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

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.).