1. Introduction
The etiological agent of Chagas disease,
Trypanosoma cruzi, has a complicated life cycle that alternates between intermediate invertebrates and definitive mammal hosts [
1], and Chagas disease is considered a neglected disease worldwide [
2,
3].
Iron (Fe) is one nutritional element that controls
T. cruzi growth and differentiation during its life cycle since it is a necessary micronutrient for all forms of life and a cofactor of many enzymes in a considerable number of metabolic pathways [
4]. Fe is also hazardous because of its potential to accelerate the creation of reactive oxygen species (ROS), and all biological systems have evolved mechanisms for managing Fe intake, metabolism, and storage [
5].
Fe is essentially physiologically inaccessible due to the limited solubility of its thermodynamically stable +3 oxidation state in the presence of O
2 at neutral pH [
6,
7]. The concentration of free Fe in the environment ranges between 10
−9 and 10
−18 M, which is lower than the concentration necessary for microbial development [
8]. Fe is required for DNA synthesis [
9,
10], energy generation [
11], and oxidative stress in trypanosomes [
12]. Furthermore, mammalian hosts sequester free Fe into proteins such as transferrin and lactoferrin [
13,
14,
15], resulting in a free Fe concentration in serum of roughly 10
−24 M [
13,
14,
15]. Thus, there are three essential sources of Fe in mammals’ bodies that a pathogenic microbe may use: (i) transferrin, (ii) ferritin, and (iii) heme-containing proteins like hemoglobin [
16].
Trypanosoma cruzi requires iron (Fe) for growth, in vitro proliferation of epimastigotes forms (mobilizing heminic or non-heminic Fe), and pathogenicity in mice [
17].
Trypanosoma cruzi can hijack Fe-proteins from mammalian hosts. In culture, adding deferoxamine, a Fe chelator, or transferrin-free serum can reduce amastigotes cell multiplication, demonstrating that Fe is an essential nutrient [
18]. This parasite has evolved human transferrin receptors that bind exogenous transferrin. Acid treatment does not remove transferrin attached to amastigote cells, indicating that this transferrin may be internalized and used [
18]. Transferrin is taken up by the cytostome, a specialized structure consisting of a profound membrane invagination in the anterior area near the flagellar pocket [
19].
Trypanosoma cruzi also uses heme as a Fe source; it can boost
T. cruzi growth in culture in a dose-dependent way [
20]. Furthermore,
T. cruzi epimastigotes internalize heme/porphyrin through a process that might be mediated by an ABC transporter protein [
21]. However, because no heme oxidase gene is indicated on the
T. cruzi genome, the first heminic ring hydrolysis for Fe release is the limiting step for pathogenic trypanosomatids using heme [
5].
Since Fe is present in aerobic conditions as Fe
3+, it must be converted to Fe
2+ by the Fe-reductase enzyme [
22] to be transported across the plasma membrane. There is much evidence that Fe
3+ reduction is frequently linked to Fe
2+ transport in bacteria, yeast, plant, and animal cells [
23]. As a result, the identification of Fe-reductase activity in
Leishmania chagasi [
24],
L. amazonensis [
22], and, subsequently,
T. cruzi [
25] was a strong signal of the presence of a Fe
2+ transport mechanism in trypanosomatids.
The discovery of Fe-reductase activity in trypanosomatids reinforced the hypothesis of a two-step Fe transport mechanism: first, the reduction of Fe
3+ to Fe
2+, followed by the absorption of Fe
2+ by specialized transporters [
26]. In addition, the finding of a Fe transporter (LIT, [
27]) in the plasma membrane of
L. amazonensis, which belongs to the zinc and iron transporter family (ZIP family), gave early support to this idea. ZIP family members are said to be capable of transporting Zn
2+; however, some members of this family can also transport Fe
2+ [
28]. Recently, the discovery of the TcIT Fe transporter in
T. cruzi [
29] supports the hypothesis of a functional link between TcFR [
25] and TcIT for Fe
2+ uptake in this parasite.
Due to its low redox potential, Fe is a suitable element for redox catalysis processes [
30,
31], acting as an electron donor and receptor and being able to catalyze the formation of Once internalized, free Fe must be stored or processed as it enters the cytosol to avoid the generation of ROC. Corrêa et al. [
32] discovered Fe in the acidocalcisomes of
T. cruzi blood trypomastigotes. A putative Fe transporter in the form of a metal ion in the acidocalcisome of
T. cruzi supports the hypothesis of Fe storage in this organelle [
33]. This metabolic element has lately gained prominence due to the discovery that Fe mobilization and oxidative stress play a critical role in the parasite’s persistence and survival in the tissues of the mammalian host—a role that had not previously been proven [
34].
Although Fe is a critical micronutrient for trypanosomatids, as previously stated [
9,
10,
11,
12], there are still many unknowns about its involvement in the life cycle and pathogenicity of these organisms, as well as the ways in which it is acquired and used. The main aim of the present work was to investigate whether exogenous ionic Fe modulates
T. cruzi’s redox status and metabolic pathways, as well as the proliferation and differentiation of the parasite. The study revealed molecular mechanisms and intracellular processes modulated by exogenous ionic Fe that had not previously been reported.
2. Materials and Methods
2.1. Epimastigote Growth and Metacyclogenesis
Trypanosoma cruzi (Dm28c strain) epimastigotes were grown in stationary phase at 28 °C in Brain Heart Infusion (BHI) medium supplemented with 10% FBS, 30 µM hemin, and 1% penicillin-streptomycin (P/S) cocktail (referred to hereafter as Regular Media, RM). Iron-Depleted Media (IDM) was prepared using an iron-free BHI medium, as described by Dick et al. [
25]. Briefly, BHI medium without hemin addition was treated with Chelex (5 g/100 mL) for 1 h at room temperature and sterilized by filtration using 0.22 µm pore-size filters. To this Iron-Free BHI medium was added 1% P/S cocktail and 10% Iron-Free FBS. The iron-free FBS was prepared by adding 10 mM ascorbic acid for 6–7 h at 37 °C until the optical density at 405 nm had decreased by 50%. Then, the solution was supplied with 5 g of Chelex resin per 100 mL and incubated at room temperature under stirring at 50 rpm for 3–4 h, filtered to remove the resin, and dialyzed (with a cutoff of 2000 Da) against 4 l of cold, sterile PBS for 6 h, changing the solution every 2 h. The iron-free FBS was also sterilized by filtration using 0.22 µm pore-size filters and stored at −20 °C. Iron-Free FBS Iron-Depleted Media with Fe (IDM + Fe) was made the same way as IDM but with 8 µM Fe-citrate added. The parasites were inoculated (10
6 cells/mL) into the BHI medium on the sixth day of growth to test epimastigotes proliferation (RM, IDM, or IDM + Fe). Every day, cell proliferation was measured by counting the number of cells in a hemocytometer. Dm28c is a strain that differentiates “in vitro” and was previously used in our previous studies that demonstrated the existence of a Fe-reductase and a Fe-transporter in
T. cruzi [
25,
29]. This strain is deposited in the Collection of Trypanosoma from Wild and Domestic Mammals and Vectors (COLTRYP), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Metacyclogenesis was induced according to Koeller et al. [
35]. Epimastigotes in the transition from the logarithmic to the stationary phase were adjusted to 5 × 10
8 parasites/mL in triatomine artificial urine (TAU) medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl
2, 2 mM CaCl
2, 0.035% (
w/
v) NaHCO
3, and 8 mM phosphate buffer at pH 6.0). After 2 h at 28 °C, the cultures were diluted 100-fold in 10 mL TAU medium supplemented with 10 mM proline and 250 mM glucose (TAU-P) plus 500 g/mL G418 (Sigma-Aldrich, Saint Louis, MO, USA) and transferred to T25 flasks—lying at a 45° angle to increase the area in contact with O
2—and kept at 28 °C to promote metacyclogenesis. Following 3–5 days, parasites were counted using hemocytometry, and the proportion of metacyclic epimastigotes (Tryp) was determined using their morphology after Giemsa staining.
2.2. Intracellular Fe Concentration Determination
A colorimetric test based on ferrozine was used to assess the quantity of intracellular ionic Fe accumulated under different circumstances, as described before [
29]. Briefly, suspensions containing 10
8 parasites were obtained from various cultures and washed three times with PBS pretreated with 5 g/100 mL Chelex resin (Sigma-Aldrich). The cells were lysed with 100 µL of 50 mM NaOH, and then 100 µL of 10 mM HCl was added; the release of ionic Fe bound to intracellular structures was induced by adding 100 µL of a mixture containing 1.4 M HCl and 4.5% (
w/
v) KMnO
4 (1:1) to the cell lysate, followed by incubation at 60 °C for 2 h. Then, 30 µL Fe detection reagent was added (6.5 mM ferrozine, 6.5 mM neocuproin, 2.5 M ammonium acetate, and 1 M ascorbic acid). The sample’s absorbance at 550 nm was measured after 30 min of incubation at room temperature. A standard curve with known FeCl
3 concentrations (0–75 µM; [
36]) (Merck, Darmstadt, Germany) was used to calculate the Fe content.
2.3. Real-Time-PCR
Total
T. cruzi RNA was extracted using a Direct-zol RNA Miniprep Kit (Zymo Research, Orange, CA, USA) from epimastigotes kept at RM, IDM, or IDM + Fe for 5 days (as indicated in the figure legends). The high-capacity cDNA reverse transcription kit was used to reverse-transcribe whole RNA (Thermo Fisher Scientific, Waltham, MA, USA). For RT-PCR, 100 ng/µL cDNA per well (15 µL total volume) was utilized, coupled with a 5 µM primer mix and a 7 µL PowerUp SYBR green master solution (Thermo Fisher Scientific). Primers were designed using the Primer3 software, with predicted amplicon sizes of 100 pb each [
37]. The primers for amplification are shown in
Table 1. Gene expression data were normalized to an endogenous reference, β-tubulin. The expression ratios were determined using the threshold cycle (ΔΔCT) [
38].
2.4. Endocytosis Assay
Epimastigotes were submitted to endocytosis with 30 µg/mL of transferrin-FITC, hemoglobin-FITC, or BSA-FITC in Roswell Park Memorial Institute (RPMI) medium for 30 min at 28 °C. The parasites were fixed with 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS, pH 7.2) for 1 h for flow cytometry and imaging using the fluorescence microscope AxioObserver (Zeiss, Oberkochen, Germany) after staining with DAPI.
2.5. Cell Cytometry
After the endocytosis assay, tracer uptake was measured on a BD Accuri C6 flow cytometer (Becton Dickinson Bioscience, BDB, San José, CA, USA), counting 10,000 events at the FL2 channel. The data were analyzed by the BD Accuri C6 software. This analysis was performed in three independent experiments.
2.6. Transmission Electron Microscopy
Samples were fixed with 2.5% (
v/
v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature. After a wash in cacodylate buffer, cells were post-fixed using an osmium-thiocarbohydrazide-osmium (OTO) protocol as already described [
39]. Samples were washed in water, dehydrated in an acetone series, and embedded in epoxy resin (EMBED 812 resins, EMS, Hatfield, PA, USA). Ultrathin sections were cut with a UC7 ultramicrotome (Leica, Wetzlar, Germany), stained with 5% (
w/
v) uranyl acetate and lead citrate, and observed in an HT7800 transmission electron microscope (Hitachi, Tokyo, Japan) operating at 100 kV.
2.7. Protein Kinase A (PKA) Activity
Epimastigotes cells (5 × 107 cells/mL) were washed twice in ice-cold phosphate buffer saline (PBS, pH 7.2) and lysed in 0.5 mL radioimmunoprecipitation assay buffer (RIPA buffer) for 30 min. PKA activity was assayed in the presence of 4 mM Hepes-Tris (pH 7.0), 0.4 mM MgCl2, 1 mM CaCl2, 1 µM ATP, and 50 µg of lysed cells in a final volume of 50 µL, in the presence or absence of 5 µM 3-isobutyl-1-methylxanthine (IBMX, a PKA activator), in MTS-11C mini tubes (Axygen Scientific, Union City, CA, USA). The reaction was triggered by adding 50 μL of the Kinase-Glo luminescent kit, and after 10 min at 37 °C, the samples were placed in a GloMax Multi JR detection system (Promega Corporation, Fitchburg, WI, USA). PKA activity was quantified as the difference between the reading in the presence of IBMX and in the absence of the activator.
2.8. High-Resolution Respirometry in Different Respiratory States
Oxygen consumption of intact epimastigotes (5 × 10
7 parasites/chamber) was measured using an O2k-system high-resolution oxygraph (Oxygraph-2K; Oroboros Instruments, Innsbruck, Austria) at 28 °C with continuous stirring. The cells were suspended in a 2 mL respiration solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris–HCl (pH 7.2), and 50 μM digitonin was added to permeabilize the parasites. Following that, 10 mM succinate and 200 μM ADP were added. Uncoupled respiration was induced with 3 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and then blocked with 2.5 μg/mL antimycin A to assess residual O
2 consumption [
29]. Oxygen concentrations and O
2 consumption were recorded using DatLab software coupled to Oxygraph-2K.
2.9. Succinate-Cytochrome C Oxidoreductase Activity
The activity of succinate-cytochrome c oxidoreductase (complex II/III) was determined by the increase in absorbance due to the reduction of ferricytochrome c at 550 nm [
40,
41]. Frozen-thawed parasite homogenates (50 µg) were incubated with 25 mM potassium phosphate (pH 7.4), 10 mM succinate, 1 mM KCN, and 5 mM MgCl
2 for 20 min to allow for complete activation of succinate dehydrogenase, after which the reaction was initiated with 50 µM horse heart cytochrome c and monitored for 2 min. Protein concentrations were determined by the Bradford method, using bovine serum albumin as a standard [
42].
2.10. Mitochondrial Membrane Potential
Mitochondrial membrane potential was analyzed in the
T. cruzi epimastigotes using the MitoProbe JC-1 assay kit (Molecular Probes; Thermo Fisher Scientific). Epimastigotes (1 × 10
7 cells/mL) were loaded with 10 µM 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) and incubated for 40 min at room temperature. The fluorescence intensity ratio of red (540 nm excitation and 590 nm emission) to green (490 nm excitation and 540 nm emission) was measured using a multi-well fluorescence reader [
43].
2.11. Intracellular ATP Quantification
Intracellular ATP (ATP
i) was measured using an ATP bioluminescent somatic cell test kit (Sigma-Aldrich). In brief, epimastigotes (1 × 10
7 parasites per tube) were incubated in a solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris-HCl in 0.1 mL (pH 7.2 adjusted with HCl). Cellular extracts were prepared by combining them with 0.1 mL of somatic cell ATP-releasing reagent and then chilling the mixture for 1 min. The mixture was transferred to MTS-11C mini tubes containing 0.1 mL ATP assay mix (
v:
v; Axygen) and swirled for 10 s at room temperature. A GloMax Multi JR detection system (Promega) measured the overall quantity of light emitted. In each experiment, the total intracellular ATP concentration per 10
7 cells was determined using a standard ATP curve [
29].
2.12. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Activity
The reduction of NAD
+ to NADH was used to assess the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglicerate, as described before [
44], with slight modifications. Total epimastigote lysates were incubated for 15 min at 37 °C in reaction media containing 100 mM Triethalonamine:HCl buffer (pH 7.5) containing 1.0 mM EDTA, 5 mM MgSO
4, 1.0 mM dithiothreitol, 1.5 mM NAD
+, and 30 mM KH
2AsO
4 [
45,
46]. The reaction was started using 2 mM glyceraldehyde-3-phosphate, and the absorbance at 340 nm was measured every 1 min for 5 min. Total NADH generation was determined using the NADH standard curve.
2.13. Glucokinase Activity
Cellular extracts of epimastigotes were incubated in a reaction buffer containing 20 mM Tris-HCl (pH 7.4), 5 mM MgCl
2, 1 mM glucose, 1 unit/mL glucose-6-phosphate dehydrogenase (G6PDH) (
Leuconostoc mesenteroides), 0.1% Triton X-100, 1 mM NaF, 5 mM NaN
3, 1 mM ATP, and 50–100 μg/mL protein [
47]. After 3 min of incubation, the reactions were started by the addition of 0.5 mM β-NADP
+, and quantified spectrophotometrically following the reduction of β-NADP
+ to β-NADPH (λ = 340 nm) for 10 min. Total NADPH generation was determined using the NADPH standard curve.
2.14. Western Blotting
For western blotting detection, epimastigotes were lysed with 1 mL RIPA buffer supplemented with 1 mM phenylmethanesulfonyl fluoride and 5 mM leupeptin, for 30 min at 4 °C. Then, homogenates were centrifuged at 16,000 rpm for 10 min. Aliquots of the supernatants (containing 100 µg total protein) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA), which were blocked with 5% milk in PBS plus 0.1% (w/v) Tween 20, probed overnight at 4 °C with the primary mouse anti-GAPDH antibody (1:500, Sigma-Aldrich), and detected using horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA). The loading control was probed with a primary rabbit anti-tubulin antibody (1:500, Sigma-Aldrich) and detected using an HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000, Santa Cruz Biotechnology). Luminescence was detected using an ImageQuant LAS 4000 digital imaging system (GE Healthcare Life Sciences, Amersham, UK) after the reaction with LuminataTM Forte Western HRP Substrate (Millipore, Billerica, MA, USA). Densitometric analysis was performed using ImageJ software version 1.50i (NIH Image, Bethesda, MD, USA) with background correction.
2.15. Superoxide Dismutase (SOD) Activity
Total SOD activity was assessed as previously described [
29], based on SOD inhibiting the reduction of nitro blue tetrazolium (NBT) by O
2•−. Epimastigote cells were harvested by centrifugation, washed three times in cold PBS, and disrupted by freeze-thaw. Centrifugation as described above was used to collect epimastigote cells, which were washed three times in cold PBS and disrupted by freeze-thaw. The Bradford method [
42] was used to determine the protein content in the whole homogenate. In a final volume of 200 µL, the homogenates (using known amounts of protein in the range of 10–50 µg) were incubated in a reaction medium containing 45 mM potassium phosphate buffer (pH 7.8), 6.5 mM EDTA, and 50 mM NBT. The reaction began with the addition of 2 mM riboflavin. The sample’s absorbance at 560 nm was measured after 15 min in a lightbox. SOD activity was expressed as the quantity of enzyme-blocking NBT reduction by 50% for each amount of protein.
2.16. Amplex® Red Peroxidase Assay
The rate of H
2O
2 reduction was assayed by the production of H
2O
2 in epimastigotes to H
2O, which is stoichiometrically coupled (1:1) to the simultaneous oxidation of the non-fluorescent Amplex
® Red probe to the fluorescent resorufin [
48]. Briefly, 10
7 parasites/mL were incubated with 5 mM Tris-HCl (pH 7.4), 1.7 μM Amplex
® Red (Invitrogen, Carlsbad, CA, USA), and 6.7 U/mL horseradish peroxidase (Sigma-Aldrich) in a final volume of 100 µL, for 30 min at room temperature. Fluorescence evolution was observed at excitation/emission wavelengths of 563/587 nm. H
2O
2 concentration was determined using a standard curve.
2.17. Statistical Analysis
The data are provided as mean ± standard error of the mean (SEM). The unpaired Student’s t-test was used to compare two means. When comparing more than two means, a one-way ANOVA with Tukey’s test was applied, as specified in the text or the figure captions. The normal distribution was assessed before each ANOVA analysis. When results were expressed as a percentage of the RM group, the SEM was obtained from the absolute values. A comparison of the absolute results was carried out using the parametric test. Significance was set at p < 0.05. Except when otherwise indicated, different lowercase letters in superscripts indicate statistical differences among means in the same line of tables. Asterisks (also indicating p < 0.05) were used in figures and for comparing values from different lines in figures. GraphPad Prism 7.0 was used for statistical analysis and the preparation of figures (GraphPad Software, San Diego, CA, USA).
4. Discussion
The central findings in the present study reveal a key role of medium ionic Fe in the proliferation of T. cruzi epimastigotes, with Fe depletion promoting increased oxidative stress, selective modifications in the intracellular ATP content, alterations in the HRI→eIF2α and PKA signaling pathways, increased lipid accumulation in the reservosomes, decreased mitochondrial function, and inhibition of differentiation toward trypomastigotes, in a metabolic condition shifted from respiration to glycolysis. This ensemble of results points to a pleiotropic function of ionic Fe in connected processes and pathways in T. cruzi epimastigotes. Using the Dm28c strain, which differentiates from trypomastigotes, allowed us to investigate the influence of exogenous Fe on the evolution of epimastigotes to trypomastigotes and, therefore, on a vital step of the parasite’s life cycle.
The Fe depletion-induced lower intracellular content of Fe (
Table 2). Notably, in the IDM + Fe medium, the expression of TcFR increased without a parallel increase in TcIT transcription. Even though TcFR and TcIT are coupled in the process of Fe uptake by the parasite, their expression is differentially modulated by the intracellular Fe content. Free Fe could regulate the TcIT transcription, so in the IDM + Fe medium, TcIT is downregulated relative to the TcIT transcript in IDM. Differently, TcFR uses Fe-containing proteins for the reaction Fe
3+→Fe
2+, and since these proteins (hemin and transferrin) were neither added to IDM nor to IDM + Fe, TcFR is upregulated in both cases (
Table 2).
The decreased succinate-cytochrome c oxidoreductase activity, the dropped O
2 consumption in the presence of normal partial pressure of O
2, and the lower intracellular ATP (
Table 4) are indicative of mitochondrial damage, as proposed several years ago [
63]. The impairment of the mitochondrial function seems to be functional because the ultrastructure of the organelle is preserved (
Figure 3). Therefore, it could be hypothesized that Fe starvation promotes dysfunction at the level of the iron-sulfur clusters in the heterodimeric SDH2
N:SDH2
C subunit described in the mitochondrial complex II from
T. cruzi [
64], as well as in the FoF
1-ATPase [
65], a possibility that emerges from the accentuated inhibition of respiration in the presence of ADP (the oxphos state) and the increased ΔΨ
m (
Table 4). Although succinate-cytochrome c oxidoreductase activity is restored by Fe-citrate supplementation (
Table 4), mitochondrial function impairment seems linked to Fe-protein depletion, especially hemin. It has been demonstrated that, in epimastigotes, heme changes mitochondrial physiology [
40]. NADH-ubiquinone oxidoreductase gene (0.8-fold) and succinate dehydrogenase (1.40-fold) are upregulated in the presence of heme. Besides, heme influences
T. cruzi epimastigote energy metabolism. The contribution to ATP synthesis may depend on glycosomal fermentation, which provides energy support for the parasite’s growth, an establishment inside the vector [
66], and differentiation into trypomastigotes (
Figure 5).
The proposal that Fe and hemin depletion promotes a shift from oxidative metabolism to a glycolytic one is reinforced by the increased GADPH expression and activity (
Figure 4A,B) and the increased glucokinase activity (
Figure 4D). The upregulated glycolytic and pentose phosphate pathways, which are considered central for the glucose metabolism in
T. cruzi [
57], likely provide acetyl-CoA and NADPH, respectively, for the proposed increase of fatty acid synthesis accumulation within the reservosomes (
Figure 3). As mentioned earlier, these organelles present a varied repertoire of enzymes that catalyze different lipid metabolism pathways [
50], and for this reason, lipid accumulation in the reservosomes deserves special discussion in the context of the other enzyme modifications and proliferation.
The impairment of the IDM in the life cycle of epimastigotes could be linked to the alterations encountered in HRI→eIF2α and PKA signaling. The increased HRI, which phosphorylates eIF2α [
54], together with the downregulation of eIF2α itself (
Table 3), likely culminate in repressed gene expression and overall protein translation, thus compromising the evolution of the parasite in the Fe-deprived medium. The pronounced downregulation of PKA activity (and expression) could be associated with the downregulation of lipase activity and fatty acid release and oxidation. It may be that decreased PKA activity in IDM parasites (
Table 3) results in the inhibition of a PKA-modulated lipase and functional immobilization of the lipids in the reservosomes. This proposal is supported by the fact that PKA recovery and expression increased to levels even higher than in RM conditions after Fe supplementation (
Table 3), which ensures lipid turnover and recovery of parasite evolution as discussed above. As additional support for this view, it is noteworthy that the genetic inhibition of PKA is lethal for
T. cruzi [
67].
Lipolysis in
T. cruzi is associated with glucose metabolism [
57]. For this reason, the upregulation of central enzymes of the glycolytic pathway, GADPH, and glucokinase, in both IDM and IDM + Fe media (
Figure 4), leads us to hypothesize that the absence of hemin is central to the upregulation of glycolysis and the pentose phosphate pathway, but that replenishing of ionic Fe is responsible for the possible stimulation of lipid hydrolysis, glycerol release, formation of glycerol 3-phosphate catalyzed by a Tc-glycerol kinase [
68], and further feeding of the glycolytic pathway. The other metabolic branch after Fe-stimulated lipid turnover, the β-oxidation of fatty acids [
69], can feed the acetyl-CoA pool in the epimastigotes cell, further stimulating the formation of ATP via its condensation with succinate, synthesis of succinyl-CoA, and recycling of succinate with the release of CoA, as proposed in genomic studies carried out in
T. cruzi [
57] and earlier demonstrated in
T. brucei [
70].
Ionic Fe and heme depletion lead to a downregulation of total FeSOD, regardless of FeSOD origin. It is possible that SOD activity is higher in epimastigotes maintained at Fe/heme or heme depletion, demonstrating a compensating mechanism, probably due to higher activity of cytosolic and mitochondrial FeSOD (FeSODB and FeSODA). While FeSODB has a crucial role in the defense of parasites against O
2•− [
71], FeSODA is related to mitochondrial redox balance and generates the signaling molecule for amastigote differentiation, H
2O
2 [
72]. A downregulation of H
2O
2 levels in Fe depletion conditions probably deregulates parasite differentiation. These low H
2O
2 levels could be due to a non-enzymatic system besides the glutathione ascorbate cycle. Recently, it was demonstrated that
T. cruzi trypomastigotes employ ROS as a signaling molecule to differentiate, whereas epimastigotes use ROS to proliferate rather than differentiate [
72].
Finally,
Figure 6 presents a hypothetical mechanistic model regarding the overall mechanisms occurring during Fe depletion or Fe supplementation. Although the metabolic shift occurs in both cases, the ROS formation and pathway signaling present slight differences that culminate in differentiation/proliferation impairment (
Figure 6A), which is restored by Fe supplementation (
Figure 6B). In conclusion, although heme (or Fe-containing proteins) is essential for a functional mitochondrial metabolism, exogenous Fe is required for proper signaling to control parasite proliferation and H
2O
2 formation, which stimulate parasite differentiation, thus interfering with parasite virulence. These related mechanisms and processes modulated by exogenous ionic Fe have implications for human health because, by providing energy support for parasite growth and differentiation, they ensure the continuity of the
T. cruzi life cycle and the propagation of Chagas disease.