Mycobacterium tuberculosis partitions the Krebs cycle under iron starvation
Authors/Creators
- 1. Università degli Studi di Padova
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2.
The Francis Crick Institute
- 3. Bind Research
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4.
University of Padua
- 5. The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology
Description
ABSTRACT. In this study, we investigated how iron limitation alters central metabolism in Mycobacterium tuberculosis using metabolomics and stable isotope tracing. Our findings reveal a well-orchestrated metabolic program to enable Krebs cycle activity despite the inefficient action of its iron-dependent enzymes. Under such conditions, carbon flux through the oxidative branch of the Krebs cycle is stalled, resulting in the accumulation of metabolites that are partially secreted. As a result, carbon flux from glycolysis is partially diverted to the reductive branch of the Krebs cycle to support the production of oxaloacetate and malate through the activity of phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Both branches terminate with the synthesis of malate, which is secreted. This unprecedented split of the Krebs cycle and malate secretion in a bacterial pathogen facilitates the continuous flow of carbon through the core of carbon metabolism, overcoming the metabolic stalling triggered by iron starvation.
Methods
METHODS. Intracellular polar metabolites were extracted by mechanical rupture in an acetonitrile:methanol:water (2:2:1, v/v/v) solution as previously described (Serafini et al. 2019). Extracellular metabolites were extracted by diluting the culture-filtrate 1:5 using cold acetonitrile/methanol (1:1) supplemented with 0.1% formic acid; the samples were vigorously vortexed and stored at -20 °C for 2 hours; they were spun down for 10 minutes at 14,000 rpm and 4 °C, then filtered through 0.22 µm filter tubes. 2 µl of pellet extract and 10 μL of culture-filtrate extract were injected in a 1200 Liquid Chromatography system (Agilent) coupled to an Accurate Mass 6220 TOF (Agilent). Polar elution was performed as previously described using a gradient of two solvents, A (mQ water and 0.1% of formic acid) and B (acetonitrile and 0.1% of formic acid) (Serafini A, et al. 2019. Mycobacterium tuberculosis requires glyoxylate shunt and reverse methylcitrate cycle for lactate and pyruvate metabolism. Mol Microbiol 112: 1284-1307. DOI: 10.1111/mmi.14362, PMID: 31389636
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Additional details
Funding
Dates
- Accepted
-
2026-05