Survival to Fe3+ starvation in Mtb H37Rv and Erdman strains.

Cells were exposed to 50 μM FeCl3 (HI: High Iron), 0 μM FeCl3 (LI: Low Iron,) or 0 μM FeCl3+ DFO (DFO). Growth was monitored for 3 weeks by measuring OD600 (A, B), and survival was monitored for more than 4 weeks by CFU/mL (C, D, E, F). The charts show one experiment representative of 2-3 independent experiments. The CFU/mL charts show average and average deviation of two technical replicates of one independent experiment. G, H) ATP levels and growth (OD600) after 1, 2, 3 and 8 days in DFO. ATP levels were normalized to the optical density. The data are the average and standard deviation of three independent experiments and three technical replicates each. I) NAD/NADH ratio detected after 3 days of exposure to DFO. The data are the average and standard deviation of two independent experiments and two technical replicates. The p-values were calculated against the HI condition. * = p value < 0.05.

Intracellular and extracellular levels of metabolites in H37Rv.

Cells were exposed to 50 μM FeCl3 (HI: High Iron), 0 μM FeCl3 (LI: Low Iron,) or 0 μM FeCl3+ DFO (DFO). The analyzed metabolites are shown in black in the schematic pathways. A) Intracellular polar metabolites levels at 8 days. B) Extracellular polar metabolites levels at 1, 3 and 8 days. The plots show the normalized levels of metabolites. The data represent the average and the standard deviation of four biological replicates from an independent experiment, representative of two independent experiments. An arbitrary unit is reported on the y axis. The y axis is in Log10 scale and the numbers from 0 to 5 (panel A) indicate the exponent. C) Viability of H37Rv from one independent experiment. Cells were grown in liquid medium in HI and DFO conditions and in the presence of 2 mM of succinate. Aliquots of cells were collected after 0, 3 and 8 days and diluted to same final OD600; 5 μL of 10-fold serial dilutions were plated on 7H10. Growth was recorded after 19-25 days.

The p-values were calculated against the HI condition and independently for the two experiments, the highest p-value was reported. n.s.f.= non-significant fold change, the observed trend change was different between independent experiments; n.s.= non-significant, p value >0.05; n.d.= non-detected; * =p value <0.05; ** =p value <0.01.

Ac-CoA: acetyl-CoA; CIT: citrate; FUM: fumarate; α-KG: α-ketoglutarate; ISO: isocitrate; (ISO)CIT: isocitrate and citrate; MAL: malate; OAA: oxaloacetate; PYR: pyruvate; SUCC: succinate; SUCC-CoA: succinyl-CoA.

Percentage of labeled (13C) and unlabeled (12C) metabolites in H37Rv.

Metabolites were extracted from cells fed with 13C3-glycerol for 8 days in 50 μM FeCl3 (HI: High Iron), 0 μM FeCl3 (LI: Low Iron) or 0 μM FeCl3+ DFO (DFO). The analyzed metabolites are shown in black in the schematic pathways. For each metabolite, but fumarate, two plots are shown. The stacked column plot shows the total percentage of labeled and unlabeled molecules per each metabolite pool; the clustered column plot shows the abundance in percentage of each isotopologue. The data represent the average and the standard deviation of four biological replicates from an independent experiment, representative of two independent experiments. The p-values were calculated independently for the two experiments and the highest p-value was reported. DFO/LI vs HI condition: * = p value<0.05; ** = p value <0.01; n.s.= non-significant. n.d.= non-detected. M2 vs M1: # = p value<0.05; ## = p value<0.01.

Ac-CoA: acetyl-CoA; CIT: citrate; FUM: fumarate; α-KG: α-ketoglutarate; ISO: isocitrate; (ISO)CIT: isocitrate and citrate; MAL: malate; OAA: oxaloacetate; PYR: pyruvate; SUCC: succinate; SUCC-CoA: succinyl-CoA.

Percentage of labeled (13C) and unlabeled (12C) metabolites in the Erdman strain.

Metabolites were extracted from cells fed with 13C3-glycerol for 8 days in 50 μM FeCl3 (HI: High Iron), 0 μM FeCl3 (LI: Low Iron) or 0 μM FeCl3+ DFO (DFO). The analyzed metabolites are shown in black in the schematic pathways. For each metabolite two plots are shown. The stacked column plots show the total percentage of labeled and unlabeled molecules per each metabolite pool; the clustered column plots show the abundance in percentage of each isotopologue. The data represent the average and the standard deviation of four biological replicates from an independent experiment, representative of four independent experiments (only two for LI condition). The p-values were calculated independently between experiments and the highest value is reported. DFO/LI vs HI: ** = p value <0.01; n.s.= non-significant; n.s.f.= non-significant fold change, the observed trend change was different between independent experiments. n.d.= non-detected. M2 vs M1: ## = p value <0.01. Ac-CoA: acetyl-CoA; CIT: citrate; FUM: fumarate; α-KG: α-ketoglutarate; ISO: isocitrate; (ISO)CIT: isocitrate and citrate; MAL: malate; OAA: oxaloacetate; PYR: pyruvate, SUCC: succinate; SUCC-CoA: succinyl-CoA.

Analysis of iron-independent metabolic routes.

Cells were exposed to 50 μM FeCl3 (HI), or 0 μM FeCl3+ DFO (DFO). A) Enzymatic activity of phosphoenolpyruvate carboxykinase (PCK), reaction from phosphoenolpyruvate to oxaloacetate; B) Enzymatic activity of isocitrate lyase (ICL); the plots show the activity in (mM of NADH × min-1)/mg of total protein detected in cell-free extracts after 3 days of exposure to DFO or HI condition. Data show average and standard deviation from two (H37Rv) or three (Erdman strain) independent experiments and two technical replicates each (A) or from one independent experiment and three technical replicates (B). C, D) Isotopologue distribution of intracellular malate (C) and isocitrate (D) in Erdman-derived ΔpckA::pckA and ΔpckA strains after 8 days of exposure to DFO or HI conditions and fed with 13C3-glycerol. The plots show the abundance in percentage of each isotopologue. The histograms represent average and standard deviation from four biological replicates from one experiment, representative of three independent experiments. E, F) Enzymatic activity of pyruvate carboxylase (PCA), reaction from pyruvate to oxaloacetate; the plots show the activity in (mM of NADH × min-1)/mg of total protein detected in cell-free extracts after 3 days of exposure to DFO or HI condition. Data show average and standard deviation from three independent experiments and two technical replicates each. G, H) Erdman strain cells were exposed to HI or DFO condition for 8 days with or without 200 μM of inhibitor 3-nitropropionate (3NP) and fed with 13C3-glycerol. (G) The plots show the normalized levels of metabolites; an arbitrary unit is reported on the y axis; the y axis is in Log10 scale and the numbers from 0 to 5 indicate the exponent. (H) The plots show the isotopologue distribution in percent abundance. The data represent the average and the standard deviation of four biological replicates from an independent experiment, representative of two independent experiments. The p-values were calculated independently between experiments and the highest value is reported. The p-values were calculated as follows. DFO vs HI for A, B, E, F; HI+3NP vs HI and DFO+3NP vs DFO for H; mutant vs complemented for C, D. n.s.f. = non-significant fold change, the observed trend change was different between independent experiments. ** = p value <0.01.

Analysis of GABA shunt metabolites in H37RV and Erdman strains.

Cells were exposed to 50 μM FeCl3 (HI: High Iron), 0 μM FeCl3 (LI: Low Iron,) or 0 μM FeCl3+ DFO (DFO), and fed with 13C3-glycerol for 8 days. A) Schematic representation of the GABA shunt and the Krebs cycle. B, C, D) H37Rv; E, F, G) Erdman strain. B, E) glutamate; C, F) gamma-aminobutyric acid (GABA); D, G) succinate. Plots with one color-filled histograms represent the normalized levels of metabolites in Log10 scale, the numbers from 0 to 5 on the y axis indicate the exponent; plots with two color-stacked histograms represent the total abundance in percentage of labeled and unlabeled metabolite pools; plots with clustered histograms represent the abundance in percentage of each isotopologue. H37Rv data are from one experiment representative of two independent experiments and four biological replicates each; Erdman-derived strain data are from one experiment representative of four independent experiments (only two of them for the LI condition) and four biological replicates each. The histograms show average and standard deviation of four biological replicates from an independent experiment. The p-values were calculated against the HI condition; the highest p-value from the two independent experiments is shown. The differences between M+1 and M+2 isotopologues in the Erdman strain (E, F) are not significant between the three experiments, the trend varied between independent experiments.

a.u.: arbitrary unit (see methods paragraph). n.s.f.= non-significant fold change, the observed trend change was different between independent experiments.

DFO vs HI or LI vs HI: * =p value <0.05; ** =p value <0.01; “n.s.f.” above the individual M+1 and M+2 bars of succinate and GABA.

Re-modeling of CCM under iron starvation in Mtb.

The picture depicts a schematic representation of the CCM pathways active in Mtb exposed to Fe3+ deprivation in the presence of D-glucose and glycerol as carbon sources and asparagine as sole nitrogen source. The thickening arrows indicate the increase of levels of the metabolite, thicker arrows indicate a preferred route. Bold and larger font indicates accumulated metabolites. Under iron starvation, the pool of iron-dependent enzymes (denoted by Fe in parentheses in the figure) contains a reduced number of fully active molecules, which then slows the carbon flux through the Krebs cycle. The reduction in the transcript levels of iron-independent enzymes of the CCM pathways is likely a consequence of this. The disparity in efficiency between iron-dependent and iron-independent enzyme pools gives rise to the accumulation of (iso)citrate, pyruvate, and α-ketoglutarate. Mtb responds to these accumulations by expelling these metabolites from the cell and splitting the carbon flux from PEP/pyruvate (via PCK, PCA, and PDH activities) into both oxidative and reductive branches of the Krebs cycle. Both fluxes terminate in malate synthesis, which is then secreted. To maintain malate synthesis by succinate oxidation Mtb limits its secretion. Malate secretion relieves the slowdown of carbon flux through the oxidative branch of Krebs cycle. PCK and PCA anaplerotic reactions control pyruvate levels and recycle carbon dioxide stoichiometric to the α-ketoglutarate accumulation.

GDH: glutamate dehydrogenase. GADB: glutamate decarboxylase. GABT: 4-aminobutyrate aminotransferase. GABD: succinate-semialdehyde dehydrogenase. For the other enzyme acronyms see main text.

Intracellular and extracellular levels of metabolites in the Erdman strain.

Cells were exposed to 50 μM FeCl3 (HI: High Iron), 0 μM FeCl3 (LI: Low Iron,) or 0 μM FeCl3+ DFO (DFO). The analyzed metabolites are shown in black in the schematic pathways. A) Intracellular polar metabolites levels at 8 days. The slight decrease in succinate levels (0.66 FC) was not observed in the other four independent experiments in which no changes were observed in LI and DFO conditions compared to HI condition. B) Extracellular polar metabolites levels at 1,3 and 8 days. The plots show the normalized levels of metabolites. The data represent the average and the standard deviation of four biological replicates from an independent experiment, representative of four independent experiments for the HI and DFO conditions, and two independent experiments for the LI condition. An arbitrary unit is reported on the y axis. The y axis is in Log10 scale and the numbers from 0 to 5 (panel A) indicate the exponent. G) Viability of cells exposed to DFO in the presence of 2 mM of malate (DFO+M), succinate (DFO+S), pyruvate (DFO+P) or a combination of the three (DFO+M+S+P). The plot reports the viability measured as CFU/mL in Log10 scale. The data are representative of one independent experiment (average and average deviation of two technical replicates).

The p-values were calculated against the HI condition and independently for the four experiments; the highest p-value is reported. * = p value < 0.05; ** = p value <0.01. n.d. = not detected. n.s.f. = not significant fold change, the observed trend change was different between independent experiments.

Ac-CoA: acetyl-CoA; CIT: citrate; FUM: fumarate; α-KG: α-ketoglutarate; ISO: isocitrate; (ISO)CIT: isocitrate and citrate; MAL: malate; OAA: oxaloacetate; PYR: pyruvate SUCC: succinate; SUCC-CoA: succinyl-CoA.

Viability of Mtb exposed to severe Fe3+ starvation and in the presence of 2 mM or 5 mM Krebs cycle intermediates (A, H37Rv) or 200 μM 3NP (B, Erdman).

Cells were exposed to 50 μM FeCl3 (HI: High Iron), or 0 μM FeCl3+ DFO (DFO) in liquid medium for 8 days. Aliquots of cells were collected after 0, 1, 3 and 8 days, diluted to a final OD600 of 0.1 and 5 μL of a 10-fold serial dilution were plated on 7H10/ADC. Growth was recorded after 19-25 days.

Analysis of metabolites in the H37Rv-derived Δicl1::icl1 complemented strain.

Cells were exposed to 50 μM FeCl3 (HI: High Iron), 0 μM FeCl3 (LI: Low Iron,) or 0 μM FeCl3+ DFO (DFO), and fed with 13C3-glycerol. Intracellular metabolites were determined after 8 days; extracellular metabolites were determined after 1,3 and 8 days. The plots show the normalized levels of metabolites. The data represent average and standard deviation from one experiment and four biological replicates. The plots of intracellular abundance are in Log10 scale, the numbers from 0 to 5 on the y axis indicate the exponent. The column headers contain the labels of the y axis. The p-values were calculated against HI condition. n.s. = non-significant, p value >0.05; * = p value <0.05; ** = p value <0.01. a.u. = arbitrary unit.

Analysis of metabolites in the H37Rv-derived icl1 mutant (Δicl1) compared to its complemented strain (Δicl1::icl1) in the DFO condition.

Cells were exposed to 0 μM FeCl3+ DFO (DFO), and fed with 13C3-glycerol for 8 days. Intracellular metabolites were extracte after 8 days; extracellular metabolites were extracted after 1,3 and 8 days. A) Normalized abundance of intracellular metabolites in Log10 scale, the numbers from 0 to 4 on the y axis indicate the exponent. An arbitrary unit is reported. B) Total percentage of labeled and unlabeled metabolites. C) Isotopologue distribution of metabolites. D) Isotopologue distribution of the α-ketoglutarate pool in the low iron (LI; 0 μM FeCl3) condition. E) Normalized abundance of extracellular metabolites in Log10 scale. A, B, C, E: data from one experiment representative of two independent experiments and four biological replicates. D: data from one independent experiment and four biological replicates each. The data represent average and standard deviation of four biological replicates from an independent experiment. n.s.f. = not significant fold change, the observed trend change was different between independent experiments. a.u. = arbitrary unit.

Schematic representation of 13C-phosphoenolpyruvate, 13C-pyruvate and 13C-carbon dioxide assimilation through PDH, PCK, the Krebs cycle and the glyoxylate shunt.

Atoms of 13C are marked in green. The panels A-C include the activity of pyruvate dehydrogenase (PDH), the Krebs cycle and the glyoxylate shunt. The panels D-S include the activity of phosphoenolpyruvate carboxykinase (PCK), the Krebs cycle and the glyoxylate shunt. Labeled acetyl-CoA is assumed to derive from PDH activity. The bottom of each panel indicates which input metabolite is labeled. The labeling profiles of metabolites downstream to phosphoenolpyruvate (PEP) are identical if PCK is replaced by pyruvate carboxylase and PEP is replace by pyruvate. To simplify, only one of the enzymes for α-ketoglutarate degradation and citrate synthesis are reported. Acn: aconitase. GlcB: malate synthase. GltA: citrate synthase. Icd: isocitrate dehydrogenase. Icl: isocitrate lyase. Kor: αketoglutarate ferredoxin-oxidoreductase. Mdh: malate dehydrogenase. Sdh: succinate dehydrogenase.

Schematic representation of 13C-phosphoenolpyruvate, 13C-pyruvate and 13C-carbon dioxide assimilation through PCK, MEZ and the Krebs cycle.

The panels A-H include the activity of phosphoenolpyruvate carboxykinase (PCK) and the Krebs cycle. Labeled acetyl-CoA is assumed to derive from PDH activity. The panels I and L include the anaplerotic reaction of PCK and the reduction of oxaloacetate to malate by MDH. The labeling profiles of metabolites downstream to phosphoenolpyruvate (PEP) are identical if PCK is replaced by pyruvate carboxylase and PEP is replaced by pyruvate (A-L). The panels M-N include the anaplerotic reaction of malic enzyme (MEZ) from pyruvate to malate. To simplify, only one of the enzymes for α-ketoglutarate degradation and citrate synthesis are reported. The bottom of each panel indicates which input metabolite is labeled.

Acn: aconitase. GltA: citrate synthase. Icd: isocitrate dehydrogenase. Kor: α-ketoglutarate oxidoreductase. Sdh: succinate dehydrogenase.