*3.7. Differentiation of Epimastigotes Is Lower in Fe-Depleted Medium and Is Recovered after Fe Supplementation*

Differentiation is a crucial step in the life cycle of *T. cruzi* because it ensures the success of infection and because the redox status of the parasitic environment is an important modulator [62]. Figure 5 demonstrates that, in culture, differentiation is critically dependent on the presence of Fe in the medium, and this dependence is more evident at initial times. While differentiation was barely detectable along the first day in IDM (filled circles), it exhibited a linear behavior in RM (empty circles) and depicted a burst in IDM + Fe (gray circles). Remarkably, the initial differentiation rate slop doubled in this group regarding RM parasites, but it seemed to tend toward a lower plateau from the 3rd day of culture.

**Figure 5.** Fe depletion arrests *T. cruzi* differentiation to trypomastigotes. Epimastigotes cultivated in RM (empty circles), IDM (black circles), or IDM + Fe (gray circles) were submitted to in vitro differentiation media TAU. Each day, epimastigotes and trypomastigotes were differentially counted to determine the percentage of trypomastigotes in culture (*n* = 4); \* *p* < 0.05 comparing time-matched determinations with respect to RM. Using one-way ANOVA with Tukey's test, we assessed differences between mean values.

### **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 Fe3+→Fe2+, 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 O2 consumption in the presence of normal partial pressure of O2, 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 SDH2N:SDH2C subunit described in the mitochondrial complex II from *T. cruzi* [64], as well as in the FoF1-ATPase [65], a possibility that emerges from the accentuated inhibition of respiration in the presence of ADP (the oxphos state) and the increased ΔΨ<sup>m</sup> (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 O2 •− [71], FeSODA is related to mitochondrial redox balance and generates the signaling molecule for amastigote differentiation, H2O2 [72]. A downregulation of H2O2 levels in Fe depletion conditions probably deregulates parasite differentiation. These low H2O2 levels could be due to a nonenzymatic 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 H2O2 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.

**Figure 6.** A proposed model describing the main events occurring on *T. cruzi* submitted to (**A**) Fe depletion or (**B**) Fe supplementation in low heme concentration (left outside part of the figure), which culminate in decreased or restored intracellular Fe2+, as demonstrated by the opposite direction of the orange arrow near the upper part of the figure. The decrease in intracellular Fe in (**A**) was 50% in parasites grown in IDM (Table 2). The vesicles near the cell membrane (cytostome-cytopharynx region) indicate the selective modifications in the endocytic protein and lipid uptake. The left part of the arrow ensembles represents the modifications of the HRI→eIF2α and PKA signaling pathways and the different modifications in expression and translation. The blue circles in the middle of the panels present the quantitative differences in GAPDH abundance and activity. The blunt black arrows (in the right part of both panels) point to the impaired mitochondrial function provoked by Fe depletion, which is not sufficiently restored by Fe supplementation. The red stars in the lower right corner depict the opposite effects of Fe depletion and Fe supplementation on H2O2 formation. The figure was designed using BioRender.com.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12050984/s1. Table S1: Primer Information. Figure S1: Cytometry demonstrates selective modifications of protein uptake induced by ionic Fe depletion.

**Author Contributions:** Conceptualization, C.F.D. and A.V.; methodology, C.F.D., L.F.C.-K., C.L.A. and M.A.L.-A.; validation, C.F.D., L.F.C.-K., C.L.A. and M.A.L.-A.; formal analysis, C.F.D., C.L.A. and A.V.; investigation, C.F.D., L.F.C.-K., C.L.A. and M.A.L.-A.; resources, J.R.M.-F., N.L.C.-e.-S. and A.V.; writing—original draft preparation, C.F.D. and A.V.; writing—review and editing, C.F.D., C.L.A., N.L.C.-e.-S., J.R.M.-F. and A.V.; visualization, C.F.D., L.F.C.-K., C.L.A. and M.A.L.-A.; supervision, J.R.M.-F. and A.V.; project administration, C.F.D. and A.V.; funding acquisition, J.R.M.-F. and A.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Brazilian National Research Council/CNPq to J.R.M.- F. (grant # 401134/2014-8), and A.V. (grant # 307605/2015-9). The support of the Rio de Janeiro State Research Foundation/FAPERJ to J.R.M.-F. (grant # E-26/201.300/2014), N.L.C.-e.-S. (grant # E-26/210.449/2019), and A.V. (grant # E-26/2012.963/2017) is also acknowledged.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The original contributions presented in the study are included in the article and the supporting material. Further inquiries can be directed to the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


