**3. Results**

#### *3.1. Trypanosoma cruzi Epimastigotes Depend on Exogenous Fe Content for Fe Proliferation*

The proliferation of *T. cruzi* epimastigotes during the exponential growth phase (Figure 1) depends on Fe in a concentration-dependent manner. In Fe-depleted medium (IDM, black circles), the maximum number of protozoa after 7 days decreased from 4.4 × 107 to 1.0 × 107 parasites/mL, compared to epimastigotes maintained in regular medium (RM, empty circles). The proliferative capacity of the cells was recovered with 8 μM Fe citrate added to the Fe-depleted medium (IDM + Fe, gray circles): the number of protozoa after 7 days increased from 1.0 × <sup>10</sup><sup>7</sup> to 5.1 × <sup>10</sup><sup>7</sup> parasites/mL, a condition similar to the control. In addition, Fe citrate supplementation was enough to restore intracellular Fe concentration compared to RM, while cells maintained at IDM presented low intracellular Fe content (Table 2). Although in IDM and in IDM + Fe there was an increase in the expression of Fe reductase (TcFR), the expression of the Fe transporter (TcIT) in cells maintained in IDM + Fe remained at levels similar to the RM condition (Table 2).

**Figure 1.** The effect of exogenous Fe on *T. cruzi* growth. *Trypanosoma cruzi* epimastigotes were harvested, washed twice, seeded in fresh medium, and grown for the indicated times in Regular Media (RM: Brain Heart Infusion medium (BHI) supplemented with 30 μM hemin and 10% Fetal Bovine Serum (FBS)) (white circles), Iron-Depleted Media (IDM: BHI without hemin supplementation and treated with Chelex for Fe depletion, supplied with 10% Iron-depleted FBS) (black circles), or in Iron-Depleted Media supplemented with Fe-citrate (IDM + Fe: IDM described before, plus 8 μM Fe-citrate) (gray circles), (n = 6). Using one-way ANOVA with Tukey's test, we assessed differences between mean values at time-matched determinations; \* *p* < 0.05 with respect to RM.

**Table 2.** Intracellular Fe content and quantification of the TcFR and TcIT transcripts \*.


*\* Trypanosoma cruzi* epimastigotes were maintained in Regular Media (RM), Iron-Depleted Media (IDM), or Iron-Depleted Media plus 8 μM Fe-citrate (IDM + Fe). Intracellular Fe content determination (*n* = 5) and quantitative PCR for TcFR (*n* = 5) or TcIT (*n* = 5) (using 100 ng cDNA) were carried out in epimastigotes in a mid-log phase when maintained in the different media. Using one-way ANOVA with Tukey's test, we assessed differences between mean values. Different lower-case letters as superscripts in the same line indicate different mean values (*p* < 0.05).

#### *3.2. Exogenous Fe Selectively Modulates the Endocytic Capacity of Epimastigote Cells*

Figure 2 shows that ionic Fe modulates both the function and structure of the endocytic pathway in *T. cruzi* epimastigotes. Figure 2A (see also representative cytometric analyses in Supplementary Figure S1) demonstrates that parasites from IDM and IDM + Fe upregulate transferrin (Tf) uptake compared to control parasites grown in RM. However, hemoglobin (HB) uptake is upregulated only in IDM, compared to RM and IDM + Fe, indicating that free Fe in the culture medium controls hemoglobin uptake, in contrast with that observed with the transferrin uptake mechanism. Fe depletion stimulates bovine serum albumin (BSA) uptake in parasites grown in IDM, and the stimulation persists in IDM + Fe, indicating that this endocytic event is higher in both conditions. The fluorescence images in Figure 2B visually represent what we found in the cell cytometry data. Parasites upregulate the uptake of the endocytic tracers in IDM and IDM + Fe conditions.

**Figure 2.** Fe exogenous content alters endocytosis in epimastigotes. Epimastigotes were incubated in RPMI medium containing transferrin–FITC (Tf), hemoglobin–FITC (HB), or BSA–FITC (BSA) for 30 min at 28 ◦C. (**A**) Endocytosis assay evaluated by flow cytometry in epimastigotes maintained at RM (white bars), IDM (black bars), or IDM + Fe (gray bars) (n = 4 from different cultures); \* *p* < 0.05; (**B**) immunofluorescence of epimastigotes at different culture conditions and endocytic tracers incubation. Endocytic content staining with fluorescence macromolecules transferrin– FITC (Tf), hemoglobin–FITC (HB), or BSA–FITC (BSA) (green). Nuclei staining with DAPI (blue). Bars: 10 μm.

Ultrastructural analysis of epimastigotes grown in the three different culture media revealed opposite trends depending on the organelles analyzed (Figure 3). While the general morphological aspect remained unmodified in IDM and IDM + Fe when compared with the parasites maintained in RM, important alterations can be seen in the reservosomes, lysosome-like organelles that are also involved in the storage of exogenous lipids, especially cholesterol, and enzymes involved in lipid synthesis from acetyl-CoA [49–52]. The homogeneous content of the reservosomes encountered in RM epimastigotes (upper line) contrasts with that seen in the other two groups. The depletion of Fe (middle line) provoked a marked increase in lipid accumulation, which was not reversed by the supplementation with Fe citrate (bottom line), indicating significant metabolic modifications.
