*3.3. Free Iron Supplementation-Induced Modifications in the Heme-Regulated Eukaryotic Initiation Factor 2α Kinase-Signaling Pathway*

Facing the epimastigotes proliferation results presented in Figure 1 and considering that Fe depletion downregulates cytosolic and mitochondrial ribosomal proteins [53] via the heme-regulated inhibitor (HRI) of the translation through phosphorylation of the eukaryotic initiation factor 2α (eIF2α), we investigated two key elements of this signaling pathway (Table 3). Removal of heme and free Fe (IDM) increased the expression of HRI by approximately 90% and decreased by 50% the expression of eIF2α. In the case of HRI, the supplementation with free Fe restored the levels of HRI and upregulated those of eIF2α.

**Figure 3.** Ultrastructural changes in *T. cruzi* parasites submitted to different exogenous Fe contents. Epimastigotes were processed and observed by transmission electron microscopy. (Upper line) Epimastigotes maintained in RM show normal morphology of major cellular organelles such as the kinetoplast (K), nucleus (N), golgi complex (Gc), cytostome-cytopharynx complex (Cy), reservosomes (R), mitochondria (M), and flagellum (F). (Middle line) Epimastigotes were maintained in IDM. (Bottom line) Epimastigotes maintained in IDM + Fe. Epimastigotes maintained at IDM or IDM + Fe media present typical organelles morphology and positioning compared to those maintained at RM, except for reservosomes, which present a higher content of lipid inclusions (asterisks) compared to the ones in RM. Bars: 500 nm. Five culture samples were examined.

**Table 3.** Influence of PKA pathway on the response to exogenous ionic Fe in *T. cruzi* epimastigotes \*.


\* Quantification of the TcHRI (*n* = 5), elF2α (*n* = 5), and TcPKA (*n* = 5) transcripts in *T. cruzi* epimastigotes. Quantitative PCR was done using 100 ng of cDNA from epimastigotes in a mid-log phase when maintained in RM, IDM, or IDM + Fe. PKA activity was measured, as described in Materials and Methods (*n* = 5), in epimastigotes maintained in RM, IDM, or IDM + Fe. In all cases, differences were assessed using one-way ANOVA with Tukey's test. Different lower-case letters as superscripts in the same line indicate different mean values.

The faster response mediated by the HRI→eIF2α is integrated with the PKA pathway in several eukaryotic organisms [54], and the PKA pathway participates in the sensing of and response to nutrients, including Fe [55]. Table 3 also shows a decrease in PKA expression in epimastigotes grown in IDM and upregulation in IDM + Fe when both conditions are compared to RM. The impact of Fe depletion/supplementation was better seen with PKA activity: a pronounced decrease in parasites from IDM and an upregulation of more than 100% in IDM + Fe conditions (Table 3).

## *3.4. Exogenous Fe Alters Mitochondrial Respiratory Rates in T. cruzi Epimastigotes*

As mentioned above, mitochondrial proteins decrease when the HRI→eIF2α pathway is dysfunctional [53], as encountered in the case of Fe depletion (Table 3). Fe concentration in the culture altered mitochondrial O2 consumption by *T. cruzi,* as observed in Table 4. The digitonin permeabilization of parasites did not alter the basal respiration profile. There was a difference in the O2 consumption after adding succinate (the consumption of O2 in the LEAK state) to IDM compared to RM and IDM + Fe (line 1). The O2 consumption in the presence of succinate without further additions matched the succinate-cytochrome c oxidoreductase activity (line 5). However, after additions of ADP (oxidative phosphorylation state-OXPHOS) (line 2) and H+ ionophore FCCP (electron transfer system uncoupled from phosphorylation-ETS) (line 3), there was a significant and similar decrease in O2 consumption in both IDM and IDM + Fe. After adding antimycin A to inhibit the mitochondrial complex III, the residual respiration (ROX) was almost completely abolished under all conditions (line 4). The impact of Fe depletion (even after Fe citrate supplementation) on mitochondrial internal membrane potential (ΔΨm) can be seen in line 6. The ratio between the safranin A fluorescence in the absence and presence of FCCP (F/FFCCP) increased by 600% in parasites grown in IDM and attained an intermediary level ~200% higher in IDM + Fe conditions.

**Table 4.** Removal of exogenous Fe leads to mitochondrial alterations in epimastigotes \*.


\* *Trypanosoma cruzi* epimastigotes maintained in RM, IDM, or IDM + Fe media were tested for respiration of intact epimastigotes. The mitochondrial respiration, the succinate cytochrome c oxidoreductase activity, and the ATPi are expressed in pmol of O2 consumed per million of cells, % of the activity in RM, and nmol of intracellular ATP per 10<sup>7</sup> cells, respectively. Epimastigotes were digitonin-permeabilized, as described in Nogueira et al. [40], to measure mitochondrial respiration after the successive additions of succinate, ADP, FCCP, and antimycin A (*n* = 3) (lines 1–4). Succinate-cytochrome c oxidoreductase activity was measured as the rate of ferricytochrome c reduction upon adding succinate in epimastigotes (*n* = 3) (line 5). The F/FFCCP ratio was used to estimate the ΔΨm, where F is the mean fluorescence intensity in the absence of the uncoupler FCCP and F/FFCCP is the mean fluorescence in the presence of FCCP (*n* = 3) (line 6). Intracellular ATP in *T. cruzi* epimastigotes was measured in the absence or presence of oligomycin or iodoacetamide (*n* = 3) (lines 7–9). We assessed differences between mean values using a one-way ANOVA with Tukey's test. Different lower-case letters as superscripts indicate statistical differences among the mean values in the same line (*p* < 0.05). Asterisks in lines 8 and 9 indicate a statistical difference (*p* < 0.05) with respect to RM in the absence of inhibitors.

Parasites maintained in the RM medium presented intracellular ATP content (ATPi) more than twice as high when compared to parasites grown in IDM or IDM + Fe media (Table 4, line 7). The addition of oligomycin to block the FoF1-ATP synthase [56] decreased ATPi content by more than 50% with respect to the RM group without inhibitors (line 8), indicating that, under these conditions, the cellular ATP supply comes from mitochondrial activity. Moreover, the addition of iodoacetamide (line 9), an inhibitor of glyceraldehyde 3-phosphate dehydrogenase (GAPDH): (i) reduced ATPi in parasites grown in RM to levels that are similar to those encountered in IDM and IDM + Fe conditions in the absence or presence of oligomycin; (ii) provoked a further 50% decrease in ATPi from IDM and IDM + Fe parasites when compared to that grown in RM, indicating that the glycolytic pathway is responsible for ATP production in IDM and IDM + Fe conditions.

#### *3.5. Upregulation of Glycolytic Enzymes in Parasites Grown in Fe-Depleted Media*

The activity and abundance of two glycolytic enzymes were evaluated in the experiments depicted in Figure 4. Epimastigotes grown in IDM and IDM + Fe presented, respectively, with GAPDH activities two and three times higher than those grown in RM (Figure 4A), an increase that matches the higher expression of the enzyme (Figure 4B). The abundance of the enzyme was also investigated; the levels increased by more than 100% in IDM + Fe parasites compared with the RM group without modification in the IDM group (Figure 4C). The shift toward a glycolytic metabolic profile was confirmed by the more than 200% increase in the activity of glucokinase (Figure 4D), the enzyme that connects the pentose phosphate pathway and the glycolytic pathway, which are the two major pathways for glucose metabolism in *T. cruzi* [57].

**Figure 4.** Exogenous Fe leads to glycolytic changes in epimastigotes. (**A**) GAPDH activity in the absence or presence of iodoacetamide (IAA, 10 μM), as indicated on the abscissa, in epimastigotes in a mid-log phase when maintained in RM (empty bar), IDM (black bar), or IDM + Fe (gray bar). The absorbance due to the formation of NADH was monitored at 340 nm (*n* = 4); (**B**) quantification of the GAPDH transcript in *T. cruzi* epimastigotes. Quantitative PCR was done using 100 ng cDNA from epimastigotes maintained at RM (empty circles), IDM (black circles), or IDM + Fe (gray circles), (*n* = 4); (**C**) densitometric analysis of Western blot results (*n* = 4), with the inset showing representative Western blotting analysis. Membranes were probed with primary mouse anti-GAPDH antibody (1:500) as described in the Materials and Methods section; (**D**) glucokinase activity was measured as described in Materials and Methods in epimastigotes maintained at RM (empty bar), IDM (black bar), or IDM + Fe (gray bar). The absorbance due to the formation of NADPH was monitored at 340 nm (*n* = 4); \* *p* < 0.05 in all cases with respect to RM. We used a one-way ANOVA with Tukey's test to assess differences between mean values.
