Fatty acid oxidation participates of the survival to starvation, cell cycle progression and differentiation in the insect stages of Trypanosoma cruzi 1 2 3 4 Fatty acid oxidation participates of the survival to starvation, cell cycle progression 5 and differentiation in the insect stages of Trypanosoma cruzi 6 7 Rodolpho Ornitz Oliveira Souza¹, Flávia Silva Damasceno¹, Sabrina Marsiccobetre1, Marc Biran2, 8 Gilson Murata3, Rui Curi4, Frédéric Bringaud5, Ariel Mariano Silber¹* 9 10 11 ¹ University of São Paulo, Laboratory of Biochemistry of Tryps – LaBTryps, Department of 12 Parasitology, Institute of Biomedical Sciences – São Paulo, SP, Brazil 13 14 2 Centre de Résonance Magnétique des Systèmes Biologiques (RMSB), Université de Bordeaux, 15 CNRS UMR-5536, Bordeaux, France 16 17 3 University of São Paulo, Department of Physiology, Institute of Biomedical Sciences – São Paulo, 18 SP, Brazil 19 20 4 Cruzeiro do Sul University, Interdisciplinary Post-Graduate Program in Health Sciences - São Paulo, 21 SP, Brazil 22 23 5 Laboratoire de Microbiologie Fondamentale et Pathogénicité (MFP), Université de Bordeaux, 24 CNRS UMR-5234, Bordeaux, France 25 26 27 28 *Corresponding author 29 E-mail: asilber@usp.br (AMS) 30 31 32 33 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 34 Abstract 35 During its complex life cycle, Trypanosoma cruzi colonizes different niches in its insect and 36 mammalian hosts. This characteristic determined the types of parasites that adapted to face 37 challenging environmental cues. The primary environmental challenge, particularly in the insect 38 stages, is poor nutrient availability. These T. cruzi stages could be exposed to fatty acids originating 39 from the degradation of the perimicrovillar membrane. In this study, we revisit the metabolic fate of 40 fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose 41 concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the 42 parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to 43 CO2 production via beta-oxidation. Lastly, we also show that fatty acids are degraded through beta- 44 oxidation. Additionally, through beta-oxidation, electrons are fed directly to oxidative 45 phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid cycle, which can be used to 46 feed other anabolic pathways such as the de novo biosynthesis of fatty acids. 47 48 Author Summary 49 Trypanosoma cruzi is a protist parasite with a life cycle involving two types of hosts, a 50 vertebrate one (which includes humans, causing Chagas disease) and an invertebrate one (kissing 51 bugs, which vectorize the infection among mammals). In both hosts, the parasite faces environmental 52 challenges such as sudden changes in the metabolic composition of the medium in which they 53 develop, severe starvation, osmotic stress and redox imbalance, among others. Because kissing bugs 54 feed infrequently in nature, an intriguing aspect of T. cruzi biology (it exclusively inhabits the 55 digestive tube of these insects) is how they subsist during long periods of starvation. In this work, we 56 show that this parasite performs a metabolic switch from glucose consumption to lipid oxidation, and 57 it is able to consume lipids and the lipid-derived fatty acids from both internal origins as well as 58 externally supplied compounds. When fatty acid oxidation is chemically inhibited by etomoxir, a very 59 well-known drug that inhibits the translocation of fatty acids into the mitochondria, the proliferative 60 insect stage of the parasites has dramatically diminished survival under severe metabolic stress and 61 its differentiation into its infective forms is impaired. Our findings place fatty acids in the centre of 62 the scene regarding their extraordinary resistance to nutrient-depleted environments. 63 64 65 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 66 Introduction 67 T. cruzi, a flagellated parasite, is the causative agent of Chagas disease, a neglected health 68 problem endemic to the Americas [1]. The parasite life cycle is complex, alternating between 69 replicative and non-replicative forms in two types of hosts, mammalians and triatomine insects [2]. 70 In mammalian hosts, two primary forms are recognized: replicative intracellular amastigotes and 71 nondividing trypomastigotes, which are released from infected host cells into the extracellular 72 medium. After being released from infected cells, trypomastigotes can spread the infection by 73 infecting new cells, or they can be ingested by a triatomine bug during its blood meal. Once inside 74 the invertebrate host, the ingested trypomastigotes differentiate into epimastigotes, which initiate 75 their proliferation and colonization of the insect digestive tract [3]. Once the epimastigotes reach the 76 final portion of the digestive tube, they initiate differentiation into non-proliferative, infective 77 metacyclic trypomastigotes. These forms will be expelled during a new blood meal and will be able 78 to infect a new vertebrate host [2,4–6]. 79 The diversity of environments through which T. cruzi passes during its life cycle (i.e., the 80 digestive tube of the insect vector, the bloodstream and the mammalian cells cytoplasm) subjects it 81 to different levels of nutrient availability [3,7]. Therefore, this organism evolved a robust, flexible 82 and efficient metabolism [5,8]. As an example, it was recognized early on that epimastigotes are able 83 to rapidly switch their metabolism, allowing the consumption of carbohydrates and different amino 84 acids [9,10]. Several studies identified aspartate, asparagine, glutamate [11], proline [12–14], 85 histidine [15], alanine [11,16] and glutamine [11,17] as oxidisable energy sources. Despite the 86 quantity of accumulated information on amino acid and carbohydrate consumption, little is known 87 about how T. cruzi uses fatty acids and how these compounds contribute to the parasite´s metabolism 88 and survival. In this study, we explore fatty acid metabolism in T. cruzi. We also address fatty acid 89 regulation by external glucose levels and the involvement of their oxidation in the replication and 90 differentiation of T. cruzi insect stages. 91 92 Methods 93 Parasites 94 Epimastigotes of T. cruzi strain CL clone 14 were maintained in the exponential growth phase 95 by sub-culturing them for 48 h in Liver Infusion Tryptose (LIT) medium at 28 °C [18]. Metacyclic 96 trypomastigotes were obtained through the differentiation of epimastigotes at the stationary growth 97 phase in TAU-3AAG (Triatomine Artificial Urine supplemented with 10 mM proline, 50 mM 98 glutamate, 2 mM aspartate and 10 mM glucose) as previously reported [19]. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 99 100 Fatty acid oxidation assays 101 Preparation of palmitate-BSA conjugates. Sodium palmitate at 70 mM was solubilized in water by 102 heating it up to 70 °C. BSA free fatty acids (FFA BSA) (Sigma®) was dissolved in PBS and warmed 103 up to 37 °C with continuous stirring. Solubilized palmitate was added to BSA at 37 °C with 104 continuous stirring (for a final concentration of 5 mM in 7% BSA). The conjugated palmitate-BSA 105 was aliquoted and stored at −80 °C [20]. 106 107 CO2 production from oxidisable carbon sources. To test the production of CO2 from palmitate, 108 glucose or histidine, exponentially growing epimastigotes (5x107 mL-1) were washed twice in PBS 109 and incubated for different times (0, 30, 60 and 120 min) in the presence of 0.1 mM of palmitate 110 spiked with 0.2 µCi of 14C-U-substrates. To trap the produced CO2, Whatman paper was embedded 111 in 2 M KOH solution and was placed in the top of the tube. The 14CO2 trapped by this reaction was 112 quantified by scintillation [15,16]. 113 114 1H-NMR analysis of the exometabolome. Epimastigotes (1x108 mL-1) were collected by 115 centrifugation at 1,400 x g for 10 min, washed twice with PBS and incubated in 1 mL (single point 116 analysis) of PBS supplemented with 2 g/L NaHCO3 (pH 7.4). The cells were maintained for 6 h at 27 117 °C in incubation buffer containing [U-13C]-glucose, non-enriched palmitate or no carbon sources. The 118 integrity of the cells during the incubation was checked by microscopic observation. The supernatant 119 (1 mL) was collected and 50 µl of maleate solution in D2O (10 mM) was added as an internal 120 reference. 1H-NMR spectra were collected at 500.19 MHz on a Bruker Avance III 500 HD 121 spectrometer equipped with a 5 mm Prodigy cryoprobe. The measurements were recorded at 25 °C. 122 The acquisition conditions were as follows: 90° flip angle, 5,000 Hz spectral width, 32 K memory 123 size, and 9.3 sec total recycling time. The measurements were performed with 64 scans for a total 124 time of close to 10 min and 30 sec. The resonances of the obtained spectra were integrated and the 125 metabolite concentrations were calculated using the ERETIC2 NMR quantification Bruker program. 126 127 Oxygen consumption. To evaluate the importance of internal fatty acid sources in O2 consumption, 128 exponentially growing parasites were treated or not treated with 500 µM ETO (the inhibitor of 129 carnitine palmitoyltransferase 1), washed twice in PBS and resuspended in Mitochondrial Cellular 130 Respiration (MCR) buffer. The rates of oxygen consumption were measured using intact cells in a 131 high-resolution oxygraph (Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria). Oligomycin A 132 (0.5 µg/mL) and FCCP (0.5 µM) were sequentially added to measure the optimal non-coupled .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 133 respiration and the respiration leak state, respectively. The data were recorded and treated using 134 DatLab 7 software [15,16,21]. 135 136 Mitochondrial activity assays 137 MTT and Alamar Blue. The parasites were washed twice and incubated in PBS supplemented with 138 0.1 mM palmitate in 0.35% FFA BSA, 0.35% FFA BSA alone, and 5 mM glucose, and 5 mM histidine 139 or not supplemented media were used as controls (positives and negative, respectively). The cell 140 viability was evaluated at 24 h and 48 h after incubation using the MTT assay, as described in [15,16]. 141 Alamar Blue. The parasites were washed twice and incubated in PBS or PBS supplemented with 500 142 μM ETO in 96-well plates. The plates were maintained at 28 °C during all the experiments. After 143 every 24 h, the cells were incubated with 0.125 μg.mL-1 of Alamar Blue reagent and kept at 28 °C for 144 2 h under protection from light. The fluorescence was accessed using the wavelengths λexc = 530 nm 145 and λem = 590 nm in the SpectraMax® i3 (Molecular Devices) plate reader. 146 147 Measurement of intracellular ATP content 148 The intracellular ATP levels were assessed using a luciferase assay kit (Sigma-Aldrich ®), as 149 described in [15–17]. In brief, the parasites were incubated in PBS supplemented (or not) with 0.1 150 mM palmitate, 0.35% FFA BSA, 5 mM glucose or 5 mM histidine for 24 h at 28 °C. The ATP 151 concentrations were determined by using a calibration curve with ATP disodium salt (Sigma), and 152 the luminescence at 570 nm was measured as indicated by the manufacturer. 153 154 Enzymatic activities 155 Carnitine palmitoyltransferase 1 (CPT1). The epimastigotes were washed twice in PBS (1,000 x 156 g, 5 min at 4 °C), resuspended in buffered Tris-EDTA (100 mM, 2.5 mM and 0.1% Triton X-100) 157 containing 1 µM phenylmethyl-sulphonyl fluoride (PMSF), 0.5 mM N-alpha-p-tosyl-lysyl- 158 chloromethyl ketone (TLCK), 0.01 mg aprotinin and 0.1 mM trans-epoxysuccinyl-L-leucyl amido 159 (4-guanidino) butane (E-64) as a protease inhibitors (Sigma Aldrich®) and lysed by sonication (5 160 pulses for 1 min each, 20%). The lysates were clarified by centrifugation at 10,000 x g for 30 min at 161 4 °C. The soluble fraction was collected and the proteins were quantified by Bradford method [22] 162 and adjusted to 0.1 mg/mL protein. The reaction mixture contained 0.5 mM L-carnitine, 0.1 mM 163 palmitoyl-CoA and 2.5 mM DTNB in Tris-EDTA buffer (pH = 8.0). The CPT1 activity was measured 164 spectrophotometrically at 412 nm by DTNB reaction with free HS-CoA, forming the TNB- ion. To 165 calculate the specific activity, the absorbance values were converted into molarity by using the TNB- 166 extinction molar coefficient of 12,000 M-1.s-1 [23]. As a blank, we performed the same assay without .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 167 adding the substrate. All the enzymatic assays were performed in 96-well plates at a final volume of 168 0.2 mL in the SpectraMax® i3 (Molecular Devices). 169 170 Acetyl-CoA carboxylase (ACC). The ACC activity was measured spectrophotometrically by 171 coupling its enzymatic reaction with that of citrate synthase (CS), which uses oxaloacetate and acetyl- 172 CoA to produce citrate. Measurements were performed at the end-points in two steps. First, the 173 reaction mixture contained 100 mM potassium phosphate buffer (pH = 8.0), 15 mM KHCO3, 5 mM 174 MnCl2, 5 mM ATP, 1 mM acetyl-CoA and 0.1 μM biotin. The reaction was initiated by adding 0.1 175 mg of cell extract and developed using 15 min incubations at 28 °C. The reaction was stopped by 176 adding perchloric acid 40% (v/v) and centrifuged 10,000 x g for 15 min at 4 °C. The second reaction 177 was performed by using 0.1 mL of the supernatant from the first reaction, 20 mM oxaloacetate and 178 0.5 mM of DTNB in 100 mM potassium phosphate buffer (pH = 8.0). The reaction was initiated by 179 adding 0.5 units of CS (Sigma Aldrich©). To calculate the specific activity of ACC, we converted 180 the absorbance values to molarity by using the TNB- extinction molar coefficient of 12,000 M-1.s-1. 181 For the blank reaction, we performed the same assay without acetyl-CoA [24]. 182 183 Hexokinase (HK). The HK activity was measured as described in [25]. Briefly, the activity was 184 measured by coupling the hexokinase activity with a commercial glucose-6-phosphate 185 dehydrogenase, which oxidizes the glucose-6-phosphate (G6PD, SIGMA) resulting from the HK 186 activity with the concomitant reduction of NADP+ to NADPH. The resulting NADPH was 187 spectrophotometrically monitored at 340 nm. The reaction mixture contained 50 mM Triethanolamine 188 buffer pH 7.5, 5 mM MgCl2, 100 mM KCl, 10 mM glucose, 5 mM ATP and 5 U of commercial 189 G6PD. To calculate the specific activity, the absorbance values were converted to molarity using the 190 NADP(H) extinction molar coefficient of 6,220 M-1.s-1. 191 Serine palmitoyltransferase (SPT). The SPT activity was measured through the reduction of the 192 DTNB reaction by the free HS-CoA, forming the TNB- ion, which was measured 193 spectrophotometrically at 412 nm as previously described [23]. In brief, the epimastigotes were 194 washed twice in PBS, resuspended in Tris-EDTA buffer (100 mM/2.5 mM) containing Triton X-100 195 0.1% and lysed by sonication (20% of potency, during 2 min). The reaction mixture contained 0.1 196 mg of protein free-cell extract, 0.5 mM L-serine, 0.1 mM palmitoyl-CoA and 2.5 mM DTNB in Tris- 197 EDTA buffer (100 mM/2.5 mM) pH = 8.0 [26]. To calculate the specific activity, we converted the 198 absorbance values to molarity using the TNB- extinction molar coefficient of 12,000 M-1.s-1. For the 199 blank reaction, we performed the same assay without adding palmitoyl-CoA. All the enzymatic assays 200 were performed in 96-well plates in a final volume of 0.2 mL in the SpectraMax® i3 (Molecular 201 Devices). .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 202 203 Glucose and triglyceride quantification 204 Spent LIT medium from epimastigote cultures was collected by recovering the supernatants 205 from a centrifugation (10,000 x g for 15 min at 4 °C). Each sample of spent LIT was analysed for its 206 glucose and triglyceride contents using commercial kits (triglyceride monoreagent and glucose 207 monoreagent by Bioclin Brazil) according to the manufacturer’s instructions. These kits are based on 208 colorimetric enzymatic reactions, and the absorbance of each assay was measured in 96-well plates 209 at a final volume of 0.2 mL in the SpectraMax® i3 (Molecular Devices). 210 211 Proliferation assays 212 Exponentially growing T. cruzi epimastigotes (5x107 mL-1) were treated with different 213 concentrations of ETO or not treated (negative control) in LIT medium. As a positive control for 214 growth inhibition, we used a combination of rotenone (60 µM) and antimycin (0.5 µM) [27]. The 215 parasites (2.5x106 mL-1) were transferred to 96-well plates and then incubated at 28 °C. The cell 216 proliferation was quantified by reading the optical density (OD) at 620 nm for eight days. The OD 217 values were converted to cell numbers using a linear regression equation previously obtained under 218 the same conditions. Each experiment was performed in quadruplicate [28]. 219 220 Flow cytometry analyses 221 Cell death. Epimastigotes in the exponential phase of growth were maintained in LIT and treated 222 with ETO 500 µM for 5 days. After the incubation time, the parasites were analysed as described in 223 [28]. The cells were analysed by flow cytometry (FACScalibur BD Biosciences). 224 225 Cell cycle (DNA content). Epimastigotes in the exponential phase of growth were maintained in LIT 226 and treated with ETO 500 µM over 5 days. After the incubation time, the parasites were washed twice 227 in PBS and resuspended in lysis buffer (phosphate buffer Na2HPO4 7.7 mM; KH2PO4 2.3 mM; pH = 228 7.4) and digitonin 64 µM. After incubating on ice for 30 min, propidium iodide 0.2 μg/mL was added. 229 The samples were analysed by flow cytometry (Guava) adapted from [29]. 230 231 Fatty acid staining using BODIPY® 500/510. Exponentially growing epimastigotes were kept in 232 LIT medium to reach three different cell densities (2.5x107 mL-1, 5x107 mL-1 and 108 mL-1) in 24- 233 well plates at 28 °C. Twenty-four hours before the flow cytometry analysis, the parasites were treated 234 with 1 µM C1-BODIPY® 500/510-C12. This fluorophore allows for measurements of the relationship 235 between fatty acid accumulation and consumption by shifting the fluorescence filter. The samples 236 were collected, washed twice in PBS and incubated in 4% paraformaldehyde for 15 min. After .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 237 incubation, the cells were washed twice with PBS and suspended in the same buffer. Flow cytometry 238 analysis was performed with FL-1 and FL-2 filters in a FACS Fortessa DB®. The results were 239 analysed using FloJo software. 240 241 Fluorescence microscopy 242 The parasites were maintained in LIT medium as previously reported for fatty acid staining 243 using BODIPY® 500/510. After incubation, the cells were washed twice in PBS and placed on glass 244 slides. The images were acquired with a digital DFC 365 FX camera coupled to a DMI6000B/AF6000 245 microscope (Leica). The images were analysed using ImageJ software. 246 247 Results 248 Palmitate supports ATP synthesis in T. cruzi 249 We initially investigated the ability of T. cruzi epimastigotes to oxidize fatty acids. To this 250 end, we used palmitate as a proxy for fatty acids in general. The parasites were incubated with 0.1 251 mM 14C-[U]-palmitate, which allowed us to measure the production of 1.3 nmoles of CO2 derived 252 from palmitate oxidation during the first 60 min and 1.5 extra nmoles during the following 60 min 253 (Fig 1a). This finding indicated that beta-oxidation and the further ‘burning’ of the resulting acetyl- 254 CoA is operative in epimastigote mitochondria. Because palmitate is taken up from extracellular 255 medium and oxidized to CO2, it is reasonable to assume that it could contribute to resistance to severe 256 nutritional stress. To support this idea, we tested the ability of palmitate to extend parasite survival 257 under extreme nutritional stress. Parasites were incubated for 24 and 48 h in PBS (negative control, 258 in this condition we expected the lower viability after the incubations), 0.1 mM palmitate in PBS 259 supplemented with BSA (as a palmitate carrier), 5.0 mM histidine in PBS or 5.0 mM glucose in PBS 260 (both positive controls, since it is well knowing the ability of both metabolites to extend the parasites´ 261 viability in metabolic stress conditions, see [15]). As an additional negative control, we used PBS 262 supplemented with BSA without added palmitate. The viability of these cells was assayed by 263 measuring the total reductive activity by MTT assay. Additionally, we measured the total ATP levels. 264 Cells incubated in the presence of palmitate showed higher viability than the negative controls, but 265 not as high as that of parasites incubated with glucose or histidine (Fig 1b). Consistently, parasites 266 incubated in the presence of palmitate showed higher ATP contents than both negative controls. 267 However, the intracellular ATP levels in the cells incubated with palmitate were diminished by half 268 when compared to parasites incubated with histidine. Interestingly, the palmitate kept the ATP 269 content at levels comparable to glucose (Fig 1c). 270 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 271 Figure 1. Palmitate oxidation promotes ATP production and viability in epimastigote forms 272 under starvation. Schematic representation of 14C-U-palmitate metabolism. The metabolites 273 corresponding to labelled palmitate metabolism are presented in green. A) 14CO2 production from 274 epimastigotes incubated in PBS with 14C-U-palmitate 100 µM. The 14CO2 was captured at 0, 30, 60 275 and 120 min. B) Viability of epimastigote forms after incubation with different carbon sources and 276 palmitate. The viability was assessed after 24 and 48 h by MTT assay. C) The intracellular ATP 277 content was evaluated following incubation with different energy substrates or not (PBS, negative 278 control). The ATP concentration was determined by luciferase assay and the data were adjusted by 279 the number of cells. A statistical analysis was performed with one-way ANOVA followed by Tukey's 280 post-test at p < 0.05 using the GraphPad Prism 8.0.2 software program. We represent the level of 281 statistical significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; * p value < 282 0.05. For a p value > 0.05 we consider the differences to be not significant (ns). 283 284 Epimastigote forms excrete acetate as a primary end-product of palmitate oxidation 285 Because the epimastigotes were able to oxidize 14C-U-palmitate to 14CO2, we were interested 286 in analysing their exometabolome and comparing it with that of parasites exclusively consuming 287 glucose, palmitate or without any carbon source. Thus, we subjected exponentially growing parasites 288 to 16 h of starvation and then incubated them for 6 h in the presence of 0.3 mM palmitate, 10 mM 289 13C-U-glucose or without any carbon source. For the control, we analysed a sample of non-starved 290 parasites. After the incubations, the extracellular media were collected and analysed by 1H-NMR 291 spectrometry. As expected, all the incubation conditions produced different flux profiles for excreted 292 metabolites (Fig 2 and S1 Fig). Under our experimental conditions, the non-starved parasites 293 primarily excreted succinate and acetate in similar quantities, and alanine and lactate to a lesser extent. 294 Parasites starved for 16 h in PBS and left to incubate in the absence of other metabolites had 295 diminished succinate production (~7-fold) but increased acetate production three-fold compared to 296 the non-starved parasites. It is relevant to stress that the only possible origin for these metabolites are 297 internal carbon sources (ICS). Notably, no other excreted metabolites were detected under these 298 conditions, indicating that under starvation, most of the ICS are transformed into acetate as an end 299 product, which is compatible with the oxidation of internal fatty acids. These results raise the question 300 about the metabolic fates of glucose or fatty acids in previously starved parasites. Starved 301 epimastigotes that recovered in the presence of glucose exhibited a profuse excretion of succinate 302 (450-fold the quantity excreted by the starved cells) and roughly equivalent quantities of acetate 303 compared with the starved cells. Interestingly, lactate and alanine were also excreted at similar levels. 304 As expected, the recovery with glucose produced an increase in all the secreted metabolites. However, 305 analysing their distribution is a reconfiguration of the metabolism towards a majority production of 306 succinate. Finally, in epimastigotes incubated with palmitate, we observed an increase in the acetate 307 and alanine production of approximately 2.5 times to the levels in parasites that recovered in the 308 presence of glucose. Interestingly, succinate is excreted in a smaller quantity than acetate and alanine, 309 but still at 10-fold the rate observed in the starved non-recovered cells. Surprisingly, there was also a .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 310 significant production of pyruvate (not previously described in the literature, and not observed under 311 any other conditions) and a small amount of lactate derived from palmitate. 312 313 Figure 2. Excreted end products of glucose and palmitate metabolism in epimastigote forms of 314 T. cruzi. A) The extracellular medium of epimastigote forms incubated under different conditions 315 was analysed by 1H-NMR spectrometry to detect and quantify the end-products. The resulting data 316 were expressed in nmoles/h/108 cells. Means ± SD of three independent experiments. ICS is internal 317 carbon sources; nd is non-detectable. B) and C) Schematic representation of the contribution of 318 glucose and palmitate to the metabolism of epimastigote forms of T. cruzi. The glycosomal 319 compartment and TCA cycle are indicated. The amount of end-product determined by the font size. 320 Numbers indicates enzymatic steps. 1. Glycolysis; 2. pyruvate dehydrogenase; 3. citrate synthase; 4. 321 aconitase; 5. isocitrate dehydrogenase; 6. α-ketoglutarate dehydrogenase; 7. succinyl-CoA 322 synthetase; 8. Succinate dehydrogenase/complex II/fumarate reductase NADH-dependent; 9. 323 fumarate hydratase; 10. malate dehydrogenase; 11. Malic enzyme; 12. alanine dehydrogenase/alanine 324 aminotransferase; 13. lactate dehydrogenase; 14. acetate:succinyl-CoA transferase; 15. acetyl-CoA 325 hydrolase; 16. succinyl-CoA synthetase; 17. Glycosomal fumarate reductase and 18. Palmitate 326 oxidation by beta-oxidation, resulting in FADH2, NADH and acetyl-CoA; Abbreviations: Cit: Citrate, 327 Aco: Aconitate, IsoC: Isocitrate, α-kg: α-Ketoglutarate, Suc-CoA: Succinyl-CoA, Suc: Succinate, 328 Fum: Fumarate, Mal: Malate, and Oxa: Oxaloacetate. 329 330 Glucose metabolism represses the fatty acid oxidation in epimastigotes 331 Glucose is the primary carbon source for exponentially proliferating epimastigotes, and after 332 its exhaustion from the culture medium, the parasites change their metabolism to use amino acids as 333 carbon sources preferentially [10]. Therefore, we were interested in analysing if this preference for 334 glucose is maintained in relation to the consumption of lipids. To determine if glucose metabolism 335 interferes with the consumption of fatty acids, we created a 48 h proliferation curve using parasites 336 with an initial concentration adjusted to 2.5 x 107 mL-1 and quantified them for 24 h each. Under these 337 conditions, the parasites from the beginning of the experiment, at 0 h, are at mid-exponential phase, 338 they are at late exponential phase at 24 h, and at 48 h they reached stationary phase at a concentration 339 of 10 x 107 mL-1 (Fig 3A). At 0 h, 24 h and 48 h, the culture medium was collected to measure the 340 remaining glucose and triacylglycerol (TAGs) concentrations (Figs 3B and 3C). Most of the glucose 341 was consumed during the first 24 h (during proliferation), while the concentration of TAGs remained 342 the same. After 48 h of proliferation (stationary phase), the TAG levels and lipid contents of the 343 droplets were decreased by 1.5-fold and 2-fold, respectively, suggesting that glucose is preferentially 344 consumed relative to fatty acids. These data show a decrease in the extracellular TAGs between 24 345 and 48 h, while the glucose was already almost entirely consumed, suggesting that glucose is 346 negatively regulating the fatty acid catabolism. 347 348 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 349 Figure 3. Changes in glucose and triacylglycerol contents in LIT medium. A) Growth curve of 350 epimastigote forms. B) Glucose quantification over 48 h. C) Triacylglycerol levels over 48 h. In each 351 experiment, we collected each medium at different times and subjected it to quantification according 352 to the manufacturer's instructions. All the experiments were performed in triplicates. Statistical 353 analysis was performed with one-way ANOVA followed by Tukey's post-test p < 0.05 using the 354 GraphPad Prism 8.0.2 software program. We represent the levels of statistical significance in this 355 figure as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p value > 0.05, 356 we consider the differences not significant (ns). 357 358 Epimastigote forms use endogenous fatty acids to support growth after glucose exhaustion 359 From the previous results, we learned that under glucose deprivation, TAGs are taken up by 360 the epimastigotes, and internally stored fatty acids are mobilized. However, to date, we did not 361 provide any evidence pointing to their use as reduced carbon sources. To confirm this idea, 362 exponentially proliferating epimastigotes were incubated in PBS supplemented with palmitate and 363 14C-U-glucose, or reciprocally, glucose and 14C-U-palmitate. In both cases, the production of 14C- 364 labelled CO2 was quantified. The presence of 5 mM glucose diminished the release of 14CO2 from 365 14C-U-palmitate by 90% while the presence of palmitate did not interfere with the production of 14CO2 366 from 14C-U-glucose (Fig 4). Taken together, our results show that glucose inhibits TAGs and fatty 367 acid consumption, and after glucose exhaustion, a metabolic switch occurs towards the oxidation of 368 internally stored fatty acids. 369 370 Figure 4. Glucose metabolism inhibits FAO. Parasites were incubated in the presence of 14C-U- 371 palmitate + 5 mM glucose and 14C-U-glucose + 0.1 mM palmitate in PBS. 14CO2 production from 372 epimastigotes incubated in PBS. The 14CO2 was captured after 120 min of incubation. The 373 experiments were performed in triplicates. Statistical analysis was performed with one-way ANOVA 374 followed by Tukey's post-test p < 0.05 using the GraphPad Prism 8.0.2 software program. We 375 represent the level of statistical significance in this figure as follows: *** p value < 0.001; ** p value 376 < 0.01; and * p value < 0.05. For p value > 0.05, we consider the differences not significant (ns). 377 378 To monitor the dynamics of use or accumulation of fatty acids in lipid droplets, we used as a 379 probe a fluorescent fatty acid analogue called BODIPY 500/510 C1-C12. BODIPY shifts its 380 fluorescence from red to green upon the uptake and catabolism of fatty acids, and from green to red 381 when fatty acids are accumulated in the lipid droplets. Parasites collected at the mid and late 382 exponential proliferation phases and the stationary phase were incubated with 1 μM BODIPY 383 500/510 C1-C12 for 16 h, before fluorescence determination by flow cytometry (Figs 5A, 5B and 5C). 384 The fluorescence values increased with the harvesting time (and therefore, with the glucose 385 depletion), indicating the increased uptake and use of fatty acids as substrates by a fatty acyl-CoA 386 synthetase. These data were confirmed by fluorescence microscopy (Fig 5D). Interestingly, parasites 387 in stationary phase showed an accumulation of activated fatty acids in spots along the cell. However, 388 the number of lipid droplets increased upon parasite proliferation (Figs 6A, 6B 6C). This observation .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 389 indicates that not only fatty acids metabolism is activated after glucose exhaustion, but also the 390 parasite storage of fatty acids into lipid droplets. 391 392 393 Figure 5. Flow cytometry reveals distinct patterns in fatty acid pools during epimastigote 394 growth. The epimastigotes were treated with 1 µM of BODIPY C1-C12 (500/510) and analysed by 395 flow cytometry and fluorescence microscopy. A) 0 h. B) 24 h. C) 48 h. In the flow cytometry 396 histograms, dashed peaks represent unstained parasites. Green-filled peaks represent stained 397 parasites. D) Mean fluorescence per cell. The fluorescence for each cell was calculated using ImageJ 398 software. All the experiments were performed in triplicates. Statistical analysis was performed with 399 one-way ANOVA followed by Tukey's post-test p < 0.05 using the GraphPad Prism 8.0.2 software 400 program. We represent the level of statistical significance in this figure as follows: *** p value < 401 0.001; ** p value < 0.01; and * p value < 0.05. For p value > 0.05, we consider the differences not 402 significant (ns). 403 404 405 Figure 6. Epimastigote forms accumulates fatty acids into lipid droplets during growth. The 406 epimastigotes were treated with 1 µM BODIPY C1-C12 (500/510) and analysed by flow cytometry 407 and fluorescence microscopy. A) 0 h. B) 24 h. C) 48 h. In the flow cytometry histograms, dashed 408 peaks represent unstained parasites. Yellow filled peaks represent positively stained parasites. The 409 number of green/yellow spots for each cell was calculated using ImageJ software. All the experiments 410 were performed in triplicates. 411 412 To find if the increase in fatty acid pools is accompanied by a change in the levels of enzymes 413 related to fatty acid metabolism, we evaluated the specific activities of the enzymes hexokinase (HK), 414 which is responsible for the initial step of glycolysis and an indicator of active glycolysis; acetyl-CoA 415 carboxylase (ACC), which produces malonyl-CoA for fatty acid synthesis and carnitine 416 palmitoyltransferase 1 (CPT1), the complex that plays a central role in fatty acid oxidation (FAO) by 417 controlling the entrance of long-chain fatty acids into the mitochondria [30]. For the control, we 418 selected the enzyme serine palmitoyltransferase 1 (SPT1), a constitutively expressed protein in T. 419 cruzi [31] (Fig 7). The hexokinase activity diminished up to 30% with the progression of the 420 proliferation curve and the correlated depletion of glucose (Fig 7A). In addition, the ACC activity is 421 no more detectable in the stationary phase cells (Fig 7B). By contrast, the CPT1 activity is increased 422 by ~4-fold when the stationary phase is reached (Fig 7C), which confirms that fatty acid degradation 423 occurs in the absence of glucose. It is noteworthy that the high levels of ACC activity in the presence 424 of glucose supports the idea that under these conditions, fatty acids are probably synthesized instead 425 of being catabolized. As expected, SPT1 did not change during the analysed time frame (Fig 7D). 426 427 Figure 6. Activities of enzymes related to lipid and glucose metabolism during T. cruzi growth 428 curves. A) (HK) Hexokinase B) (ACC) acetyl-CoA carboxylase, C) (CPT1) carnitine- 429 palmitoyltransferase, and D) (SPT) serine palmitoyltransferase. All these activities were measured in .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 430 crude extracts from epimastigote forms at different moments of the growth curve. All the experiments 431 were performed in triplicates. Time course activities and controls shown in Fig S2. Statistical analysis 432 was performed with one-way ANOVA followed by Tukey's post-test at p < 0.05, using the GraphPad 433 Prism 8.0.2 software program. We represent the level of statistical significance in this figure as 434 follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p value > 0.05 we consider 435 the differences not significant (ns). 436 437 438 Etomoxir, a CPT1 inhibitor, affects T. cruzi proliferation and mitochondrial activity 439 To investigate the role of FAO in T. cruzi, we tested the effect of a well characterized inhibitor 440 of CPT1, etomoxir (ETO), on the proliferation of epimastigotes. Among the ETO concentrations 441 tested here (from 0.1 to 500 µM), only the higher concentration arrested parasite proliferation (Fig 442 8A). Importantly, the ETO effect was manifested when the parasites reached the late exponential 443 phase (a cell density of approximately 5x107 mL-1). This result is consistent with our previous 444 findings showing that FAO (and thus CPT1 activity) acquires an important role at this point in the 445 proliferation curve. To confirm that CPT1 is in fact a target of ETO in T. cruzi, we assayed the drug's 446 effect on the enzyme activity in free cell extracts. Our results showed that 500 µM ETO diminished 447 the CPT1 activity by almost 80% (Fig 8B). To confirm the interference of ETO with the beta- 448 oxidation of fatty acids, parasites incubated in PBS containing 14C-U-palmitate were treated with 500 449 µM ETO to compare their production of 14CO2 with that of the untreated controls. Palmitate-derived 450 CO2 production diminished by 80% in ETO-treated cells compared to untreated parasites (Fig 8C). 451 In addition, ETO treatment did not affect the metabolism of 14C-U-glucose or 14C-U-histidine, ruling 452 out a possible unspecific reaction of this drug with CoA-SH as described by [32]. Other compounds 453 described as FAO inhibitors were also tested, but none of them inhibited epimastigote proliferation 454 or 14CO2 production from 14C-U-palmitate (S3 Fig). In addition, the BODIPY cytometric analysis of 455 cells treated with 500 µM ETO showed a strong decrease in the CoA acylation levels (activation of 456 fatty acids) with respect to the untreated controls (Fig 8D), as confirmed by fluorescence microscopy 457 (Fig 8D). To reinforce the validation of ETO for further experiments, a set of controls are offered in 458 S3 Fig. Our preliminary conclusion is that ETO inhibited beta-oxidation by inhibiting CPT1, 459 confirming that the breakdown of fatty acids is important to proliferation progression in the absence 460 of glucose. 461 462 Figure 8. ETO inhibits CPT1 and interferes with cell proliferation in epimastigote forms. (A) 463 Proliferation of epimastigote forms in the presence of 0.1 to 500 µM ETO. For the positive control 464 of dead cells, a combination of antimycin (0.5 µM) and rotenone (60 µM) was used. (B) Inhibition of 465 CPT1 activity in crude extracts using 250 and 500 µM of ETO. C) 14CO2 capture from 14C-U- 466 palmitate oxidation. D) Flow cytometry analysis and fluorescence microscopy of epimastigote forms 467 treated (or not) with ETO. In the histograms, dashed peaks represent unstained parasites and green- 468 filled peaks represent parasites stained with BODIPY C1-C12. All the experiments were performed in .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 469 triplicates. Statistical analysis was performed with one-way ANOVA followed by Tukey's post-test 470 at p < 0.05 using the GraphPad Prism 8.0.2 software program. We represent the level of statistical 471 significance in this figure as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. 472 For p values > 0.05, we consider the differences not significant (ns). 473 474 Etomoxir treatment affects cell cycle progression 475 The metabolic interference of ETO diminished epimastigote proliferation; however, this 476 finding could be due to a decrease in the parasite proliferation rate or an increase in the death rate. 477 Therefore, we checked if this compound could induce cell death through programmed cell death 478 (PCD) or necrosis. PCD is characterized by biochemical and morphological events such as exposure 479 to phosphatidylserine, DNA fragmentation, decreases (or increases) in the ATP levels, and increases 480 in reactive oxygen species (ROS), among others [33]. The parasites were treated with 500 µM of 481 ETO for 5 days, followed by incubation with propidium iodide (PI) for cell membrane integrity 482 analysis and annexin-V FITC to evaluate the phosphatidylserine exposure. Parasites treated with ETO 483 showed negative results for necrosis or programmed cell death markers (Fig 9A), indicating that the 484 cell proliferation was arrested but cell viability was maintained. Because the multiplication rates 485 seemed to be diminished, we performed a cell cycle analysis. Noticeably, the treated parasites were 486 enriched in G1 (85.9%) with respect to non-treated cells (43.6%), suggesting that ETO prevented the 487 entry of epimastigotes into the S phase of the cell cycle (Fig 9B). Last, we noticed that after washing 488 out the ETO, the parasites recovered their proliferation at rates comparable to our untreated controls 489 (Figs 9C). 490 491 Figure 9. Analysis of extracellular phosphatidylserine exposure, membrane integrity and cell 492 cycle after ETO treatment. Parasites in the exponential growth phase were treated with 500 µM of 493 ETO for 5 days. (A) Following the incubation period, the parasites were labelled with propidium 494 iodide (PI) and annexin V-FITC (ANX) and analysed by flow cytometry. (B) The cell cycle was 495 assessed using PI staining. (C) Growth curves of epimastigote forms before and after removing the 496 treatment. All the experiments were performed in triplicates. Statistical analysis was performed with 497 one-way ANOVA followed by Tukey's post-test p < 0.05, using the GraphPad Prism 8.0.2 software 498 program. We represent the level of statistical significance in this figure as follows: *** p value < 499 0.001; ** p value < 0.01; and * p value < 0.05. For p values > 0.05, we consider the differences not 500 significant (ns). 501 502 Inhibition of FAO by ETO affects energy metabolism, impairing the consumption of 503 endogenous fatty acids 504 The evidence obtained to date suggests that parasites resist metabolic stress by mobilizing and 505 consuming stored fatty acids. Therefore, it is reasonable to hypothesize that ETO, which blocks the 506 mobilization of fatty acids into the mitochondria for oxidation, probably perturbs the ATP levels in .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 507 late-exponential or stationary phase cells. Parasites growing for 5 days under 500 µM ETO treatment 508 or no treatment were collected to evaluate the ability of parasites that were treated or not with ETO 509 to trigger oxygen consumption. The rates of O2 consumption corresponding to basal respiration were 510 measured in cells resuspended in MCR respiration buffer. We then measured the leak respiration by 511 inhibiting the ATP synthase with oligomycin A. Finally, to measure the maximum capacity of the 512 electron transport system (ETS), we used the uncoupler FCCP [21]. Our results demonstrate that 513 compared to no treatment, ETO treatment diminishes the rate of basal oxygen consumption, the leak 514 respiration and the ETS capacity. In general, respiratory rates diminished in parasites treated with 515 ETO when compared to the untreated ones. As expected, ETO treatment led to a 75% decrease in the 516 levels of total intracellular ATP compared to untreated parasites (Fig 10A). To complement this 517 result, because all these experiments were conducted in the complete absence of an oxidizable 518 external metabolite, our results show that the parasite is able to oxidize internal metabolites (Figs 10B 519 and 10C). Taking into account that treating parasites with ETO diminished the basal respiration rates 520 of these parasites by approximately one-half (Figs 10B and 10C), it is reasonable to conclude that a 521 relevant part of the respiration in the absence of external oxidisable metabolites is based on the 522 consumption of internal lipids. This is consistent with the confirmation that epimastigotes maintain 523 their viability in the presence of non-fatty acid carbon sources in the presence of ETO (S4 Fig). In 524 summary, these results confirm that ETO is interfering with ATP synthesis through oxidative 525 phosphorylation in epimastigote forms. 526 527 Figure 10. Effects of ETO on respiration and ATP production in epimastigote forms of T. cruzi. 528 (A) Oxygen consumption of epimastigote forms after normal growth in LIT medium. (B) Oxygen 529 consumption after ETO 500 μM treatment. Parasite growth in LIT medium with the compound until 530 the 5th day. In black, a time-course register of the concentration (pmols) of O2 in the respiration 531 chamber. In blue, negative of the concentration derivative (pmols) of O2 with respect to time (velocity 532 of O2 consumption in pmoles per second). The parasites were washed twice in PBS and kept in MRC 533 buffer at 28 °C during the assays (see Materials and Methods for more details). (C) The basal 534 respiration (initial oxygen flux values, MRC), respiration leak after the sequential addition of 0.5 535 µg/mL of oligomycin A (2 µg/mL), and electron transfer system (ETS) capacity after the sequential 536 addition of 0.5 µM FCCP (2 µM) were measured for each condition. (D) Intracellular levels of ATP 537 after treating with 500 µM ETO. The intracellular ATP content was assessed following incubation 538 with different energy substrates or not (PBS, negative control). The ATP concentration was 539 determined by luciferase assay and the data were adjusted by the number of cells. All the experiments 540 were performed in triplicates. Statistical analysis was performed with one-way ANOVA followed by 541 Tukey's post-test at p < 0.05 using GraphPad Prism 8.0.2 software. We represent the level of statistical 542 significance as follows: *** p value < 0.001; ** p value < 0.01; and * p value < 0.05. For p values > 543 0.05, we consider the differences not significant (ns). 544 545 Endogenous fatty acids contribute to long-term starvation resistance in epimastigote forms .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 546 As previously demonstrated, ETO interferes with the consumption of endogenous fatty acids, 547 and this impairment causes ATP depletion and cell cycle arrest. One intriguing characteristic of the 548 insect stages of T. cruzi is their resistance to starvation. To observe the importance of internal fatty 549 acids in this process, we incubated epimastigotes in PBS in the presence (or absence) of 500 μM ETO. 550 The mitochondrial activity of these cells was followed for 24 h with Alamar blue®. Our results 551 showed that the mitochondrial activity of the parasites in the presence of ETO was reduced by 31% 552 after 48 h of starvation, and 65% after 72 h of starvation (Fig. 11) compared to the controls (untreated 553 parasites). These data confirmed our hypothesis that the breakdown of accumulated fatty acids 554 partially contributes to the resistance of the parasite under severe starvation. 555 556 Figure 11. Internal fatty acid consumption contributes to parasite viability under severe 557 nutritional starvation. Viability of epimastigote forms after incubation in PBS with or without ETO. 558 The viability was assessed every 24 h using Alamar Blue®. Statistical analysis was performed with 559 one-way ANOVA followed by Tukey's post-test p < 0.05 using GraphPad Prism 8.0.2 software. We 560 represent the levels of statistical significance as follow: *** p value < 0.001, and for p values > 0.05, 561 we consider the differences not significant (ns). 562 563 Inhibition of CPT1 impairs metacyclogenesis 564 Considering that the FAO increases in the epimastigotes during the stationary phase, and that 565 differentiation into infective metacyclic trypomastigotes (metacyclogenesis) is triggered in the 566 stationary phase of epimastigote parasites, one might expect a possible relationship between the 567 consumption of fatty acids and metacyclogenesis. To approach this possibility, we initially compared 568 the CPT1 activity of stationary epimastigote forms before and after a 24 h incubation in the 569 differentiation medium TAU-3AAG. As observed, there is an increase in CPT1 activity after 570 submitting the parasites to the metacyclogenesis in vitro (Fig. 12A). Parasites were then submitted to 571 differentiation with TAU-3AAG medium in the presence of the probe BODIPY. The probe was 572 incorporated into lipid droplets, confirming that fatty acids metabolism was active during the 573 beginning of metacyclogenesis (Fig 12B). To address the importance of FAO during differentiation, 574 metacyclogenesis was induced in vitro on ETO-treated or untreated (control) parasites. ETO 575 treatment interfered with differentiation, diminishing the number of metacyclic forms present in the 576 culture (Fig 12C). In addition, this inhibition was dose-dependent, with an IC50 = + 32.96 µM (Fig 577 12D). Importantly, we ruled out that the variation found in the differentiation rates was due to a 578 selective death of treated epimastigotes, since their survival during this experiment in the presence or 579 absence of ETO (from 5 to 500 µM) was not significantly different (S5 Fig). Based on these data, we 580 could conclude that fatty acid oxidation, at the level of the CPT1, was also participating in the 581 regulation of metacyclogenesis. 582 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 583 Figure 12. ETO inhibits metacyclogenesis. A) CPT1 activity of epimastigote forms in stationary 584 phase and 24h after incubated in TAU-3AAG medium (for triggering metacyclogenesis). B) 585 Fluorescence microscopy of cells incubated in TAU-3AAG in the presence of BODIPY® 500-510 586 C1-C12. C) Effects of different ETO concentrations on metacyclogenesis. The differentiation was 587 evaluated by counting the cells in a Neubauer chamber each day for 6 days. This experiment was 588 performed in triplicate. D) Percentage of differentiation at the 5th day of differentiation. Inset: IC50 of 589 metacyclogenesis inhibition by ETO. The enzymatic activities were measured in duplicate. All the 590 other experiments were performed in triplicates. 591 592 593 Discussion 594 During the journey of T. cruzi inside the insect vector, the glucose levels decrease rapidly after 595 each blood meal [34], leaving the parasite exposed to an environment rich in amino and fatty acids in 596 the digestive tube of Rhodnius prolixus [35,36]. Because the digestive tract of triatomine insects 597 possesses a perimicrovillar membrane, which is composed primarily of lipids and is enriched by 598 glycoproteins [37], it has been speculated that its degradation could provide lipids for parasite 599 metabolism [38]. In this study, we showed that the insect stages of T. cruzi coordinate the activation 600 of fatty acid consumption with the metabolism of glucose. Our experiments corroborate early studies 601 about the relatively slow use of palmitate as an energy source by proliferating epimastigotes [39,40]. 602 In addition, our results shed light on the end product excretion by epimastigote forms during 603 incubation under starvation conditions, and during their recovery from starvation using glucose or 604 palmitate. First, we showed that non-starved and starved parasites recovered in the presence of 605 glucose, excreting succinate as their primary metabolic waste, as expected [41–43]. After 16 h of 606 nutritional starvation, the consumption of internal carbon sources produces acetate as the primary 607 end-product. In the presence of glucose after 16 h of starvation, we found that glucose-derived 608 carbons contribute to the excreted pools of acetate and lactate. Interestingly, palmitate metabolism 609 contributed to the increase in acetate production, followed by the production of alanine, pyruvate, 610 succinate and lactate. The unexpected production of alanine, pyruvate and lactate can be explained 611 by an increase in the TCA cycle activity, producing malate, which can be converted into pyruvate by 612 the decarboxylative reaction of the malic enzyme (ME) [44]. Pyruvate can be converted into alanine 613 through a transamination reaction by an alanine- [45], a tyrosine- [46] an aspartate aminotransferase 614 [47], or a reductive amination by an alanine dehydrogenase [48]. The excretion of lactate could be a 615 consequence of lactate dehydrogenase activity. However, it should be noted that this enzymatic 616 activity has not been observed to date. In relation to the succinate production, a relevant factor 617 favouring this process is the production of NADH by the third step of the beta-oxidation (3- 618 hydroxyacyl-CoA dehydrogenase). This NADH can be oxidized through the activity of NADH- .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 619 dependent mitochondrial fumarate reductase [49], which concomitantly converts NADH into NAD+ 620 and fumarate into succinate. This succinate can be excreted or re-used by the TCA cycle, and the 621 resulting NAD+ can be used as a cofactor for other enzymes. 622 As previously mentioned, it is well known that during the initial phase of proliferation, 623 epimastigotes preferentially consume glucose, and during the stationary phase, a metabolic switch 624 occurs towards the consumption of amino acids [8,10,42]. Our results show that this switch 625 constitutes a broader and more systemic metabolic reprogramming, which also includes FAO. We 626 detected this switch through changes in the enzymatic activities of key enzymes responsible for the 627 regulation of FAO, such as CPT1 and ACC, which have increased and decreased activities, 628 respectively, in the presence of glucose. Our findings showed that the inhibition of CPT1 affects the 629 late phase of proliferation of epimastigotes when the switch to FAO has already occurred. 630 An interesting question about T. cruzi epimastigotes is how they survive long periods of 631 starvation. Early data showed high respiration levels in epimastigotes incubated in the absence of 632 external oxidisable carbon sources. This oxygen consumption was attributed to the breakdown of 633 TAGs into free fatty acids and their further oxidation [50]. Here, we confirmed this finding by 634 inhibiting the internal fatty acid consumption, which in turn diminished the oxidative phosphorylation 635 activity, internal ATP levels and the total reductive activity of parasites under severe nutritional stress. 636 Even more notably, we showed that under these conditions, the lipids stored in lipid droplets [51,52] 637 are consumed. Unlike what has been observed in procyclic forms of T. brucei, in which the function 638 of lipid droplets is not clear [53], our results show that in T. cruzi, they are committed to epimastigote 639 survival under extreme metabolic stress. Of course, the contribution of other metabolic sources and 640 processes such as autophagy in coping with nutritional stress cannot be ruled out [54]. 641 Multiple metabolic factors has been involved in metacyclogenesis, such as the proline, aspartate, 642 glutamate [55], glutamine [17] and lipids present in the triatomine digestive tract [56]. Interestingly, 643 the occurrence of metacyclic trypomastigotes in culture leads to an increase in CO2 production from 644 labelled palmitate [39]. The ETO treatment inhibited metacyclogenesis in vitro, showing that the 645 consumption of internal fatty acids is important for cell differentiation. Consequently, we propose 646 that lipids are not only external signals of metacyclogenesis, as previously suggested [56], but they 647 also have a central role in the bioenergetics of metacyclogenesis. As in the oxidation of several amino 648 acids, the acetyl-CoA produced from beta-oxidation and probably the reduced cofactors resulting 649 from these processes are contributing to the mitochondrial ATP production necessary to support this 650 differentiation step. 651 In conclusion, fatty acids are important carbon sources for T. cruzi epimastigotes in the 652 absence of glucose. Palmitate can be taken up by the cells and fuel the TCA cycle by producing 653 acetyl-CoA, the oxidation of which generates CO2. However, in the absence of external carbon .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 654 sources, lipid droplets become the primary sources of fatty acids, helping the organism to survive 655 nutritional stress. Importantly, FAO supports endogenous respiration rates and ATP production and 656 powers metacyclogenesis. 657 658 659 Acknowledgements 660 We thank the Core Facility for Scientific Research at the University of Sao Paulo (CEFAP- 661 USP/FLUIR) for the flow cytometry analysis and Dr. Mauro Javier Veliz Cortez (Department of 662 Parasitology, ICB-USP) for the microscopy work. We thank the Core Facility for Scientific Research 663 at the University of Sao Paulo (CEFAP-USP/FLUIR) for the flow cytometry analysis and Dr. Mauro 664 Javier Veliz Cortez (Department of Parasitology, ICB-USP) for the microscopy support. 665 666 References 667 [1] WHO | Chagas disease (American trypanosomiasis), WHO. (2018). 668 https://www.who.int/chagas/en/ (accessed January 29, 2019). 669 [2] J.A. Perez-Molina, I. Molina, Chagas disease, Lancet. 391 (2018) 82–94. 670 https://doi.org/10.1016/S0140-6736(17)31612-4. 671 [3] R. de F.P. Melo, A.A. Guarneri, A.M. 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Patti, Identifying off-target 880 effects of etomoxir reveals that carnitine palmitoyltransferase I is essential for cancer cell 881 proliferation independent of β-oxidation, PLOS Biol. 16 (2018) e2003782. 882 https://doi.org/10.1371/journal.pbio.2003782. 883 [63] S.N. Rampersad, Multiple applications of Alamar Blue as an indicator of metabolic function 884 and cellular health in cell viability bioassays, Sensors (Basel). 12 (2012) 12347–60. 885 https://doi.org/10.3390/s120912347. 886 [64] J.J. Homsy, B. Granger, S.M. Krassner, Some factors inducing formation of metacyclic 887 stages of Trypanosoma cruzi, J. Protozool. 36 (1989) 150–3. 888 http://www.ncbi.nlm.nih.gov/pubmed/2657033 (accessed January 29, 2019). 889 890 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 891 Supporting information 892 S1 893 S1 Fig. 1H-NMR analysis of excreted end products from glucose and threonine metabolism. The 894 metabolic end products (succinate, acetate, alanine and lactate) excreted by the epimastigote cells that 895 were incubated after 6 h in PBS (A), PBS after 16 h of starvation without (B) or with D-[U-13C]- 896 glucose (C) or palmitate (D) were determined by 1H-NMR. Each spectrum corresponds to one 897 representative experiment from a set of at least 3. A part of each spectrum ranging from 0.5 ppm to 898 4 ppm is shown. The resonances were assigned as indicated: A12, acetate; A13, 13C-enriched acetate; 899 Al12, alanine; Al13, 13C-enriched alanine; G13, 13C-enriched glucose; L12, lactate; L13, 13C-enriched 900 lactate; P12, palmitate; S12, succinate; and S13, 13C-enriched succinate. 901 902 S2 903 904 S2 Fig. Time course activities of enzymes measured in this work. A) (ACC) acetyl-CoA 905 carboxylase, B) (CPT1) carnitine-palmitoyltransferase, and C) (SPT) serine palmitoyltransferase. All 906 the activities were measured in cell-free extracts of epimastigote forms at different moments of the 907 growth curve as indicated in the main text. All the measurements were performed in triplicates. 908 S3 909 910 To check if other well-known FAO inhibitors have the same effect on the proliferation of T. cruzi 911 epimastigotes, we performed the same assay as described in Materials and Methods by evaluating 912 different concentrations of valproic acid (AV) [57], trimetazidine [58,59] and β-hydroxybutyrate [60], 913 which are inhibitors of 3-ketothiolase. Because they did not affect the proliferation of the epimastigote 914 forms, we used the higher concentration evaluated in these assays to know if the compounds inhibit 915 FAO by 14CO2 trapping by using U-14C-palmitate as a substrate. As observed, none of these 916 compound inhibited the 14CO2 production from palmitate, confirming that they are not inhibiting FAO 917 in T. cruzi. 918 919 S3 Fig. Other FAO inhibitors did not affect cell proliferation and FAO in the epimastigote 920 forms. The compounds were evaluated at concentrations between 0.1 and 1000 µM. For positive 921 controls of dead cells, a combination of antimycin (0.5 µM) and rotenone (60 µM) were used. The 922 maximum concentration tested for these compounds does not diminish CO2 liberation from FAO. A) 923 Valproic Acid (AV). B) Trimetazidine (TMZ). C) β-hydroxybutyrate (βHOB). 924 925 S4 926 927 In this study, we showed that the epimastigote forms of T. cruzi present low sensitivity in response 928 to ETO treatment. Recently, some groups described off-target effects when ETO is used at 929 concentrations of up to 200 μM [61,62]. To validate ETO as an FAO inhibitor of T. cruzi, the parasites 930 were incubated for 24 h in PBS (negative control), 0.1 mM palmitate supplemented with BSA, 5.0 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ 931 mM histidine, 5 mM glucose, 0.1 mM carnitine and BSA without adding palmitate in the presence 932 (or not) of 500 μM ETO. The viability of these cells was inferred from the measured total reductive 933 activity using MTT assays (see Material and Methods section for more details). As expected, ETO 934 treatment did not affect the viability of cells incubated in glucose or histidine but did affect the 935 viability of the cells incubated with palmitate or carnitine. Surprisingly, we also observed an ETO 936 effect on parasites under metabolic stress, such as those incubated with PBS or BSA. This finding 937 could be explained by the fact that under metabolic stress, the parasite mobilizes and consumes its 938 internal lipids. 939 940 941 S4 Fig. ETO did not affect the viability of epimastigote forms in the presence of other carbon 942 sources. The viability of epimastigote forms after incubation with different carbon sources and 943 palmitate. The viability was assessed after 24 h using MTT. 944 945 S5 946 947 Because metacyclogenesis occurs in chemically defined conditions, we performed a viability assay 948 to define the maximum tolerated concentration that allows the parasites to survive under ETO 949 treatment. Stationary epimastigotes in TAU-3AAG media were treated with different concentrations 950 of ETO (range 5 to 500 μM) during 24 h. The viability of these cells was inferred by measuring the 951 total reductive activity using an Alamar blue assay [63]. Briefly, after 24 h in the presence or absence 952 of ETO, the cells were incubated with 0,125 μg.mL-1 of Alamar blue reagent in accordance with the 953 protocol by [17]. Under these conditions, the parasites were 10 times more sensitive to ETO 954 treatment, surviving when subjected to ETO concentrations between 5-50 µM (Fig. S3 A). This range 955 of concentrations used to treat the parasites was maintained in TAU-3AAG medium and to follow 956 the differentiation by daily counts, based on the percentage of metacyclic trypomastigotes collected 957 in culture supernatant. To confirm that the parasites were still alive after 5 days under differentiation, 958 we checked the viability of cells that were treated (or not, control) using the same assay. As shown 959 above (Figure S3 B), the parasites were viable under all the tested conditions. Considering that TAU- 960 3AAG contains glucose in its composition, we performed an in vitro metacyclogenesis using only 961 proline as a metabolic inducer [64]. As observed, even in the absence of glucose, ETO treatment 962 affects metacyclogenesis. 963 964 965 966 Fig S5. Viability of epimastigote forms subjected to metacyclogenesis under different ETO 967 concentrations. A) Cell viability under metacyclogenesis after 24 h of treatment with different ETO 968 concentrations. B) Cell viability under metacyclogenesis after 5 days in the presence of ETO. C) 969 Effect of ETO on the metacyclogenesis induced by proline. 970 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425864doi: bioRxiv preprint https://doi.org/10.1101/2021.01.08.425864 http://creativecommons.org/licenses/by/4.0/ .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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