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. 2022 Jul 9;5(1):685.
doi: 10.1038/s42003-022-03650-z.

Iron limitation in M. tuberculosis has broad impact on central carbon metabolism

Monique E Theriault  1 , Davide Pisu  1 , Kaley M Wilburn  1 , Gabrielle Lê-Bury  1 , Case W MacNamara  2 , H Michael Petrassi  2 , Melissa Love  2 , Jeremy M Rock  3 , Brian C VanderVen  1 , David G Russell  4
Affiliations

Iron limitation in M. tuberculosis has broad impact on central carbon metabolism

Monique E Theriault et al. Commun Biol. .

Abstract

Mycobacterium tuberculosis (Mtb), the cause of the human pulmonary disease tuberculosis (TB), contributes to approximately 1.5 million deaths every year. Prior work has established that lipids are actively catabolized by Mtb in vivo and fulfill major roles in Mtb physiology and pathogenesis. We conducted a high-throughput screen to identify inhibitors of Mtb survival in its host macrophage. One of the hit compounds identified in this screen, sAEL057, demonstrates highest activity on Mtb growth in conditions where cholesterol was the primary carbon source. Transcriptional and functional data indicate that sAEL057 limits Mtb's access to iron by acting as an iron chelator. Furthermore, pharmacological and genetic inhibition of iron acquisition results in dysregulation of cholesterol catabolism, revealing a previously unappreciated linkage between these pathways. Characterization of sAEL057's mode of action argues that Mtb's metabolic regulation reveals vulnerabilities in those pathways that impact central carbon metabolism.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Growth inhibition of Mtb upon treatment with sAEL057 in various conditions.
a, b Mtb were grown in broth (7H9 + OADC, 7H9 + Oleate, 7H9 + Acetate, 7H9 + Glucose, and 7H9 + Cholesterol) and treated with sAEL057 at 50 µM down to 0.097 µM in a dose curve-dependent manner. Alamar blue was added on day 9 and results were measured via fluorescence on day 10. HMDMs were infected at MOI 2:1 with mCherry-expressing Mtb and fluorescence values were measured at 7dpi. a Representative graph of percent inhibition curves. Percent inhibition was determined relative to DMSO (0% inhibition) and 20 µM RIF (100% inhibition) controls. n = 2 technical replicates from a representative experiment. b EC50 calculated using nonlinear regression analyses (log inhibitor vs response) of percent inhibition curves. n = 3–5 replicate experiments c, d Mtb was grown in 7H9 + OADC (c) or 7H9 + cholesterol (d), and replicate plates were used for plating CFUs on day 5 and day 10 posttreatment. n = 4 from two replicate experiments. e Mtb-infected HMDMs were lysed via 0.01% SDS in water at 4 dpi and 8 dpi to plate for CFU. n = 4 from two replicate experiments. Statistical significance and P values were assessed using a student’s unpaired t-test. Error bars indicate standard deviation. Source data and P values for all main figures are available in Supplementary Data 1.
Fig. 2
Fig. 2. sAEL057 treatment leads to dysregulation of genes involved in iron homeostasis and cholesterol metabolism in Mtb.
a PCA analysis of Mtb transcriptomes from infected HMDMs. b, c Heatmaps showing relative expression of genes in the KstR1 regulon. d, e Heatmaps showing relative expression of genes involved with iron homeostasis. a, b, d, f, h, j, l HMDMs were infected at MOI 2:1 for 2 days with mCherry-expressing Mtb prior to administration of sAEL057 at 10 µM. Infected macrophages were sorted for mCherry positivity 2 days after sAEL057 treatment was commenced, at 4 dpi. Samples were sorted into triazole prior to RNA extraction. c, d, g, i, k, m Mtb was pre-grown in cholesterol-supplemented media (7H9 + cholesterol) for 2 days prior to the addition of sAEL057 at 10x MIC (23 µM). Samples were collected at 4 h posttreatment for RNA extraction. Normalized counts were used for the generation of all heatmaps. f–m Violin plots showing expression (in log normalized counts) of genes encoding for HsaC, FadE29, BfrB, and MbtI in the two environmental conditions. Statistical significance values based on adjusted P values (padj) in Supplementary Data 1 for respective genes.
Fig. 3
Fig. 3. Gallium addition synergizes with sAEL057 treatment to inhibit Mtb growth in cholesterol media but not in rich broth.
Mtb were grown in cholesterol-supplemented media (a–c) or rich broth (d–f) and treated with sAEL057 at 50 µM down to 0.097 µM in a dose curve-dependent manner. Alamar blue was added at day 9 and results were measured via fluorescence on day 10. a, d Representative graphs of percent inhibition curves. Percent inhibition was determined relative to DMSO (0% inhibition) and 20 µM RIF (100% inhibition) controls. n = 2 technical replicates from a representative experiment. b, e EC50 values calculated using nonlinear regression analyses (log inhibitor vs response) of percent inhibition curves. n = 4 from two replicate experiments. c, f % Fitness defect was calculated by dividing gallium only (without sAEL057) controls by DMSO controls and multiplying by 100 in the given media condition. n = 4–6 from two replicate experiments. Statistical significance was assessed using a student’s unpaired t-test. Error bars indicate standard deviation. Source data and P values for all main figures are available in Supplementary Data 1.
Fig. 4
Fig. 4. sAEL057 treatment leads to altered cholesterol metabolism in Mtb.
a, b Mtb was pre-grown for 6 days in cholesterol-supplemented media prior to the addition of sAEL057 at 10 µM or DMSO control. [4-14C] radiolabeled cholesterol was added at 24 h posttreatment and 14CO2 was measured 6 h later. n = 6 from two replicate experiments. c, d The bacterial reporter, prpD’::GFP smyc’::mCherry, was used to assess levels of propionyl-CoA with sAEL057 treatment in cholesterol-supplemented media. c % GFP-positive bacteria were determined by gating on mCherry positive bacteria and measuring the GFP-positive signal using a BD Symphony analyzer. d Bacteria positive for both mCherry and GFP fluorescence were analyzed to determine the GFP median fluorescence intensity (MFI). n = 2 from replicate experiments. Statistical significance was assessed using a student’s unpaired t-test. Error bars indicate standard deviation. Gating strategy displayed in Supplemental Fig. 7. Source data and P values for all main figures available in Supplementary Data 1.
Fig. 5
Fig. 5. Growth phenotype with knockdown of ideR is more pronounced in cholesterol vs rich broth conditions.
IdeR knockdown strain was generated in Erdman Mtb using a CRISPRi construct. The knockdown strain was grown in media supplemented with OADC (a) or cholesterol (b) and OD600 values were measured every 2 days. +ATc = induced ideR knockdown, no ATc = uninduced controls. Red lines: DMSO controls (no sAEL057). Black lines: sAEL057 treated at 10 µM in OADC (a) or 5 µM in cholesterol media (b). Statistical significance was assessed using a student’s unpaired t-test. Source data and P values for all main figures are available in Supplementary Data 1.
Fig. 6
Fig. 6. Proposed model for sAEL057 mode of action.
Mycobactin (MB) chelates ferric iron (Fe3+) from host storage proteins inside the phagosome. Iron-laden mycobactin is imported into the bacterial cytosol through the IrtA/B transporter. Ferric iron is reduced to ferrous iron (Fe2+) to facilitate release from mycobactin. Nitrogen atoms of sAEL057 molecules form coordinate covalent bonds with Fe2+ through the donation of electrons to Fe2+. The binding of sAEL057 to iron makes it inaccessible to Mtb, resulting in a reduced availability of intracellular iron. This leads to an iron deprivation transcriptional response, driven by the iron-sensing transcription factor IdeR, and is demonstrated by the upregulation of mycobactin and iron import genes and downregulation of iron storage genes. Additionally, we predict that catabolism of certain carbon substrates (such as cholesterol and glucose) have an increased reliance on iron-dependent proteins as compared to catabolism of other carbon substrates (such as long-chain fatty acids). Therefore, a secondary consequence of sAEL057’s iron chelation is altered cholesterol catabolism leading to reduced metabolic output and a compensatory upregulation of genes involved in the early steps of the enzymatic breakdown of cholesterol. Ultimately, this results in reduced growth and survival when Mtb is restricted to specific single carbon substrates. Created with BioRender.com, publishing license in Supplementary Material.
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