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• W2602442885 abstract "Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite with a complex life cycle involving a triatomine insect and mammals. Throughout its life cycle, the T. cruzi parasite faces several alternating events of cell division and cell differentiation in which exponential and stationary growth phases play key biological roles. It is well accepted that arrest of the cell division in the epimastigote stage, both in the midgut of the triatomine insect and in vitro, is required for metacyclogenesis, and it has been previously shown that the parasites change the expression profile of several proteins when entering this quiescent stage. However, little is known about the metabolic changes that epimastigotes undergo before they develop into the metacyclic trypomastigote stage. We applied targeted metabolomics to measure the metabolic intermediates in the most relevant pathways for energy metabolism and oxidative imbalance in exponentially growing and stationary growth-arrested epimastigote parasites. We show for the first time that T. cruzi epimastigotes transitioning from the exponential to the stationary phase exhibit a finely tuned adaptive metabolic mechanism that enables switching from glucose to amino acid consumption, which is more abundant in the stationary phase. This metabolic plasticity appears to be crucial for survival of the T. cruzi parasite in the myriad different environmental conditions to which it is exposed during its life cycle. Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite with a complex life cycle involving a triatomine insect and mammals. Throughout its life cycle, the T. cruzi parasite faces several alternating events of cell division and cell differentiation in which exponential and stationary growth phases play key biological roles. It is well accepted that arrest of the cell division in the epimastigote stage, both in the midgut of the triatomine insect and in vitro, is required for metacyclogenesis, and it has been previously shown that the parasites change the expression profile of several proteins when entering this quiescent stage. However, little is known about the metabolic changes that epimastigotes undergo before they develop into the metacyclic trypomastigote stage. We applied targeted metabolomics to measure the metabolic intermediates in the most relevant pathways for energy metabolism and oxidative imbalance in exponentially growing and stationary growth-arrested epimastigote parasites. We show for the first time that T. cruzi epimastigotes transitioning from the exponential to the stationary phase exhibit a finely tuned adaptive metabolic mechanism that enables switching from glucose to amino acid consumption, which is more abundant in the stationary phase. This metabolic plasticity appears to be crucial for survival of the T. cruzi parasite in the myriad different environmental conditions to which it is exposed during its life cycle. Cell growth, both in natural environments or in in vitro culture, usually presents in two phases: the exponential phase where cells divide at a roughly constant rate, and the stationary phase where cells slow down or stop the cell cycle and division. During the exponential phase, cells go through the well described canonical cell cycle of G1 → S → G2 → M. In the stationary phase, cells leave the regular cell cycle to enter a resting state known as quiescence, which is highly relevant because it corresponds to the physiological state in which most cells from both unicellular and multicellular organisms spend most of their life (1Gray J.V. Petsko G.A. Johnston G.C. Ringe D. Singer R.A. Werner-Washburne M. Sleeping beauty: quiescence in Saccharomyces cerevisiae.Microbiol. Mol. Biol. Rev. 2004; 68: 187-206Crossref PubMed Scopus (458) Google Scholar). Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite with a complex life cycle involving a triatomine insect and mammals. Throughout its life cycle, the T. cruzi parasite faces several alternated events of cell division and cell differentiation. Briefly, insects are infected during a blood meal from a mammal having non-dividing forms of the parasite, denominated trypomastigotes, present in their blood. Once in the midgut of the triatomine insect, the trypomastigotes differentiate to replicative non-infective epimastigotes, which proliferate and colonize the digestive tube. After the blood meal, nutrients are consumed by the intestinal epithelium, the parasite, and the intestinal microbiota population; therefore, the replicative epimastigotes eventually face nutrient starvation. Under this situation of metabolic stress, cell division is arrested, and the parasites adhere to the intestinal epithelium along the midgut. In the terminal portion of the digestive tube, epimastigotes start the process to differentiate to infective, non-dividing metacyclic trypomastigotes (2Brener Z. Life cycle of Trypanosoma cruzi.Rev. Inst. Med. Trop. Sao Paulo. 1971; 13: 171-178PubMed Google Scholar). Interestingly, this differentiation process, called metacyclogenesis, is fueled by amino acids present in the urine at the distal portion of the triatomine's intestine such as proline (Pro), aspartate (Asp), and glutamate (Glu), among others (3Barrett F.M. Friend W.G. Differences in the concentration of free amino acids in the haemolymph of adult male and female Rhodnius prolixus.Comp. Biochem. Physiol. B. 1975; 52: 427-431Crossref PubMed Scopus (15) Google Scholar). In a new blood meal on a mammalian host the infected triatomine, which usually defecates near the biting point, will contaminate the skin or mucosa with metacyclic trypomastigotes expelled along with their feces, allowing the parasite to internalize and establish infection in a new host (2Brener Z. Life cycle of Trypanosoma cruzi.Rev. Inst. Med. Trop. Sao Paulo. 1971; 13: 171-178PubMed Google Scholar). It is well accepted that arrest of the cell division in the epimastigote stage, both in vivo and in vitro, is required for metacyclogenesis, and it has been previously shown that the parasites change the expression profile of several proteins when entering this quiescent stage (4Hernández R. Cevallos A.M. Nepomuceno-Mejía T. López-Villaseñor I. Stationary phase in Trypanosoma cruzi epimastigotes as a preadaptive stage for metacyclogenesis.Parasitol. Res. 2012; 111: 509-514Crossref PubMed Scopus (16) Google Scholar). In addition, a previous work comparing exponential and stationary phases of T. cruzi epimastigotes (EPE 5The abbreviations used are: EPEexponential phase epimastigoteSPEstationary phase epimastigoteTCAtricarboxylic acidP5CΔ1-pyrroline-5-carboxylateP5CDHP5C dehydrogenaseTSHtrypanothioneRTPreverse trans-sulfuration pathwayMRMmultiple-reaction monitoringPCAprincipal component analysisBCAAbranched-chain amino acidγ-GSγ-glutamate semi-aldehydeCBScystathionine β-synthasePyrpyruvateα-KGα-ketoglutarateESIelectrospray ionization. and SPE, respectively) has assessed the physiological changes occurring at each stage and how some genes are regulated during the epimastigote growth to understand their possible participation in the metacyclogenesis (4Hernández R. Cevallos A.M. Nepomuceno-Mejía T. López-Villaseñor I. Stationary phase in Trypanosoma cruzi epimastigotes as a preadaptive stage for metacyclogenesis.Parasitol. Res. 2012; 111: 509-514Crossref PubMed Scopus (16) Google Scholar, 5de Godoy L.M. Marchini F.K. Pavoni D.P. Rampazzo Rde C. Probst C.M. Goldenberg S. Krieger M.A. Quantitative proteomics of Trypanosoma cruzi during metacyclogenesis.Proteomics. 2012; 12: 2694-2703Crossref PubMed Scopus (60) Google Scholar, 6Goldenberg S. Avila A.R. Aspects of Trypanosoma cruzi stage differentiation.Adv. Parasitol. 2011; 75: 285-305Crossref PubMed Scopus (47) Google Scholar). However, little is known about metabolic changes that occur along with this physiological transition, and there are no reported studies supporting a broader view of the adaptive changes that epimastigotes undergo before they develop into metacyclic trypomastigote stage. exponential phase epimastigote stationary phase epimastigote tricarboxylic acid Δ1-pyrroline-5-carboxylate P5C dehydrogenase trypanothione reverse trans-sulfuration pathway multiple-reaction monitoring principal component analysis branched-chain amino acid γ-glutamate semi-aldehyde cystathionine β-synthase pyruvate α-ketoglutarate electrospray ionization. Early studies have focused on specific enzymes involved in energy metabolism as well as a specific subset of metabolites (7Cazzulo J.J. Energy metabolism in Trypanosoma cruzi.Subcell. Biochem. 1992; 18: 235-257Crossref PubMed Scopus (18) Google Scholar). However, a systematic analysis of the metabolic changes associated with energy metabolism that occurs during exponential and stationary phases of growth in T. cruzi epimastigotes has not been performed. We hypothesized that T. cruzi epimastigotes finely orchestrate a metabolic switch, especially related to the energy metabolism, when exposed to nutritional starvation. Here, we show for the first time T. cruzi epimastigotes transitioning from exponential to stationary phase (Fig. 1) present a distinct metabolic profile regarding their energy metabolism and redox imbalance, including amino acids, carbohydrates, and the tricarboxylic acids (TCA) cycle intermediates. To assess the main metabolic changes between exponentially growing and stationary arrested epimastigote T. cruzi parasites, we focused on a set of 47 metabolic intermediates from the most relevant pathways for energy metabolism and oxidative imbalance. Selected metabolites (supplemental Table S1) were analyzed in positive and negative modes using two different chromatography separations. Our initial hypothesis was that EPE and SPE constitute two different populations in terms of energy metabolome composition due to their adaptation to different nutritional conditions. To assess this hypothesis, we performed a principal component analysis (PCA) where results are displayed as score plots, and each point represents a sample that when clustered together indicates similar metabolite composition based on the selected metabolites for the targeted analysis (Fig. 2). PCA analysis revealed a clear separation between the two growth phases of T. cruzi epimastigotes. This result strongly supports the proposed hypothesis that T. cruzi EPE and SPE present different energy and oxidative imbalance-related metabolic states. Interestingly, our results also revealed a greater metabolic heterogeneity among stationary phase cells than exponential phase cells. To further identify the underlying metabolites responsible for the segregation between both T. cruzi EPE and SPE, a heat map analysis was performed. In contrast to the scores plots, a heat map displays the actual data values that allow visualization of changing patterns in metabolite levels across samples and across experimental conditions. The heat map analysis showed that amino acids and their metabolic intermediates were increased in the stationary phase when compared with exponential phase, whereas glycolysis and TCA intermediates were reduced (Fig. 3 and supplemental Fig. 1). Consequently, we pursued a more detailed analysis for each metabolic pathway to assess specific changes that occur when parasites are exposed to nutritional starvation. To study in more detail the metabolic switch from a glucose-based to an amino acid-based metabolism in T. cruzi epimastigotes, we measured the relative levels of the first and last metabolites of glycolysis, i.e. glucose and pyruvate (Pyr), and all the intermediates of the TCA cycle, except oxaloacetate that could not be resolved by any of the two chromatographic methods used for this study (Fig. 4). As shown in the heat map (Fig. 3), both glycolysis and most of the TCA-related metabolite levels decreased during the transition of parasites from EPE to SPE. The relatively higher levels of glucose during the exponential phase as compared with the stationary phase could be explained by its uptake from the extracellular medium by the exponentially replicating cells due to its presence in high concentrations in the fresh medium (2 mg/ml). Notably, the intracellular concentration of glucose was still detected in SPE instead of being completely consumed as we would expect because extracellular glucose has been depleted at that stage (8Shaw A.K. Kalem M.C. Zimmer S.L. Mitochondrial gene expression is responsive to starvation stress and developmental transition in Trypanosoma cruzi.mSphere. 2016; 1e00051Crossref PubMed Scopus (15) Google Scholar). This finding may indicate that in SPE either amino acids are serving for de novo synthesis of glucose to maintain a certain intracellular level or its consumption is arrested. Alongside a decrease in the glucose content, the relative abundance of Pyr as well as the metabolic intermediates canonically linked to the TCA cycle, such as isocitrate, citrate, succinyl-CoA, and malate, were significantly reduced in SPE when compared with EPE (Fig. 4 and supplemental Table S2). By contrast, α-ketoglutarate (α-KG) was the only derivative from the amino acid oxidation that appeared to exhibit around six times higher abundance in the SPE. The increased levels of α-KG are compatible with an increase of amino acids catabolism in SPE (Fig. 5). T. cruzi epimastigotes depend on different catabolic routes leading to α-KG generation for energy production, such as the Pro (9Mantilla B.S. Paes L.S. Pral E.M. Martil D.E. Thiemann O.H. Fernández-Silva P. Bastos E.L. Silber A.M. Role of δ1-pyrroline-5-carboxylate-dehydrogenase supports mitochondrial metabolism and host-cell invasion of Trypanosoma cruzi.J. Biol. Chem. 2015; 290: 7767-7790Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 10Paes L.S. Suárez Mantilla B. Zimbres F.M. Pral E.M. Diogo de Melo P. Tahara E.B. Kowaltowski A.J. Elias M.C. Silber A.M. Proline dehydrogenase regulates redox state and respiratory metabolism in Trypanosoma cruzi.PLoS ONE. 2013; 8e69419Crossref PubMed Scopus (45) Google Scholar) or His (11Barison M.J. Damasceno F.S. Mantilla B.S. Silber A.M. The active transport of histidine and its role in ATP production in Trypanosoma cruzi.J. Bioenerg. Biomembr. 2016; 48: 437-449Crossref PubMed Scopus (23) Google Scholar) oxidation pathways, both rendering Glu that can be converted into α-KG through a reductive deamination or by transamination of the –NH2 to Pyr forming Ala (12Lisvane Silva P. Mantilla B.S. Barisón M.J. Wrenger C. Silber A.M. The uniqueness of the Trypanosoma cruzi mitochondrion: opportunities to identify new drug target for the treatment of Chagas disease.Curr. Pharm. Des. 2011; 17: 2074-2099Crossref PubMed Scopus (38) Google Scholar). It worth mentioning that several metabolites are present simultaneously in different subcellular compartments such as malate, which is present in the glycosome, cytoplasm, and mitochondrion (Fig. 4). Because we measured the total level of a metabolite, the detected changes in their levels are the result of all metabolic contributions independently of their localization. Glu and α-KG constitute the main metabolic bridge between amino acid metabolism and the TCA cycle. Interestingly, our results showed that the levels of Glu remained nearly constant in both growth phases. As shown in Fig. 5, it is worth stressing that Glu is a branching point for several metabolic pathways. Epimastigotes are able to acquire Glu through a variety of processes, which include its uptake from the extracellular medium, biosynthesis by reductive amination of α-KG catalyzed by glutamate dehydrogenase, which is NADH- and NADPH-dependent, transamination reactions, or by oxidizing other amino acids such as Pro, His, and glutamine (Gln). Notably, both Glu uptake and its precursors such as α-KG, Pro, His, Asp and its derivative asparagine were detected at higher levels in the SPE as compared with EPE (Fig. 5). In addition, the uptake of Pro and His was also increased in SPE. Because their main metabolic fate is Glu, our results suggest that the metabolism in SPE shifts toward Glu production and further oxidation in the TCA cycle through α-KG. However, it is also possible that the concomitant increase of Pro, His, and Glu uptake could lead to an accumulation of these precursors. In both cases, our findings are in agreement with the hypothesis that Glu or its 2-oxoacid derivative is critically involved in the parasite's survival in this phase. T. cruzi is the only trypanosomatid equipped with putative genes encoding the four enzymatic steps connecting His to Glu as follows: His ammonia-lyase, urocanate hydratase, imidazolone propionase, and formiminoglutamase. Recently, it was shown that this parasite can fully oxidize His, producing CO2 and triggering an increase of the mitochondrial membrane potential and O2 consumption (11Barison M.J. Damasceno F.S. Mantilla B.S. Silber A.M. The active transport of histidine and its role in ATP production in Trypanosoma cruzi.J. Bioenerg. Biomembr. 2016; 48: 437-449Crossref PubMed Scopus (23) Google Scholar). In addition, His is an essential amino acid in T. cruzi; therefore, its intracellular levels depend on both its uptake and degradation rates. Our results show that the intracellular levels of His and urocanate, the first metabolic intermediate in the His metabolism, were significantly increased in SPE as compared with EPE. Therefore, we measured His transport-specific activity in both EPE and SPE populations, and it was slightly increased in SPE when compared with EPE (Fig. 5 and supplemental Table S2). The small increase in His uptake could contribute to the increased intracellular levels of His and urocanate in SPE. Our results showed that the intracellular levels of Pro were significantly increased in SPE as compared with EPE (Fig. 5 and supplemental Table S2). Pro levels were shown to be critical in several biological processes in T. cruzi epimastigotes, such as differentiation, resistance to oxidative imbalance, as well as response to nutritional and thermal stress (10Paes L.S. Suárez Mantilla B. Zimbres F.M. Pral E.M. Diogo de Melo P. Tahara E.B. Kowaltowski A.J. Elias M.C. Silber A.M. Proline dehydrogenase regulates redox state and respiratory metabolism in Trypanosoma cruzi.PLoS ONE. 2013; 8e69419Crossref PubMed Scopus (45) Google Scholar, 13Contreras V.T. Salles J.M. Thomas N. Morel C.M. Goldenberg S. In vitro differentiation of Trypanosoma cruzi under chemically defined conditions.Mol. Biochem. Parasitol. 1985; 16: 315-327Crossref PubMed Scopus (333) Google Scholar, 14Tonelli R.R. Silber A.M. Almeida-de-Faria M. Hirata I.Y. Colli W. Alves M.J. l-Proline is essential for the intracellular differentiation of Trypanosoma cruzi.Cell. Microbiol. 2004; 6: 733-741Crossref PubMed Scopus (85) Google Scholar, 15Magdaleno A. Ahn I.Y. Paes L.S. Silber A.M. Actions of a proline analogue, l-thiazolidine-4-carboxylic acid (T4C), on Trypanosoma cruzi.PLoS ONE. 2009; 4e4534Crossref PubMed Scopus (55) Google Scholar, 16Sayé M. Miranda M.R. di Girolamo F. de los Milagros Cámara M. Pereira C.A. Proline modulates the Trypanosoma cruzi resistance to reactive oxygen species and drugs through a novel dl-proline transporter.PLoS ONE. 2014; 9e92028Crossref PubMed Scopus (35) Google Scholar). Differently from His, epimastigotes were able to synthesize Pro. Therefore, the balance of Pro levels is more complex than His and is determined by the interplay among its degradation, biosynthesis from Glu, and uptake rates through two active transporters. Similar to most organisms, Pro degradation to Glu occurs through two enzymatic steps where Pro is first converted into Δ1-pyrroline-5-carboxylate (P5C) by a Pro dehydrogenase with the production of FADH2 followed by conversion into Glu by P5C dehydrogenase (P5CDH) with the production of NADH (Fig. 5 and supplemental Table S2). Both enzymatic steps by themselves are able to energize the mitochondria, promoting the production of ATP without requiring the deamination of Glu and further full oxidation through the TCA cycle and oxidative phosphorylation to obtain energy. Differently from other organisms, this pathway is not branched because there is no functional ornithine aminotransferase in T. cruzi epimastigotes able to interconvert P5C and ornithine (12Lisvane Silva P. Mantilla B.S. Barisón M.J. Wrenger C. Silber A.M. The uniqueness of the Trypanosoma cruzi mitochondrion: opportunities to identify new drug target for the treatment of Chagas disease.Curr. Pharm. Des. 2011; 17: 2074-2099Crossref PubMed Scopus (38) Google Scholar, 17Silber A.M. Colli W. Ulrich H. Alves M.J. Pereira C.A. Amino acid metabolic routes in Trypanosoma cruzi: possible therapeutic targets against Chagas' disease.Curr. Drug. Targets Infect. Disord. 2005; 5: 53-64Crossref PubMed Scopus (63) Google Scholar). The lack of an ornithine aminotransferase interconverting ornithine and P5C indicates that all of the oxidized Pro have to be converted into P5C/γGS and Glu and that all the biosynthesized Pro have to be produced from Glu. Thus, the relative gene expression levels and specific enzymatic activities of P5CR (Pro biosynthesis) and P5CDH (Pro degradation) both in EPE and SPE could indicate the predominant direction of Pro metabolism (biosynthesis or degradation) in each phase (Fig. 6). Interestingly, both protein and enzymatic activity levels of P5CR were higher in the EPE than in the SPE, whereas the opposite was observed for P5CDH (Fig. 6, B and C). This suggests an up-regulation of the synthesis of Pro during the exponential phase, which could drive the metabolism to a pre-adaptive accumulation of this amino acid. It is worth mentioning that Pro can be consumed in SPE to support metacyclogenesis and can be used by metacyclic trypomastigotes to energize the invasion of the mammalian hosts (18Martins R.M. Covarrubias C. Rojas R.G. Silber A.M. Yoshida N. Use of l-proline and ATP production by Trypanosoma cruzi metacyclic forms as requirements for host cell invasion.Infect. Immun. 2009; 77: 3023-3032Crossref PubMed Scopus (57) Google Scholar). In fact, from all the selected metabolites analyzed in this study, Pro presented the highest levels, measured as metabolite/internal standard area ratio of MS signals detected by MRM normalized to the cell numbers, in both T. cruzi EPE and SPE (Fig. 5). Because we detected differences between Pro synthesis and degradation in EPE and SPE, we evaluated the contribution of its uptake in both growth phases. Previously, we described and characterized two Pro transport systems, designated A and B (19Silber A.M. Tonelli R.R. Martinelli M. Colli W. Alves M.J. Active transport of l-proline in Trypanosoma cruzi.J. Eukaryot. Microbiol. 2002; 49: 441-446Crossref PubMed Scopus (50) Google Scholar). Both systems had different kinetic and thermodynamic characteristics, showing substrate saturations at 0.75 mm (system A) and 3 mm (systems A + B). Thus, transport activities for both systems were measured at substrate-saturating concentrations. Interestingly, system A did not show variations in Pro uptake, but system B increased ∼3 times its activity (Fig. 5). The increase in the Pro uptake may be compensating for the simultaneous up-regulation of Pro consumption and down-regulation of Pro synthesis, avoiding a futile cycle, and could be responsible for Pro accumulation in SPE. Leucine (Leu), isoleucine (Ile), and valine (Val) are branched-chain amino acids (BCAA), and the only ones involved in the energy production in T. cruzi that are not directly connected to Glu (20Mancilla R. Naquira C. Lanas C. Protein biosynthesis in trypanosomidae. II. The metabolic fate of dl-leucine-1-C14 in Trypanosoma cruzi.Exp. Parasitol. 1967; 21: 154-159Crossref PubMed Scopus (16) Google Scholar). These amino acids are oxidized to acetyl- or methylmalonyl-CoA and further oxidized through the TCA cycle. Similar to most eukaryotic cells, BCAA are essential for T. cruzi. Therefore, their intracellular levels also depend on their uptake and metabolism. As described previously, all three BCAA are taken up by the same transporter (21Manchola N.C. Rapado L.N. Barisón M.J. Silber A.M. Biochemical characterization of branched chain amino acids uptake in Trypanosoma cruzi.J. Eukaryot. Microbiol. 2016; 63: 299-308Crossref PubMed Scopus (12) Google Scholar), and their specific transport activity did not change in SPE when compared with EPE (Fig. 5). The fact that the intracellular levels for Ile and Val are slightly reduced, while Leu remained constant in SPE compared with EPE, indicates that Ile and Val catabolism is more active than Leu during the stationary phase (Fig. 5). An important group of metabolites related to energy metabolism and defense against oxidative stress are polyamines and the thiol-containing amino acids. To assess potential variations of these thiol-containing metabolites between EPE and SPE, we measured the levels of intermediates involved in the different routes for cysteine (Cys) biosynthesis as well as potential variations in the levels of glutathione, spermidine, and trypanothione (TSH). The latter represents the main antioxidant agent in the redox metabolism in trypanosomatids. As shown in Fig. 7, the relative abundance of the intermediates belonging to the reverse trans-sulfuration pathway (RTP) (serine, homocysteine, and cystathionine) as well as those related to de novo synthesis of cysteine (serine and O-acetylserine) were increased in SPE as compared with EPE. These results strengthened previous findings showing that unlike most living organisms T. cruzi displays an unusual redundancy in cysteine production by the co-existence of de novo synthesis and the RTP. As schematized in Fig. 7, precursors such as serine, acetyl-CoA, and O-acetylserine are required for de novo synthesis of cysteine. This is a two-step pathway catalyzed by serine acetyltransferase and cysteine synthase, and both enzymes have been found to be functional in T. cruzi (22Nozaki T. Shigeta Y. Saito-Nakano Y. Imada M. Kruger W.D. Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine β-synthase and serine acetyltransferase from Trypanosoma.J. Biol. Chem. 2001; 276: 6516-6523Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 23Marciano D. Santana M. Nowicki C. Functional characterization of enzymes involved in cysteine biosynthesis and H(2)S production in Trypanosoma cruzi.Mol. Biochem. Parasitol. 2012; 185: 114-120Crossref PubMed Scopus (17) Google Scholar). Interestingly, cystathionine β-synthase (CBS), which catalyzes the first step of the RTP, is distinguished from its mammalian counterpart by its broader substrate specificity. In addition to catalyzing the condensation of serine with homocysteine to produce cystathionine, the T. cruzi CBS can also act as a serine sulfhydrylase and a cysteine synthase. Given that cystathionine γ-lyase is also functional in T. cruzi, cystathionine is expected to be also cleaved to produce cysteine, the end product of the RTP. In contrast, synthesis of TSH is known to be essential for neutralizing free radicals, thereby contributing to the redox homeostasis in all of the developmental stages of T. cruzi. However, our results unexpectedly showed that the availability of Glu and the higher abundance of cysteine in SPE were not reflected in an increase in the relative abundance of TSH in SPE (Fig. 7). It is possible that the synthesis of TSH is limited by the significantly lower abundance of spermidine in SPE, which is essential to accomplish the final steps in TSH biosynthesis. T. cruzi is the only eukaryotic organism unable to synthesize polyamines de novo because it lacks ornithine and arginine decarboxylases, the enzymes catalyzing the first step in polyamines biosynthesis (24Hunter K.J. Le Quesne S.A. Fairlamb A.H. Identification and biosynthesis of N1,N9-bis(glutathionyl)aminopropylcadaverine (homotrypanothione) in Trypanosoma cruzi.Eur. J. Biochem. 1994; 226: 1019-1027Crossref PubMed Scopus (72) Google Scholar, 25Carrillo C. Cejas S. González N.S. Algranati I.D. Trypanosoma cruzi epimastigotes lack ornithine decarboxylase but can express a foreign gene encoding this enzyme.FEBS Lett. 1999; 454: 192-196Crossref PubMed Scopus (64) Google Scholar). Interestingly, despite there being no evidence of the urea cycle in the parasite's genome, ornithine and citrulline were detected in both growth phases, and ornithine levels were around four times higher in SPE compared with EPE, whereas arginine and citrulline levels were similar between both phases (supplemental Fig. 1). Spermidine is involved in cell cycle control in trypanosomatids (26González N.S. Huber A. Algranati I.D. Spermidine is essential for normal proliferation of trypanosomatid protozoa.FEBS Lett. 2001; 508: 323-326Crossref PubMed Scopus (29) Google Scholar). As mentioned above, our results showed that the intracellular levels of spermidine were decreased in SPE compared with EPE (Fig. 7). This finding is in agreement with the fact that in SPE the cellular replication process, one of the most polyamines demanding cellular processes, is arrested. In this work, we applied a targeted metabolomic approach to assess the metabolic changes that occur in T. cruzi epimastigote parasites transitioning from the exponential to the stationary phase of growth. Herein, we established a correlation between the relative abundance of assessed metabolites with a metabolic process resulting from the nutritional conditions that parasites have to face during exponential and stationary growth. We carefully normalized the data, and the presented results have been obtained from independent biological replicates that also included new media preparation" @default.
• W2602442885 created "2017-04-07" @default.
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• W2602442885 date "2017-05-01" @default.
• W2602442885 modified "2023-10-12" @default.
• W2602442885 title "Metabolomic profiling reveals a finely tuned, starvation-induced metabolic switch in Trypanosoma cruzi epimastigotes" @default.
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