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

Abstract

New chemotherapeutics are urgently required to control the tuberculosis pandemic. We describe a new pathway from trehalose to alpha-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 alpha-glucan exhibited a synthetic lethal interaction with the glucosyltransferase Rv3032, which is involved in biosynthesis of polymethylated alpha-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.

Figure 1. Characterization of the M. smegmatis ΔglgE mutant

(a) M1P accumulation in the ΔglgE mutant, as revealed by TLC analysis of cell extracts from 48-h-old cultures. See Supplementary Figure 9 for a full, uncut TLC image. (b) Trehalose sensitivity of the ΔglgE mutant. (c) Abolition of M1P accumulation by suppressor mutations in the ΔglgE mutant, as revealed by TLC analysis of cell extracts from 48-h-old cultures. (d) Trehalose sensitivity of the ΔglgB mutant. (e) M1P accumulation in the ΔglgE and ΔglgB mutants determined by TLC/autoradiography analysis of extracts from cells labeled with ¹⁴C-trehalose for the indicated time intervals. (f) Maltose accumulation in Δpep2 mutant strains determined by TLC/autoradiography analysis of cell extracts from cells labeled with ¹⁴C-trehalose for 30 min. In b and d, equal dilutions of cultures were spotted onto Middlebrook 7H10 agar plates containing trehalose. ‘Comp.’ refers to mutants complemented with the respective WT genes.

Figure 2. GlgE is a new M1P-dependent maltosyltransferase

(a) Proposed reaction catalyzed by GlgE. x and y indicate number of glucose units. (b) Acceptor specificity of Mtb GlgE. Enzyme activity with maltooligosaccharide acceptor substrates was determined by monitoring phosphate release in triplicate. The bars indicate means ± s.e.m. (c) Mass spectra showing the extension of maltooligosaccharides (DP2 after 22 h; DP3, DP4 and DP5 after 1 h) by GlgE in the presence of M1P. Maltooligosaccharides were detected as [M+Na]⁺ ions. (d) Determination of the glycosidic linkage formed by GlgE monitored using ¹H NMR spectroscopy. The upper spectrum shows a control without enzyme, and the lower spectrum shows the reaction with enzyme after completion according to the phosphate release assay. Peak assignments are as indicated. (e) Release of M1P from glycogen by Mtb GlgE in the presence of inorganic phosphate analyzed by TLC. St, maltooligosaccharide standards (glucose through maltoheptaose). (f) ESI-MS analysis of M1P formed in e with an expected mass for [M-H⁺]⁻ of 421 Da. Accurate mass spectrometry in positive mode gave a mass of 467.05304 with an expected mass of 467.05371 for [M+Na₂+H]⁺, giving an error of <2 p.p.m.

Figure 3. A new prokaryotic pathway from trehalose to α-glucan

The reactions involved in conversion of trehalose to α-glucan comprise two essential steps in Mtb, catalyzed by GlgE and GlgB. n = number of glucose units.

Figure 4. M1P self-poisoning is lethal for Mtb grown in vitro and in mice

All strains were precultivated in the presence of 5 mM validamycin A to log phase and then subjected to the indicated conditions. (a) GlgE inactivation causes M1P accumulation in a recombinant Mtb H37Rv mutant strain. M1P accumulation was determined by TLC analysis of extracts from cells depleted in validamycin A for 24 or 48 h. See Supplementary Figure 9 for a full, uncut TLC image. (b) GlgE inactivation is bactericidal in vitro in liquid culture. Viability was determined by quantifying colony-forming units (CFU). (c) The conditional lethal mutant strain ΔtreS ΔglgE (pMV 361::treS) is killed in a mouse infection model. BALB/c mice were infected intravenously with 10⁶ CFU per animal. (d) TreS is dispensable for virulence in mice. BALB/c mice were infected intravenously with 5 × 10⁴ CFU per animal. Bacterial burden in organ homogenates in c and d was determined by counting CFU. The data in b, c and d represent means of triplicates ± s.d.

Figure 5. Whole-genome transcriptional profiling of the M1P-induced stress response in Mtb

The conditional lethal Mtb mutant strain ΔtreS ΔglgE (pMV 361::treS) and the vector control strain were grown to log phase in the presence of 5 mM validamycin A to suppress M1P formation. Subsequently, cells were washed to remove the inhibitor, and microarrays were performed on cells after 48 h of depletion of validamycin A. (a) Diagram showing the overlap of the M1P stress response with potassium cyanide (KCN) treatment. Microarray data for KCN treatment have been reported previously and were obtained through the US National Center for Biotechnology Information Gene Expression Omnibus (GEO dataset GSM28256). Only genes with a differential regulation ≥2 are included. (b) Induction of the GlgP-TreX-TreY-TreZ trehalose biosynthetic pathway in M1P-stressed Mtb. Gene expression level was analyzed by qRT-PCR and normalized to 16S rRNA. Expression ratio reported as mean of triplicates ± s.e.m. See also Supplementary Figure 7 for the intersections of the M1P stress response with functionally clustered groups of genes (DNA damage response, electron transport chain and ATP synthase, DosR regulon and the translational apparatus) and Supplementary Figure 8 for qRT-PCR data of other selected transcripts.

Figure 6. Synthetic lethality of treS and Rv3032

(a) Hypersensitivity of the Mtb ΔRv3032 mutant toward the TreS inhibitor validamycin A. (b) Low-level resistance of the Mtb ΔRv3032 mutant against validamycin A mediated by overexpression of the target TreS from an integrative single-copy plasmid. Cultures were inoculated at 5 × 10⁶ CFU ml⁻¹ and cultivated for 7 d. The data in a and b represent means of triplicates ± s.e.m. Viability was determined by quantifying CFU.