Self-poisoning of Mycobacterium tuberculosis by targeting GlgE in an α-glucan pathway

Abstract

New chemotherapeutics are urgently required to control the tuberculosis pandemic. We describe a new pathway from trehalose to α-glucan in Mycobacterium tuberculosis comprising four enzymatic steps mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Using traditional and chemical reverse genetics, we show that GlgE inactivation causes rapid death of M. tuberculosis in vitro and in mice through a self-poisoning accumulation of maltose 1-phosphate. Poisoning elicits pleiotropic phosphosugar-induced stress responses promoted by a self-amplifying feedback loop where trehalose-forming enzymes are upregulated. Moreover, the pathway from trehalose to α-glucan exhibited a synthetic lethal interaction with the glucosyltransferase Rv3032, which is involved in biosynthesis of polymethylated α-glucans, because key enzymes in each pathway could not be simultaneously inactivated. The unique combination of maltose 1-phosphate toxicity and gene essentiality within a synthetic lethal pathway validates GlgE as a distinct potential drug target that exploits new synergistic mechanisms to induce death in M. tuberculosis.

Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis

“…In contrast to Mtb, the M. smegmatis ΔglgE mutant can tolerate the levels of M1P, which is formed from endogenous trehalose by the sequential catalytic action of the trehalose synthase TreS and the maltokinase Pep2. However, if exogenous trehalose is present in the growth medium, this disaccharide is efficiently taken up by the cells and channeled into the same pathway in addition to endogenous trehalose, leading to hyperaccumulation of M1P and causing bacteriostasis in the M. smegmatis ΔglgE mutant (15). During the screening for mutations abolishing the trehalose-sensitivity of the M. smegmatis ΔglgE mutant, we identified a suppressor mechanism based on the metabolic blockade of reactions leading to conversion of trehalose to M1P (i.e., mutations abolishing the function of the trehalose synthase TreS or the maltokinase Pep2) (15).…”

“…However, if exogenous trehalose is present in the growth medium, this disaccharide is efficiently taken up by the cells and channeled into the same pathway in addition to endogenous trehalose, leading to hyperaccumulation of M1P and causing bacteriostasis in the M. smegmatis ΔglgE mutant (15). During the screening for mutations abolishing the trehalose-sensitivity of the M. smegmatis ΔglgE mutant, we identified a suppressor mechanism based on the metabolic blockade of reactions leading to conversion of trehalose to M1P (i.e., mutations abolishing the function of the trehalose synthase TreS or the maltokinase Pep2) (15). In the present work, to identify additional suppressor mechanisms, we performed transposon mutagenesis of the M. smegmatis ΔglgE mutant, selecting for resistance to 1 mM trehalose.…”

“…GlgE is a maltosyltransferase involved in a recently described synthetic lethal trehalose-metabolizing α-glucan pathway in mycobacteria that utilizes maltose 1-phosphate (M1P) as a substrate (15). Although glgE is strictly essential in Mtb, inactivation of the glgE homolog in M. smegmatis results in a conditional lethal mutant strain that is highly sensitive to the exogenous presence of the disaccharide trehalose [α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside].…”

“…All strains were derived from M. smegmatis mc 2 155 and M. tuberculosis H37Rv. The M. smegmatis ΔglgE mutant has been described previously (15). Cells were grown aerobically at 37°C in Middlebrook 7H9 medium supplemented with 10% (vol/vol) OADC enrichment (Becton Dickinson Microbiology Systems), 0.5% (vol/vol) glycerol and 0.05% (vol/vol) Tyloxapol.…”

“…Lipids were visualized by autoradiography. Carbohydrates were extracted from equal amounts of cells with hot water (95°C for 4 h) and analyzed by TLC on Silica gel 60 (EMD Chemicals) using the solvent system 1-propanol:ethyl acetate: water (6:1:3, vol/vol), as described (15).…”