The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs

Abstract

Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.Mycobacterium tuberculosis, the causative agent of human tuberculosis, poses an increasing threat to health. The escalating incidence of drug resistant strains provides a new impetus to understand this complex pathogen and to identify metabolic targets for novel therapeutics. As a first step toward this aim, we have been directing our efforts to identifying metabolic pathways essential for bacterial viability.

IL‐21 enhances the potential of human γδ T cells to provide B‐cell help

“…While such a role can only be postulated in humans and at best supported by indirect epidemiological evidence, infection and/or vaccination experiments in non-human primates might address the contribution of Vg9/Vd2 T cells to the generation of high-affinity, class-switched antibodies. Such studies could exploit the availability of convenient pathogen models such as specially engineered HMB-PP-deficient strains of Listeria monocytogenes [45] or HMB-PP overproducing strains of Mycobacterium tuberculosis [46].…”

“…While such a role can only be postulated in humans and at best supported by indirect epidemiological evidence, infection and/or vaccination experiments in non-human primates might address the contribution of Vg9/Vd2 T cells to the generation of high-affinity, class-switched antibodies. Such studies could exploit the availability of convenient pathogen models such as specially engineered HMB-PP-deficient strains of Listeria monocytogenes [45] or HMB-PP overproducing strains of Mycobacterium tuberculosis [46].Recent findings also point towards a more antigen-restricted role of Vg9/Vd2 T cells in providing B-cell help, given their potential to take up exogenous antigens and present them to CD4 1 and CD8 1 T cells [33,47]. Antigen-presenting gd T cells may thus be able to interact directly with T FH cells.…”

HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe

“…Several studies have been performed in recent years to determine the role of individual enzymes involved in isoprenoid biosynthetic pathways (for in-depth reviews, see Boucher & Doolittle, 2000;Rodríguez-Concepció n & Boronat, 2002;Hunter, 2007). As the majority of pathogenic bacteria use the MEP pathway for isoprenoid biosynthesis (Brown et al, 2010;Buetow et al, 2007;Campos et al, 2001), MEP pathway enzymes have received significant attention as potential drug targets (Hunter, 2007).…”

A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents

“…The cell lines described in this work have the MEP pathway downregulated by limiting the level at which DXP is available. DXS has been reported to represent a rate-limiting step of the MEP pathway in several organisms (8,14,17,29). As a result, altering the levels of DXP available for DXR would be expected to have significant effects on cell viability.…”