**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].

**Citation:** Dick, C.F.; Alcantara, C.L.; Carvalho-Kelly, L.F.; Lacerda-Abreu, M.A.; Cunha-e-Silva, N.L.; Meyer-Fernandes, J.R.; Vieyra, A. Iron Uptake Controls *Trypanosoma cruzi* Metabolic Shift and Cell Proliferation. *Antioxidants* **2023**, *12*, 984. https:// doi.org/10.3390/antiox12050984

Academic Editor: Serge Ankri

Received: 17 March 2023 Revised: 18 April 2023 Accepted: 20 April 2023 Published: 22 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Fe is essentially physiologically inaccessible due to the limited solubility of its thermodynamically stable +3 oxidation state in the presence of O2 at neutral pH [6,7]. The concentration of free Fe in the environment ranges between 10−<sup>9</sup> and 10−<sup>18</sup> 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 [13–15], resulting in a free Fe concentration in serum of roughly 10−<sup>24</sup> M [13–15]. 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 Fe3+, it must be converted to Fe2+ by the Fe-reductase enzyme [22] to be transported across the plasma membrane. There is much evidence that Fe3+ reduction is frequently linked to Fe2+ 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 Fe2+ 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 Fe3+ to Fe2+, followed by the absorption of Fe2+ 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 Zn2+; however, some members of this family can also transport Fe2+ [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 Fe2+ 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 [9–12], 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.
