id/eprint/85460

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