Fig 1.
Palmitate oxidation promotes ATP production and viability in epimastigote forms under starvation.
A) Schematic representation of 14C-U-palmitate metabolism. The metabolites corresponding to labelled palmitate metabolism are presented in green. B) 14CO2 production from epimastigotes incubated in PBS with 14C-U-palmitate 100 μM. The 14CO2 was captured at 0, 30, 60 and 120 min. C) Viability of epimastigote forms after incubation with different carbon sources and palmitate. The viability was assessed after 24 and 48 h by MTT assay. D) The intracellular ATP content was evaluated following incubation with different energy substrates or not (PBS, negative control). The ATP concentration was determined by luciferase assay and the data were adjusted by the number of cells. A statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test at p < 0.05 using the GraphPad Prism 8.0.2 software program. We represent the level of statistical significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; * p value < 0.05. For a p value > 0.05 we consider the differences to be not significant (ns).
Fig 2.
Excreted end products of glucose and palmitate metabolism in epimastigote forms of T. cruzi.
A) The extracellular medium of epimastigote forms incubated under different conditions was analysed by 1H-NMR spectrometry to detect and quantify the end-products. The resulting data were expressed in nmoles/h/108 cells. Means ± SD of three independent experiments. ICS is internal carbon sources; nd is non-detectable. B) and C) Schematic representation of the contribution of glucose and palmitate to the metabolism of epimastigote forms of T. cruzi. The glycosomal compartment and TCA cycle are indicated. The amount of end-product determined by the font size. Numbers indicates enzymatic steps. 1. Glycolysis; 2. pyruvate dehydrogenase; 3. citrate synthase; 4. aconitase; 5. isocitrate dehydrogenase; 6. α-ketoglutarate dehydrogenase; 7. succinyl-CoA synthetase; 8. Succinate dehydrogenase/complex II/fumarate reductase NADH-dependent; 9. fumarate hydratase; 10. malate dehydrogenase; 11. Malic enzyme; 12. alanine dehydrogenase/alanine aminotransferase; 13. lactate dehydrogenase; 14. acetate:succinyl-CoA transferase; 15. acetyl-CoA hydrolase; 16. succinyl-CoA synthetase; 17. Glycosomal fumarate reductase and 18. Palmitate oxidation by beta-oxidation, resulting in FADH2, NADH and acetyl-CoA; Abbreviations: Cit: Citrate, Aco: Aconitate, IsoC: Isocitrate, α-kg: α-Ketoglutarate, Suc-CoA: Succinyl-CoA, Suc: Succinate, Fum: Fumarate, Mal: Malate, and Oxa: Oxaloacetate.
Fig 3.
Changes in glucose and triacylglycerol contents in LIT medium.
A) Growth curve of epimastigote forms. B) Glucose quantification over 48 h. C) Triacylglycerol levels over 48 h. In each experiment, we collected each medium at different times and subjected it to quantification according to the manufacturer’s instructions. All the experiments were performed in triplicates. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test p < 0.05 using the GraphPad Prism 8.0.2 software program. We represent the levels of statistical significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p value > 0.05, we consider the differences not significant (ns).
Fig 4.
Glucose metabolism inhibits FAO.
Parasites were incubated in the presence of 14C-U-palmitate + 5 mM glucose and 14C-U-glucose + 0.1 mM palmitate in PBS. 14CO2 production from epimastigotes incubated in PBS. The 14CO2 was captured after 120 min of incubation. The experiments were performed in triplicates. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test p < 0.05 using the GraphPad Prism 8.0.2 software program. We represent the level of statistical significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p value > 0.05, we consider the differences not significant (ns).
Fig 5.
Flow cytometry reveals distinct patterns in fatty acid pools during epimastigote growth.
The epimastigotes were treated with 1 μM of BODIPY C1-C12 (500/510) and analysed by flow cytometry and fluorescence microscopy. A) 0 h. B) 24 h. C) 48 h. In the flow cytometry histograms, dashed peaks represent unstained parasites. Green-filled peaks represent stained parasites. D) Mean fluorescence per cell. The fluorescence for each cell was calculated using ImageJ software. All the experiments were performed in triplicates. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test p < 0.05 using the GraphPad Prism 8.0.2 software program. We represent the level of statistical significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p value > 0.05, we consider the differences not significant (ns).
Fig 6.
Epimastigote forms accumulates fatty acids into lipid droplets during growth.
The epimastigotes were treated with 1 μM BODIPY C1-C12 (500/510) and analysed by flow cytometry and fluorescence microscopy. A) 0 h. B) 24 h. C) 48 h. In the flow cytometry histograms, dashed peaks represent unstained parasites. Yellow filled peaks represent positively stained parasites. The number of green/yellow spots for each cell was calculated using ImageJ software. All the experiments were performed in triplicates.
Fig 7.
Activities of enzymes related to lipid and glucose metabolism during T. cruzi growth curves.
A) (HK) Hexokinase B) (ACC) acetyl-CoA carboxylase, C) (CPT1) carnitine-palmitoyltransferase, and D) (SPT) serine palmitoyltransferase. All these activities were measured in crude extracts from epimastigote forms at different moments of the growth curve. All the experiments were performed in triplicates. Time course activities and controls shown in S2 Fig. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test at p < 0.05, using the GraphPad Prism 8.0.2 software program. We represent the level of statistical significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p value > 0.05 we consider the differences not significant (ns).
Fig 8.
ETO inhibits CPT1 and interferes with cell proliferation in epimastigote forms.
(A) Proliferation of epimastigote forms in the presence of 0.1 to 500 μM ETO. For the positive control of dead cells, a combination of antimycin (0.5 μM) and rotenone (60 μM) was used. (B) Inhibition of CPT1 activity in crude extracts using 250 and 500 μM of ETO. C) 14CO2 capture from 14C-U-palmitate oxidation. D) Flow cytometry analysis and fluorescence microscopy of epimastigote forms treated (or not) with ETO. In the histograms, dashed peaks represent unstained parasites and green-filled peaks represent parasites stained with BODIPY C1-C12. All the experiments were performed in triplicates. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test at p < 0.05 using the GraphPad Prism 8.0.2 software program. We represent the level of statistical significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p values > 0.05, we consider the differences not significant (ns).
Fig 9.
Analysis of extracellular phosphatidylserine exposure, membrane integrity and cell cycle after ETO treatment.
Parasites in the exponential growth phase were treated with 500 μM of ETO for 5 days. (A) Following the incubation period, the parasites were labelled with propidium iodide (PI) and annexin V-FITC (ANX) and analysed by flow cytometry. (B) The cell cycle was assessed using PI staining. (C) Growth curves of epimastigote forms before and after removing the treatment. All the experiments were performed in triplicates. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test p < 0.05, using the GraphPad Prism 8.0.2 software program. We represent the level of statistical significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p values > 0.05, we consider the differences not significant (ns).
Fig 10.
Effects of ETO on respiration and ATP production in epimastigote forms of T. cruzi.
(A) Oxygen consumption of epimastigote forms after normal growth in LIT medium. (B) Oxygen consumption after ETO 500 μM treatment. Parasite growth in LIT medium with the compound until the 5th day. In black, a time-course register of the concentration (pmols) of O2 in the respiration chamber. In blue, negative of the concentration derivative (pmols) of O2 with respect to time (velocity of O2 consumption in pmoles per second). The parasites were washed twice in PBS and kept in MRC buffer at 28°C during the assays. (C) The basal respiration (initial oxygen flux values, MRC), respiration leak after the sequential addition of 0.5 μg/mL of oligomycin A (2 μg/mL), and electron transfer system (ETS) capacity after the sequential addition of 0.5 μM FCCP (2 μM) were measured for each condition. (D) Intracellular levels of ATP after treating with 500 μM ETO. The intracellular ATP content was assessed following incubation with different energy substrates or not (PBS, negative control). The ATP concentration was determined by luciferase assay and the data were adjusted by the number of cells. All the experiments were performed in triplicates. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test at p < 0.05 using GraphPad Prism 8.0.2 software. We represent the level of statistical significance as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p values > 0.05, we consider the differences not significant (ns).
Fig 11.
Internal fatty acid consumption contributes to parasite viability under severe nutritional starvation.
Viability of epimastigote forms after incubation in PBS with or without ETO. The viability was assessed every 24 h using Alamar Blue. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-test p < 0.05 using GraphPad Prism 8.0.2 software. We represent the levels of statistical significance as follow: *** p value < 0.001, and for p values > 0.05, we consider the differences not significant (ns).
Fig 12.
ETO inhibits metacyclogenesis.
A) CPT1 activity of epimastigote forms in stationary phase and 24h after incubated in TAU-3AAG medium (for triggering metacyclogenesis). B) Fluorescence microscopy of cells incubated in TAU-3AAG in the presence of BODIPY 500–510 C1-C12. C) Effects of different ETO concentrations on metacyclogenesis. The differentiation was evaluated by counting the cells in a Neubauer chamber each day for 6 days. This experiment was performed in triplicate. D) Percentage of differentiation at the 5th day of differentiation. Inset: IC50 of metacyclogenesis inhibition by ETO. The enzymatic activities were measured in duplicate. All the other experiments were performed in triplicates.