Mycobacterium tuberculosis partitions the Krebs cycle to persist under iron starvation

Reviewer #1 (Public review):

M. tuberculosis exhibits metabolic flexibility, enabling it to adapt to various environmental stresses, including antibiotic treatment. In this manuscript, Serafini et al. investigate the metabolic remodeling of M. tuberculosis used to survive iron-limited conditions by employing LC-MS metabolomics and 13C isotope tracing experiments. The results demonstrate that metabolic activity in the oxidative branch of the TCA cycle slows down, while the reductive branch is reverted to facilitate the biosynthesis of malate, which is subsequently secreted.

Overall, this study is experimentally well-designed, particularly the use of 13C isotope tracing to monitor TCA cycle remodeling under iron-limited conditions. The findings are valuable as they offer potential new targets for antibiotics aimed at non-replicating M. tuberculosis occurring in the hosts. However, despite these strengths, the reviewer has concerns regarding the mechanistic basis underlying the observed metabolic remodeling and its role in M. tuberculosis pathogenesis.

Major Comments:

The authors argue that iron starvation is a physiologically relevant stressor encountered by M. tuberculosis post-infection. Using Erdman and H37Rv strains under DFO conditions, Erdman loses viability, whereas H37Rv maintains it. Nonetheless, both strains exhibit similar metabolic remodeling in the TCA cycle based upon metabolomics and isotope tracing data. The authors should clarify the specific metabolic adaptations in H37Rv that enable it to sustain viability under DFO conditions.

The authors report no significant changes in NAD/NADH and ATP levels in H37Rv and Erdman exposed to DFO conditions. They observe TCA cycle remodeling, particularly the reversal of the reaction between OAA and MAL, catalyzed by malate dehydrogenase, an enzyme that uses NAD+ and NADH as cofactors. The directionality of this reaction likely depends on the relative levels of NAD+ and NADH. Additionally, other dehydrogenases, such as pyruvate DH and aKG DH, also require NAD+/NADH cofactors. In Figure 1I, NAD+ and NADH levels are monitored only at day 3 post-exposure to DFO conditions. Since Erdman loses viability after 2-3 weeks, the authors should include measurements of NAD+, NADH, and ATP levels at weekly intervals up to 3 weeks. Furthermore, glycine levels - which are linked to NAD+ recycling via the conversion of glyoxylate - should be measured under both HI and DFO conditions as an indirect indicator of the NAD+/NADH ratio.

In Figure 2A, it is unclear why a 100-fold accumulation of aKG does not correspond proportionally to the accumulation of (iso)citrate.

The authors state that fumarate, aKG, (iso)citrate, malate, and pyruvate are secreted under DFO conditions. While the secretion of aKG and pyruvate makes sense, given their marked intracellular accumulation, it is puzzling why (iso)citrate, malate, and fumarate are secreted even though there are no changes in their intracellular abundance. To rule out the possibility that these metabolites are released due to bacterial lysis rather than active secretion, the authors should analyze the 13C-labeled fractions of these metabolites in the culture filtrate using the M. tuberculosis culture in media containing 13C glycerol.

To validate the role of the PCK-mediated reductive TCA cycle in malate biosynthesis and secretion under DFO conditions, the authors should generate a malate dehydrogenase (MDH) knockdown strain, considering that MDH is essential, and examine the 13C labeling patterns and NAD/NADH under DFO conditions.

The authors also observe decreased GABA abundance and overall 13C labeling in DFO conditions, suggesting that the GABA shunt is the primary route for Succinate biosynthesis under DFO conditions. Thus, it is strongly recommended that the authors perform a 13C glutamate tracing experiment to directly track labeling in aKG and GABA shunt metabolites, providing more definitive evidence for the involvement of the GABA shunt.

Reviewer #2 (Public review):

Summary:

The authors investigated the effect of prolonged iron limitation (which does stop growth but does not lead to cell death), altering central metabolism in M. tuberculosis. The major tool they used is metabolomics combined with stable isotope tracing. They show that the Krebs cycle is still active, despite the fact that it is dependent on some iron-dependent enzymes. They show that carbon flux through the oxidative branch of the Krebs cycle is stalled, resulting in the accumulation of metabolites, such as malate and alpha-ketoglutarate, that are partially secreted. Apparently, the carbon flux from glycolysis is partially diverted to the reductive branch of the Krebs cycle. This is not achieved by using the glyoxylate shunt but probably through the GABA shunt. This unprecedented split of the Krebs cycle and malate secretion allows a continuous flow of carbon through the core of carbon metabolism, overcoming the metabolic stalling triggered by iron starvation.

Strengths:

Novel insight into the central metabolism of a major pathogen and its adaptation to iron starvation. Carefully conducted experimentation. The paper ends with a clear and helpful model.

Weaknesses:

The authors show some surprising and important findings, but they would need a little more effort to really substantiate these. Especially the role of the GABA shunt should be genetically tested, as they did for ICL and the glyoxylate shunt.

Also, dataset 1 is not very convincing, it is only based on transcriptomics and shown with up or down; this is not a strong base for major conclusions. As a minimum, one would want actual differences, preferably on the protein level, where it really counts.