INTRODUCTION
Catalase, one of the most efficient enzymes known, probably evolved when an oxygenated atmosphere first appeared on Earth, requiring organisms to handle and neutralize toxic oxygen radical by-products. This antioxidant enzyme, found in nearly all aerobic organisms, decomposes hydrogen peroxide (H2O2) into water and oxygen (Chelikani et al. Reference Chelikani, Fita and Loewen2004). H2O2 represents a two-electron reduction state of molecular oxygen and originates mainly from the enzymatic dismutation catalysed by superoxide dismutase (SOD) isoforms. Despite its low reactivity, H2O2 can easily diffuse across biological membranes and generate hydroxyl radicals (•OH), which can react with biomolecules and cause damage (Novo and Parola, Reference Novo and Parola2008; Winterbourn, Reference Winterbourn2008).
Surprisingly, a catalase homologous sequence has not been identified in Trypanosoma cruzi genome (El-Sayed et al. Reference El-Sayed, Myler, Bartholomeu, Nilsson, Aggarwal, Tran, Ghedin, Worthey, Delcher, Blandin, Westenberger, Caler, Cerqueira, Branche, Haas, Anupama, Arner, Aslund, Attipoe, Bontempi, Bringaud, Burton, Cadag, Campbell, Carrington, Crabtree, Darban, da Silveira, de Jong and Edwards2005). Trypanosoma cruzi is the etiologic agent of Chagas Disease, which is endemic in Latin America and is now getting disseminated to non-endemic areas due to human emigration (Hotez et al. Reference Hotez, Dumonteil, Woc-Colburn, Serpa, Bezek, Edwards, Hallmark, Musselwhite, Flink and Bottazzi2012). Given the particularities of its complex life cycle, this parasite is certainly required to deal with situations of oxidative stress (Piacenza et al. Reference Piacenza, Alvarez, Peluffo and Radi2009a ). In fact, hydroperoxide antioxidant defences in T. cruzi consist of a sophisticated system of linked pathways in which reducing equivalents from NADPH – mostly derived from the pentose phosphate pathway – are delivered to a variety of enzymatic detoxification systems through the dithiol trypanothione [T(SH)2, N1, N8-bisglutathionylspermidine] and the thioredoxin homologue tryparedoxin (TXN) or glutathione (Irigoín et al. Reference Irigoín, Cibils, Comini, Wilkinson, Flohé and Radi2008).
Five distinct peroxidases have been identified in T. cruzi, which differ in their subcellular location and substrate specificity. Two of these, namely cytosolic (cTXNPx) and mitochondrial (mTXNPx) tryparedoxin peroxidases, detoxify H2O2, peroxynitrite (Piñeyro et al. Reference Piñeyro, Arcari, Robello, Radi and Trujillo2011), and small-chain organic hydroperoxides (Wilkinson et al. Reference Wilkinson, Temperton, Mondragon and Kelly2000). Another peroxidase, the ascorbate-dependent haeme-peroxidase (APX), is located in endoplasmic reticulum and confers resistance against H2O2 challenge, using ascorbate as reducing substrate. Indeed, parasites overexpressing APX display an increased resistance to exogenous H2O2 (Wilkinson et al. Reference Wilkinson, Obado, Mauricio and Kelly2002a ). Finally, the two remaining peroxidases show extensive similarity to glutathione-dependent peroxidases: Glutathione-dependent peroxidase I (GPXI) is present in both cytosol and glycosome, and the glutathione-dependent peroxidase II (GPXI) is found in the endoplasmic reticulum. GPXI and GPXII seem to play specific roles in cellular oxidative defence since both are able to detoxify fatty acid and phospholipid hydroperoxides, although they do not exhibit activity against H2O2 (Wilkinson et al. Reference Wilkinson, Meyer, Taylor, Bromley, Miles and Kelly2002b , Reference Wilkinson, Taylor, Touitha, Mauricio, Meyer and Kelly c ). In addition, T. cruzi harbours a repertoire of four iron superoxide dismutase (Fe-SODs) – which are located in different subcellular compartments – to detoxify O2 •− (Villagrán et al. Reference Villagrán, Marín, Rodríguez-González, De Diego and Sánchez-Moreno2005; Mateo et al. Reference Mateo, Marín, Pérez-Cordón and Sánchez-Moreno2008).
In order to establish infection in the vertebrate host, T. cruzi metacyclic trypomastigotes must invade macrophages and overcome the highly oxidative conditions generated inside the phagosome. Several antioxidant enzymes, including the mitochondrial Fe-SOD, mTXNPx, cTXNPx, trypanothione synthetase and APX, are upregulated during the transformation of the insect-derived, non-infective epimastigotes into the infective metacyclic trypomastigotes. These biochemical changes may pre-adapt metacyclic forms, allowing them to develop the ability of detoxify reactive oxygen and nitrogen species (ROS and RNS, respectively) generated by macrophages during the T. cruzi-mammalian host–cell interactions (Atwood et al. Reference Atwood, Weatherly, Minning, Bundy, Cavola, Opperdoes, Orlando and Tarleton2005; Piacenza et al. Reference Piacenza, Zago, Peluffo, Alvarez, Basombrio and Radi2009b ).
Given the importance of T. cruzi antioxidant system regarding the infection process, and also considering that kinetoplastids which parasitize insects do have catalase, the lack of this antioxidant enzyme in the aetiologic agent of Chagas Disease is puzzling. In this work, we generated a T. cruzi cell line expressing catalase from Escherichia coli, and investigated how this transfected parasite responds to oxidative stress. We found that catalase heterologous expression in T. cruzi affects both the oxidative stress-induced signalling and the parasite fitness in different oxidant environments.
MATERIALS AND METHODS
Ethics statement
This study was conducted in strict accordance with recommendations found in Guide for Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation (http://www.cobea.org.br/) and in Federal Law 11.794 (8 October 2008). All animals were handled in absolute conformity with good animal practice as defined by the Internal Ethics Committee in Animal Experimentation (CETEA) from Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil (protocol number 214/11). This project was also approved by the National Technical Biosafety Commission (CTNBio) under the process number 01200.003883/97-02.
Plasmids
In order to generate a T. cruzi cell line that stably expresses catalase, the gene katE from E. coli (GenBank: M55161.1) was inserted into the integrative expression vector pROCK_HYGRO (DaRocha et al. Reference DaRocha, Silva, Bartholomeu, Pires, Freitas, Macedo, Vazquez, Levin and Teixeira2004). katE encodes catalase – also known as hydroperoxidase II (HPII) –, which is very similar to eukaryotic catalases (Von Ossowski et al. Reference Von Ossowski, Mulvey, Leco, Borys and Loewen1991). katE sequence was amplified through polymerase chain reaction (PCR) using the following primers: 5′– TCT AGA ATG TCG CAA CAT AAC GAA AAG AAC C–3′ (forward) and 5′–CTC GAG TCA GGC AGG AAT TTT GTC AAT CTT AG –3′ (reverse), and cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). The resulting pGEM-T_katE plasmid was then digested using XbaI and XhoI to release double-digested katE fragment, which was then ligated to pROCK_HYGRO – previously incubated with the same restriction enzymes – to allow the generation of pROCK_katE_HYGRO vector.
Parasite growth and transfection
Trypanosoma cruzi strain CL Brener was used for all experiments. Epimastigotes were grown in liver infusion tryptose (LIT) medium (pH 7·4) supplemented with 10% fetal bovine serum (FBS, GibcoBRL, Invitrogen, Carlsbad, CA, USA) and 1% streptomycin/penicillin, at 28 °C. Parasite transfection was performed using electroporation as described elsewhere (DaRocha et al. Reference DaRocha, Silva, Bartholomeu, Pires, Freitas, Macedo, Vazquez, Levin and Teixeira2004). Transfected parasites were cultured for 6 weeks in the presence of hygromycin (200 µg mL−1, Sigma-Aldrich, St. Louis, MO, USA) to select parasites having pROCK_katE_HYGRO stably incorporated. Cells overexpressing MTH, or expressing heterologous MutT, were previously generated by our group (Aguiar et al. Reference Aguiar, Furtado, Repolês, Ribeiro, Mendes, Peloso, Gadelha, Macedo, Franco, Pena, Teixeira, Vieira, Guarneri, Andrade and Machado2013). Cells were cultured until logarithmic growth phase before all experiments. Trypomastigotes were obtained from the supernatant from monolayers of infected LLCMK2 cell cultures growth in 2% FBS, 1% penicillin/streptomycin and 2 mm L-glutamine-supplemented Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich). Released trypomastigotes were purified by centrifuging culture supernatants at 2000 g , for 5 min. The undisturbed pellet was incubated for 2 h at 37 °C, and motile trypomastigotes were collected from the supernatant.
RNA purification and RT–PCR
Total RNA was purified from 1 × 108 T. cruzi epimastigotes using the TRIzol (Invitrogen, Life technologies, CA, USA) method, followed by the treatment with DNAse (Invitrogen) for removal of contaminant DNA, according to the manufacturer's instructions. Purified RNA was then used to perform a cDNA synthesis reaction with 500 ng oligo (dT), using the SuperScript III First-strand Synthesis System for RT–PCR (Invitrogen). The subsequent specific amplification of katE fragment was performed using the primers previously described. Negative control was processed in the same conditions as for the other samples, except for the absence of reverse transcriptase.
Total peroxidase activity
Total peroxidase activity was measured in the supernatant of 2 × 107 parasites lysed in 50 µL of ice-cold 50 mm NaCl, 20 mm Tris–HCl (pH 7·4) containing 1% Triton X-100. The activity was determined after 30 min of incubation with 2 µ m Amplex Red (Invitrogen) and 2 mm H2O2, at 25 °C (Augusto et al. Reference Augusto, Moretti, Ramos, de Jesus, Zhang, Castilho and Schenkman2015), using the UV–Vis–fluorescent microplate reader SpectraMax M3 Reader® (Molecular Devices, Sunnyvale, CA, USA), operating at 530 nm (excitation) and 590 nm (emission).
Parasite cloning
Trypanosoma cruzi trypomastigotes transfected with pROCK_katE_HYGRO were grown in solid blood agar medium [48·4% LIT, 0·75% low melting agarose and 2·5% defibrinated rabbit blood, and inactivated complement (Gomes et al. Reference Gomes, Araujo and Chiari1991)] supplemented with 200 µg mL−1 hygromycin (Sigma-Aldrich). After 30 days of selection at 28 °C, plates were examined, and six clones were selected from different plates, which were grown in liquid medium for further characterization.
Epimastigote growth and survival curves
Parasite growth curves were initiated at the density of 1 × 107 cells mL−1. In a daily basis, aliquots were withdrawn and cells were counted until the stationary phase was reached. To test the resistance to H2O2, parasite cultures containing 1 × 107 cells mL−1 were treated with 75 or 100 µ m H2O2 in phosphate-buffered saline (PBS; pH 7·4). After 20 min, parasites were centrifuged and resuspended in LIT medium. The number of cells was determined after 72 h. Alternatively, survival curves were performed by incubating the cells in the presence of 25 µ m H2O2 – i.e. H2O2-preconditioning – for 24 or 96 h before subsequent treatment with H2O2. Results were expressed as the percentage of growth in relation to control. In these experiments, the cell number was determined through a cytometry chamber using erythrosine vital stain to differentiate living from dead cells. Three independent experiments were performed in triplicate.
H2O2 detoxyfication
Hydrogen peroxide detoxification was determined by incubating 1 × 107 parasites mL−1 in PBS (pH 7·4), in the presence of 1 U mL−1 horseradish peroxidase and 2 µ m AmplexRed (Invitrogen). After 30 min, parasites were centrifuged and the resulting fluorescence was measured in the supernatants using the SpectraMax M3 Reader® (Molecular Devices), operating at 530 nm (excitation) and 590 nm (emission). Three independent experiments were carried out in triplicates.
Preparation of antibodies, protein extracts and Western blotting analysis
Anti-T. cruzi iron superoxide dismutase B (anti-Fe-SOD B) and anti-T. cruzi trypanothione reductase (anti-TR) polyclonal antibodies were obtained as previously described (Piñeyro et al. Reference Piñeyro, Parodi-Talice, Arcari and Robello2008; Peloso et al. Reference Peloso, Gonçalves, Silva, Ribeiro, Piñeyro, Robello and Gadelha2012). For Western blot analysis, parasites (1 × 108 cells mL−1) were incubated in the absence or in the presence of H2O2 (100 µ m, PBS) for 30 min. Cells were then harvested by centrifugation (2500 g , 10 min), resuspended in 80 µL of PBS containing 1 mm MgCl2, and mixed with an equal volume of lysis buffer [50 mm Tris–HCl (pH 7·4), 1% Tween 20, 150 mm NaCl, 1 mm EGTA, 1 mm Na3VO4, 1 mm NaF, 0·1 mm PMSF, aprotinin 1 mg mL−1; leupeptin 1 mg mL−1]. The resulting suspension was sonicated in a Sonopuls Ultrasonic Homogenizer (Bandelin, Berlin, Germany) for 10 cycles of 1 s, with an interval of 1 s and 30% max amperage. The material was kept for 2 h on ice, and then centrifuged (13 000 g , 4 °C, 15 min). Protein concentration was determined using the Bradford method (Bradford, Reference Bradford1976). An equal volume of loading buffer was added to the protein extract [100 mm Tris–HCl, (pH 6·8), 4% SDS, 0·02% bromophenol blue, 20% glycerol, 200 mm β-mercaptoethanol], and samples were heated at 96 °C for 4 min. Protein extracts (30 µg) were analysed by SDS–PAGE and electroblotted onto a polyvinylidene difluoride membrane using the Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (BioRad, Irvine, CA, USA). Membranes were blocked by incubation with 5% instant non-fat dry milk in PBS containing 0·05% Tween 20 (PBS-T) for 1 h, washed and incubated for 2 h in the presence of antibodies. After washing the membranes with PBS-T (3 × 10 min each), incubation with HRP-linked anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) was carried out at room temperature, for 1 h, at the end of which membranes were washed with PBS (3 × 10 min each) (Morales et al. Reference Morales, Mogi, Mineki, Takashima, Mineki, Hirawake, Sakamoto, Omura and Kita2009; Peloso et al. Reference Peloso, Vitor, Ribeiro, Piñeyro, Robello and Gadelha2011). Bands were revealed using ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences, Issaquah, WA, USA). Data were obtained from three independent experiments, analysed using the ImageJ software (National Institute of Health, Bethesda, MD, USA), and normalized by anti-tubulin signal (T9026, Sigma-Aldrich), used as loading control.
In vitro infection
Four to 8 weeks old male and female C57BL/6 mice were obtained from the CEBIO (Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil), kept in controlled dark–light cycle and temperature conditions, and fed ad libitum with a commercial diet for rodents (Labina, Purina, SP, Brazil). Peritoneal macrophages were isolated 4 days after the injection of 2 mL of thioglycollate 3% medium (BD, Le Pont de Claix, France) into the peritoneal cavity. Mice were then euthanized, and the peritoneal cells were harvested by repeated cycles of aspiration and re-injection with 10 mL of cold PBS using a 10 mL-syringe with a 24 G needle. More than 80% of cells harvested were macrophages. Cells were centrifuged at 1500 g for 10 min at 4 °C, and resuspended in DMEM supplemented with 10% FBS (Cultilab, Campinas, SP, Brazil), 1% penicillin/streptomycin and 2 mm glutamine. Macrophages were counted in a hemocytometer prior to seeding 5 × 105 cells into each well from a 24-well plate and incubated with 5% CO2 at 37 °C, for 2 h. Purified parasites were diluted in DMEM medium, and infection was performed for 2 h. In some experiments catalase–polyethylene glycol (CAT, 40 U well−1, Sigma-Aldrich) was added to the cells 2 h before the infection. Immediately after macrophage infection, cells were washed four times with PBS (pH 7·4) to remove extracellular parasites. Cells were reincubated with medium for 72 h before fixation with methanol. Coverslips with attached macrophages were stained with Panótico stain (Laborclin, Pinhais, PR, Brazil), and a minimum of 300 macrophages per coverslip was counted. Results were expressed as infection index (% infected macrophages × number of amastigotes/total number of macrophages) and the values correspond to means ± s.d. of three independent experiments.
Mouse infection
Trypanosoma cruzi trypomastigotes were maintained by blood passage in IFN-γ KO mice (Dalton et al. Reference Dalton, Pitts-Meek, Keshav, Figari, Bradley and Stewart1993) every 7 days, and obtained from heparinized blood, counted, and used to infect naïve animals. Experimental infection was performed by injection of 1 × 106 trypomastigotes into the peritoneal cavity. Parasitaemia was assessed by counting the trypomastigotes in 5 µL of tail vein blood from the third day post infection until parasites became undetectable. The number of parasites per mL of blood was calculated as previously described (Brener, Reference Brener1962).
Insect infection
Fifth-stage nymphs of Rhodnius prolixus were reared at 26 ± 1 °C and relative humidity of 65 ± 5%, with natural illumination. They were fed on chicken and mice anesthetized with an intraperitoneal injection of a ketamine (150 mg kg−1; Cristália, Itapira, SP, Brazil)/xylazine (10 mg kg−1; Bayer, São Paulo, SP, Brazil) mixture. Experimental infections were carried out using uninfected insects, which were artificially fed with heat-inactivated rabbit blood containing 1 × 107 epimastigotes mL−1 (Garcia et al., Reference Garcia, Macarini, Garcia and Ubatuba1975). Control insects were fed on the same heat-inactivated rabbit blood at the same conditions, except for the parasite presence (Elliot et al. Reference Elliot, Rodrigues, Lorenzo, Martins-Filho and Guarneri2015). In order to obtain the rectum, each infected triatomine was dissected 30 days after feeding with blood containing parasites. Samples were homogenized in PBS and examined by direct microscopic observation. Trypanosoma cruzi population density in the intestine was quantified using a Neubauer chamber. Three independent experiments were performed.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software Inc., CA, USA). Data are presented as mean ± s.d.. Results were analysed for significant differences using either ANOVA or Student's t-test, as indicated in figures legends. Minimum significance level was set at P < 0·05.
RESULTS
Catalase-expressing T. cruzi cell line
In order to better understand how T. cruzi handles oxidative stress, and the reasons why this parasite lacks catalase, we generated a T. cruzi cell line, which stably maintains the expression of catalase from E. coli (KatE), which is encoded by the gene katE. After effectively cloning katE into the expression vector pROCK_HYGRO, we transfected and selected a T. cruzi clone capable of growing in the presence of hygromycin. Then, aiming to verify if the selected T. cruzi cell line was indeed able to transcript katE, we isolated total RNA from both transfected (T. cruzikatE ) and untransfected (T. cruzi WT) epimastigotes, and performed a RT–PCR. The expected 2262-bp DNA fragment of katE was successfully amplified from the cDNA of T. cruzikatE (Fig. 1A, T. cruzikatE /+RT); in addition, the expression of KatE in T. cruzikatE population, as well as in clones #1 and #2, was further confirmed by Western blotting (Fig. 1B, lanes 2–4). We then sought to verify whether or not KatE was active in the parasite environment through the measurement of total peroxidase activity present in the supernatant of T. cruzikatE whole cell lysates. We then were able to verify that transfected parasites exhibited significant higher levels of peroxidase activity when compared with untransfected ones, demonstrating that KatE is biologically active in T. cruzi (Fig. 1C).
Expression of katE in T. cruzi. (A) RT–PCR products obtained from total RNA extracted from T. cruzi WT or T. cruzikatE , with (+RT) or without (+RT) reverse transcriptase; (B) Catalase blot in extracts from T. cruzi WT, from a selected population (T. cruzikatE pop.), and from two clones of T. cruzi katE epimastigotes (T. cruzi katE clone #1 and T. cruzi katE clone #2). Tubulin was used as loading control; (C) Total peroxidase activity from soluble extracts of 2 × 107 T. cruzi WT or T. cruzi katE parasites. Data are presented as mean ± s.d.. **P < 0·01 vs T. cruzi WT (Unpaired Student's t-test, n = 3).
T. cruzi katE exhibit similar growth rates to T. cruzi WT, but shows increased resistance to H2O2
Next, we determined the in vitro growth rate of T. cruzikatE epimastigotes in order to verify if the expression of katE could exert an impact in cellular replication. We found that such expression did not change the parasite in vitro proliferation rates under standard culture conditions as T. cruzikatE exhibits a similar growth rate to T. cruzi WT (Fig. 2A). However, we observed that T. cruzikatE does present increased survival rates when compared with those from T. cruzi WT upon exposure to increased H2O2 concentrations (Fig. 2B). This indicates that parasites expressing katE are more resistant to oxidative damage induced by H2O2 than those ones in which this enzyme is not found.
KatE promotes increased resistance to H2O2. (A) T. cruzi WT and T. cruzi katE epimastigotes were grown in LIT medium, and the number of cells was determined every day until parasites reach the stationary phase. No significant difference was found between the cells (unpaired Student's t-test, n = 3); (B) T. cruzi WT and T. cruzi katE epimastigotes were incubated with or without 75, 100 and 125 µ m H2O2 for 20 min in PBS, centrifuged, and resuspended in LIT medium. After 72 h at 28 °C, the percentage of live cells was determined. Data are presented as mean ± s.d.. *P < 0·05 and ***P < 0·001 vs T. cruzi WT (Unpaired Student's t-test, n = 3).
Expression of catalase abolishes H2O2-mediated resistance in T. cruzi
Previous studies have shown that wild-type T. cruzi cells treated with sub-lethal concentrations of H2O2 become preconditioned, and present an increased resistance to higher concentrations of this ROS, showing that H2O2 is capable of mediating improved resistance against oxidant insults in this parasite (Finzi et al. Reference Finzi, Chiavegatto, Corat, Lopez, Cabrera, Mielniczki-Pereira, Colli, Alves and Gadelha2004). In this sense, we sought to determine if the expression of katE would abolish the adaptation mediated by H2O2 in T. cruzi, since this enzyme decomposes this mediator molecule into water and oxygen. First, we verified that, under our conditions, 25 µ m H2O2 acts as a sub-lethal concentration capable of preconditioning and increasing survival rates in T. cruzi WT and T. cruzikatE further exposed to both 75 and 100 µ m H2O2 (Fig. 3A). Next, we observed that, when the oxidative challenge was carried out with 75 µ m H2O2, the presence of KatE did not prevent increased survival rates in H2O2-preconditoned T. cruzikatE when compared with those observed in H2O2-preconditoned T. cruzi WT (Fig. 3A, 75 µ m H2O2, +H2O2 pretreat., T. cruzikatE vs T. cruzi WT). However, when the oxidative challenge was conducted with 100 µ m H2O2, H2O2-preconditoned T. cruzikatE parasites exhibited a similar survival rate when compared with H2O2-preconditoned T. cruzi WT cells (Fig. 3A, 100 µ m H2O2, +H2O2 pretreat., T. cruzikatE vs T. cruzi WT), despite having elevated acquired ability in handling oxidative insults (Fig. 1C). This observation demonstrates the existence of an oxidative condition in which H2O2-preconditoned T. cruzi WT is able to present the same level of survival of H2O2-preconditoned T. cruzikatE , suggesting a concentration-dependent role of H2O2 in cellular adaptation of wild-type T. cruzi in response to oxidative stress. Then, in order to confirm that preconditioning with H2O2 is able to induce an adaptive advantage, and not simply acts selecting the most resistant cells to oxidation, we decided to pretreat T. cruzi cells with 25 µ m H2O2 and challenge the cells (with either 75 or 100 µ m H2O2) 24 or 96 h after the final of pretreatment. We then observed that, after 24 h, the ratios between preconditioned and non-preconditioned alive cells was higher in T. cruzi WT when compared with cells expressing katE (Fig. 3B, 24 h), showing that the absence of catalase increases the cellular capacity of T. cruzi to adapt to oxidant insults. We further verified that, after 96 h, T. cruzi WT cells were unable to exhibit the adaptive advantage verified in earlier times after H2O2 pretreatment (Fig. 3, open bars). Altogether, these results demonstrate that the presence of katE impairs the adaptive response triggered by oxidative stress in T. cruzi, and the oxidative signalling promoted by H2O2 is transient as viability assessment after 96 h did not exhibit increased survival rates observed in T. cruzi WT after 24 h (Fig. 3B).
KatE expression impairs T. cruzi's adaptation to oxidative stress. (A) T. cruziWT and T. cruzikatE epimastigotes preconditioned or not with 25 µm H2O2 for 24 h were challenged with 75 or 100 µ m H2O2 for 20 min in PBS (pH 7·4), and then were allowed to grow in LIT medium. After 72 h, cell viability was determined. Percentages of survival were determined in relation to non-preconditioned cells. Data are presented as mean ± s.d.. **P < 0·005 and ***P < 0·001; ns = no statistical significance (one-way ANOVA/Bonferroni, n = 3); (B) T. cruziWT and T. cruzikatE epimastigotes were preconditioned or not with 25 µ m H2O2 for either 24 or 96 h before being exposed to higher concentrations of H2O2 for 20 min. Cells were plated, and after 72 h, viability was determined. Results represent the ratio between the number of preconditioned and non-preconditioned parasites. Data are presented as mean ± s.d.. **P < 0·01, ***P < 0·001 and ****P < 0·0001 (One-way ANOVA/Bonferroni, n = 3).
KatE impairs T. cruzi's ability in maintaining basal H2O2 levels
Since both resistance and adaptation to oxidative stress generated by H2O2 differ between T. cruzi WT and T. cruzikatE (Figs 2B and 3), we decided to check whether or not these dissimilarities could be related to changes in cellular ability to decompose H2O2. We then assessed the capacity of transfected and untransfected parasites in detoxifying H2O2 by measuring the amount of H2O2 present in the systems after challenging intact cells, with this oxidant. We found that both non-H2O2-treated T. cruzi WT and T. cruzikatE showed similar residual H2O2 levels (Fig. 4). However, T. cruzikatE exhibited increased levels of residual H2O2 after the oxidant challenge compared with T. cruzi WT (Fig. 4, T. cruzikatE /+H2O2 treatment vs T. cruzi WT/+H2O2 treatment). Interestingly, these results elicit the fact that katE expression not only fails to provide T. cruzi cells an additional ability to decompose H2O2, but also impairs the parasite capacity to detoxify H2O2 (Fig. 4). Altogether, these data suggest that cellular H2O2 are maintained at basal levels in T. cruzi (Fig. 4, empty bars; T. cruzikatE /−H2O2 treatment), possibly due to the physiological requirement of this ROS in adequate quantities to properly signal cellular adaptation when the parasite faces oxidative environments.
Maintenance of basal H2O2 levels in T. cruzi is hindered by katE expression. H2O2 levels were measured by incubating 2 × 107 T. cruzi WT and T. cruzikatE epimastigotes mL−1 in PBS in the presence of 1 U mL−1 horseradish peroxidase and 2 µ m Amplex Red for 30 min (−H2O2 treatment). Alternatively, parasites were treated with 100 µ m H2O2 for 20 min in PBS before the assay (+H2O2 treatment). Parasites were centrifuged and the resulting fluorescence was measured from supernatants using a fluorescence spectrophotometer operating at 530 nm (excitation) and 590 nm (emission). Data are presented as mean ± s.d.. ***P < 0·001; ns = no statistical significance (One-way ANOVA/Bonferroni, n = 3).
Catalase expression in T. cruzi alters the levels of antioxidant enzymes
The unexpected finding that katE expression hinders T. cruzi’s ability in maintaining physiological levels of H2O2 (Fig. 4), led us to investigate if other enzymes involved in antioxidant defence could be involved in the decreased capacity in detoxifying this oxidant. We then sought to determine the levels of Fe-SOD B and trypanothione reductase (TR) in both transfected and untransfected T. cruzi cells, challenged or not with 100 µm H2O2 for 20 min. We verified that, when there is no exposure to H2O2, T. cruzikatE showed increased Fe-SOD B levels in relation to T. cruzi WT (Fig. 5A and 5B, T. cruzikatE /−H2O2 treatment vs T. cruzi WT/−H2O2 treatment). Interestingly, we also observed that challenge with H2O2 increased Fe-SOD B levels in T. cruzi WT parasites in relation to wild-type unchallenged cells (Fig. 5B, open bars), but did not alter the levels of this enzyme in T. cruzikatE (Fig. 5B, solid bars). Moreover, we verified that unchallenged T. cruzi katE exhibited a reduced expression of TR when compared with unchallenged T. cruzi WT (Fig. 5C, T. c ruzikatE /−H2O2 treatment vs T. cruzi WT/−H2O2 treatment), indicating that katE expression in T. cruzi decreases TR levels in this parasite. Interestingly, while H2O2-challenged T. cruzi WT cells showed a tendency to exhibit increased TR levels (Fig. 5C, empty bars), H2O2-challenged T. cruzikatE parasites did exhibit reduced levels of this enzyme (Fig. 5C, solid bars), indicating that the presence of KatE in fact interferes in proper antioxidant signalling in T. cruzi.
KatE expression increases Fe-SOD B and reduces trypanothione reductase levels in T. cruzi. (A) Western blots of extracts obtained from T. cruzi WT and T. cruzi katE with or without 100 µ m H2O2 treatment for 20 min. Blots were probed with anti-iron superoxide dismutase B (SOD B), trypanothione reductase (TR), or anti-α-tubulin antibodies and a similar gel stained with Coomassie Brilliant blue. In (B) and (C), each band had its signal normalized by the T.cruzi WT/−H2O2 band (set as 100). Data are presented as mean ± s.d.. For (B) and (C), *P < 0·05, ***P < 0·001 and ****P < 0·0001; ns = no statistical significance (One-way ANOVA/Bonferroni, n = 3).
The role of catalase in the in vitro infection of macrophages by T. cruzi
We showed that oxidative signalling, which is required to prepare T. cruzi parasites to deal with host conditions, is impaired by the presence of KatE (Figs 3 and 4). Therefore, we asked if the expression of KatE could, in addition, interfere with both infectivity and growth of T. cruzi inside macrophages. In fact, several studies have associated T. cruzi-induced oxidative stress during macrophage infection with stimulation of T. cruzi infection (Nogueira and Cohn, Reference Nogueira and Cohn1978; Nathan et al. Reference Nathan, Nogueira, Juangbhanich, Ellis and Cohn1979; Nogueira et al. Reference Nogueira, de Souza, de Souza Saraiva, Sultano, Dalmau, Bruno, Gonçalves, Laranja, Leal, Coelho, Masuda, Oliveira and Paes2011; Paiva et al. Reference Paiva, Feijó, Dutra, Carneiro, Freitas, Alves, Mesquita, Fortes, Figueiredo, Fantappiè, Lannes-Vieira and Bozza2012; Nogueira et al. Reference Nogueira, Saraiva, Sultano, Cunha, Laranja, Justo, Sabino, Coelho, Rossini, Atella and Paes2015;). Thus, we used T. cruzi WT and T. cruzikatE trypomastigotes to infect macrophages previously treated – or not – with commercially-available catalase in order to suppress the cellular availability of H2O2, since this ROS has proven to be a molecular mediator which triggers antioxidant signalling in T. cruzi. Therefore, we promoted infection using T. cruzi harbouring the gene mutT from E. coli (T. cruzimutT ), which encodes MutT, a protein previously shown to increase T. cruzi resistance to oxidative stress (Aguiar et al. Reference Aguiar, Furtado, Repolês, Ribeiro, Mendes, Peloso, Gadelha, Macedo, Franco, Pena, Teixeira, Vieira, Guarneri, Andrade and Machado2013). MutT – or MTH1 in T. cruzi – hydrolyses 8-oxo-dGTP in the nucleotide pool, converting it to the monophosphate form so that it cannot be incorporated into DNA by polymerases (Nakabeppu et al. Reference Nakabeppu, Kajitani, Sakamoto, Yamaguchi and Tsuchimoto2006; Setoyama et al. Reference Setoyama, Ito, Takagi and Sekiguchi2011). Interestingly, both T. cruzikatE and T. cruzimutT parasites display increased proliferation capacity when compared with T. cruzi WT in macrophages without catalase supplementation (Fig. 6A). Added-catalase decreases macrophage infection capacity in T. cruzimutT (T. cruzimutT + CAT vs T. cruzimutT −CAT; P = 0·006, unpaired Student's t-test) but does not alter T. cruzikatE infectivity capacity. Altogether, these observations demonstrate that T. cruziKatE parasites exhibit the maximum infection index amongst all the cells studied, i.e. T. cruzi WT, T. cruziKatE and T. cruzimutT (Fig. 6A); in effect, KatE plays a key role in increasing the infection by 300% in relation to the wild-type cell (Fig. 6A), in a manner independent of the presence of the oxidative mediator molecule as supplementation with commercially available catalase does not alter the infection index (T. cruzi katE/+CAT vs T. cruzikatE /−CAT, P = 0.971, unpaired Student's t-test). However, as discussed above, the same supplementation plays a crucial role in decreasing the infection index of T. cruzi expressing MutT (Fig. 6, T. cruzimutT /+CAT vs T. cruzimutT /−CAT), eliciting the fact that parasite infectivity is modulated by MutT in a manner dependent on the presence of oxidants. Since MutT expression is able, per se, to promote a two-fold increase in T. cruzi’s infectivity (Fig. 6A, T. cruzimutT vs T. cruzi WT), we conclude that oxidative environment of infected macrophages increases T. cruzi infectivity through the parasite's ability to properly signal antioxidant response in consequence of oxidative stress.
Treatment with catalase reduces the proliferation of T. cruzimutT in macrophages. (A) Inflammatory macrophages obtained from the peritoneal cavity of C57BL/6 mice were incubated with T. cruzi bloodstream trypomastigotes – T. cruzi WT, T. cruzikatE or T. cruzi mutT; (B) Alternatively, macrophages were treated with catalase-polyethylene glycol (CAT) 2 h before the infection (+CAT). Cells were washed to remove extracellular parasites and fixed after 72 h. Slides were stained and counted to determine the number of parasites per macrophage. A minimum of 200 macrophages were counted per group. Data are presented as mean ± s.d.. *P < 0·05 and ***P < 0·001 (One-way ANOVA/ Bonferroni, n = 3).
T. cruzi katE exhibits increased parasitaemia compared with T. cruzi WT
Once determined that T. cruzikatE exhibits an increased macrophages infectivity in a manner independent of the presence of the mediator molecule H2O2, as well as ability to trigger antioxidant response through MutT plays a key role in the macrophage infection process, we sought to examine the T. cruzi WT, T. cruzikatE and T. cruziMTH – a T. cruzi cell line overexpressing MTH, the T. cruzi homologue for E. coli’s MutT – abilities in infecting mice. Trypomastigotes from wild-type T. cruzi and from the two aforementioned variants were injected into the peritoneal cavity of 4–8 weeks-old mice. We then observed that T. cruzikatE cells showed higher parasitaemia levels in relation to T. cruzi WT parasites on day 4 (Fig. 7), and that T. cruziMTH cells exhibited the highest parasitaemia levels among all T. cruzi cells studied on days 3–5 (Fig. 7). Interestingly, as previously discussed, overexpression of MTH in T. cruzi is related to an increased resistance to oxidative stress similarly to MutT (Aguiar et al. Reference Aguiar, Furtado, Repolês, Ribeiro, Mendes, Peloso, Gadelha, Macedo, Franco, Pena, Teixeira, Vieira, Guarneri, Andrade and Machado2013). Altogether, these results show that capacity to mediate oxidative signalling modulates T. cruzi’s ability to parasitize its vertebrate host.
Mice infected with T. cruzikatE show a reduced parasitaemia in relation to T. cruziMTH -infected mice. C57BL/6 mice were infected via intraperitoneal route with 106 T. cruzi blood stream trypomastigotes (T. cruzi WT, T. cruzikatE and T. cruziMTH ). Parasitsemia was assessed by counting the parasites in the tail vein blood of the infected mice. Data are presented as mean ± s.d.. *P < 0·05, **P < 0·01 and ****P < 0·0001 vs T. cruzi WT; ++++ P < 0·0001 vs T. cruzikatE (two-way ANOVA/ Bonferroni, n = 5).
KatE increases T. cruzi proliferation in the invertebrate host intestine
After studying the behaviour of T. cruzikatE and T. cruziMTH cells in the vertebrate host, we went on to verify the ability of these cells in infecting the invertebrate host. Then, fifth-stage nymphs of R. prolixus were infected with either T. cruzi WT or T. cruzikatE cells. Thirty days after infection, the number of parasites found in the intestines of the invertebrate host was higher when the infection had been carried out with T. cruzikatE cells (Fig. 8). This result indicates that acquired antioxidant ability increases T. cruzi’s infectivity in R. prolixus.
KatE increases T. cruzi proliferation in the invertebrate host intestine. Fifth-stage nymphs of R. prolixus were fed with heat-inactivated rabbit blood containing 107 T. cruzi epimastigotes mL−1. Thirty days after infection, intestines were obtained through insects dissection, and parasites were quantified using a Neubauer chamber. Data are presented as mean ± s.d.. *P < 0·05 vs T. cruzi WT (unpaired Student's t-test, n = 3).
DISCUSSION
ROS produced in vivo used to be regarded almost exclusively as deleterious molecules for eukaryotic cells (Balaban et al. Reference Balaban, Nemoto and Finkel2005; Wallace, Reference Wallace2005). However, it has been becoming plausible that O2 •− and H2O2 can act as critical intermediates in cellular signalling pathways (Buetler, Reference Buetler2004; Hamanaka and Chandel, Reference Hamanaka and Chandel2010). During normal cell growth, a tight balance between these two ROS species is maintained by cellular antioxidant systems, whose impairment generates a pro-oxidant environment (Mittra and Andrews, Reference Mittra and Andrews2013). In fact, H2O2 may act as a messenger molecule since its stability and membrane diffusibility present selective reactivity towards cysteine residues, which provides an advantage with regard signaling capacity. The best characterized mechanism by which H2O2 acts as a signalling molecule is the oxidation of critical cysteine residues within redox-sensitive proteins (D'Autréaux and Toledano, Reference D'Autréaux and Toledano2007). It has been shown that mammalian cells produce H2O2 to mediate diverse physiological responses such as cellular proliferation, differentiation and migration (Sundaresan et al. Reference Sundaresan, Yu, Ferrans, Irani and Finkel1995; Rhee et al. Reference Rhee, Bae, Lee and Kwon2000). Besides that, high levels of H2O2 are involved in the activation of signalling pathways to induce proliferation, migration, and invasion in cancer cells, which correlates with low levels of catalase expression (Picardo et al. Reference Picardo, Grammatico, Roccella, Roccella, Grandinetti, Porto and Passi1996; Subapriya et al. Reference Subapriya, Kumaraguruparan, Ramachandran and Nagini2002; Yoo et al. Reference Yoo, Song, Cho, Lee, Park, Yu, Lim, Kim and Jeon2008; Sen et al. Reference Sen, Kawahara and Chaudhuri2012).
The antioxidant system of T. cruzi has been considered a target for chemotherapeutic approaches to treat Chagas disease due to its importance regarding the adaptation towards the oxidative environment to which this parasite gets exposed during its life cycle (Krauth-Siegel et al. Reference Krauth-Siegel, Bauer and Schirmer2005; Irigoín et al. Reference Irigoín, Cibils, Comini, Wilkinson, Flohé and Radi2008). In order to cope with oxidative stress and establish infection in hosts, T. cruzi exhibits an efficient and peculiar antioxidant machinery (Piacenza et al. Reference Piacenza, Alvarez, Peluffo and Radi2009a ); remarkably, an intriguing fact is that T. cruzi antioxidant system does not include catalase (El-Sayed et al. Reference El-Sayed, Myler, Bartholomeu, Nilsson, Aggarwal, Tran, Ghedin, Worthey, Delcher, Blandin, Westenberger, Caler, Cerqueira, Branche, Haas, Anupama, Arner, Aslund, Attipoe, Bontempi, Bringaud, Burton, Cadag, Campbell, Carrington, Crabtree, Darban, da Silveira, de Jong and Edwards2005), which is found in virtually all aerobic organisms. Aiming to better understand the absence of catalase in this parasite, we generated a T. cruzi cell line which stably expresses catalase from E. coli (KatE). Such approach led us to verify that cells expressing katE show increased resistance against oxidative stress generated by exposure to exogenous H2O2, and exhibit the same growth rate from untransfected cells (Fig. 2A). In fact, we previously found that T. cruzi either expressing E. coli’s MutT – an enzyme responsible for the removal of the oxidation product 7,8-dihydro-8-oxoguanine (8-oxoG) from the nucleotide pool –, or overexpressing the MutT homologue in T. cruzi, MTH, also exhibit increased resistance to H2O2, and same growth rate observed in T. cruzi wild-type cells (Aguiar et al. Reference Aguiar, Furtado, Repolês, Ribeiro, Mendes, Peloso, Gadelha, Macedo, Franco, Pena, Teixeira, Vieira, Guarneri, Andrade and Machado2013).
Previous works have shown that, at low concentrations, H2O2 acts both improving the proliferation and increasing the resistance against oxidative stress in T. cruzi (Finzi et al. Reference Finzi, Chiavegatto, Corat, Lopez, Cabrera, Mielniczki-Pereira, Colli, Alves and Gadelha2004). Similarly, Leishmasodnia chagasi promastigotes, when incubated with either sub-lethal concentrations of menadione (an O2 •− generator), or H2O2 itself, develops an increased resistance to H2O2 toxicity. It was also observed that a sub-lethal dose of menadione stimulates promastigotes to become more virulent in a BALB/c mouse model of leishmaniasis (Wilson et al. Reference Wilson, Andersen and Britigan1994). On top of that, another work involving Leishmania suggested that low concentrations of ROS, including H2O2, regulate proliferation and differentiation by modulating the activity of cellular targets by oxidation (Mittra and Andrews, Reference Mittra and Andrews2013) – this is also observed in bacteria (Demple and Halbrook, Reference Demple and Halbrook1983), yeast (Davies et al. Reference Davies, Lowry and Davies1995) and mammalian cells (Wiese et al. Reference Wiese, Pacifici and Davies1995). Altogether, these observations suggest that adaptation to oxidative stress ensues when cells are first submitted to low concentrations of H2O2 and are afterwards exposed to higher concentrations of the oxidative agent. In this work, our results show that a pretreatment with H2O2 for 24 h before exposure to higher H2O2 concentrations induces an adaptive response in T. cruzi (Fig. 3). Although these findings are in contrast with those reported by Finzi et al. (Reference Finzi, Chiavegatto, Corat, Lopez, Cabrera, Mielniczki-Pereira, Colli, Alves and Gadelha2004), we hypothesize that either the use of distinct T. cruzi strains, or the use of different methodologies, or even both, may explain the different outcomes observed.
The fact that the expression of KatE reduced the T. cruzi ability to adapt to low doses of H2O2 (Fig. 3A) suggests that the expression of this enzyme have an effect in the adaptation promoted by the pretreatment with oxidants. Besides that, we verified that the persistent oxidative signalling promoted by H2O2 is not effective in mediating increased cellular resistance against oxidative insults (Fig. 3B), suggesting that there is a gradual loss of adaptability, as observed in mammalian and yeast cells (Demple and Halbrook, Reference Demple and Halbrook1983; Wiese et al. Reference Wiese, Pacifici and Davies1995). In fact, our data also suggest that these observed differences concerning parasite resistance and adaptation to oxidative stress might be related to changes in the ability in detoxifying H2O2: T. cruzi cells expressing katE previously exposed to H2O2 exhibited increased levels of H2O2 after being challenged with this oxidant when compared with wild-type cells (Fig. 4), suggesting that katE expression impairs the cellular ability to cope with H2O2 toxicity.
Since KatE impaired the capacity of transfected parasites to overcome H2O2 toxicity, we decided to verify if other antioxidant enzymes were somehow affected by the expression of catalase. In fact, we found that Fe-SOD B levels are increased by heterologous expression of katE and pretreatment with H2O2 (Fig. 5). Strikingly, the presence of KatE prevents adaptation to oxidative stress in T. cruzi (Fig. 3). Interestingly, alterations in antioxidant enzymes levels were also found in cancer cells: Picardo et al. (Reference Picardo, Grammatico, Roccella, Roccella, Grandinetti, Porto and Passi1996) observed that the SOD levels are elevated in melanoma cells, whereas catalase levels are reduced. Besides that, catalase overexpression in a cancer breast model is able to decrease the tumour invasiveness, suggesting the overexpression of this enzyme could reduce the oxidative stress signalling, which may be important to determine tumour invasiveness (Goh et al. Reference Goh, Enns, Fatemie, Hopkins, Morton, Pettan-Brewer and Ladiges2011). Based on these findings, it has been suggested that tumour cells reduce their catalase levels to allow the H2O2 to signal an oxidative stress situation (Goh et al. Reference Goh, Enns, Fatemie, Hopkins, Morton, Pettan-Brewer and Ladiges2011).
We also observed that T. cruzi expressing katE exhibited a reduced level of TR (Fig. 5C), which is indirectly involved in the cellular redox metabolism. Additionally, wild-type parasites treated with H2O2 had their of TR levels increased in relation to untreated cells, while the same was not observed in cells expressing katE. The influence of H2O2 treatment on antioxidant enzymes involved in H2O2 metabolism was shown by Finzi et al. (Reference Finzi, Chiavegatto, Corat, Lopez, Cabrera, Mielniczki-Pereira, Colli, Alves and Gadelha2004), who observed that incubation of T. cruzi epimastigotes with H2O2 also increased the cTXNPx levels, which probably occurred to promote cell detoxification (Finzi et al. Reference Finzi, Chiavegatto, Corat, Lopez, Cabrera, Mielniczki-Pereira, Colli, Alves and Gadelha2004). Also, treatment of T. cruzi trypomastigotes with increasing concentrations of H2O2 is related to modulations of cTXNPx and mTXNPx levels in a dose-dependent way (Gadelha et al. Reference Gadelha, Gonçalves, Mattos, Alves, Piñeyro, Robello and Peloso2013). The fact that TR levels are increased after H2O2 exposure in wild-type parasites, but not in cells expressing katE, strongly suggests that catalase expression in T. cruzi changes the parasite ability to signal oxidative stress.
It is remarkable that wild-type T. cruzi is able to maintain the basal levels of H2O2 in situations of either excess or absence of this oxidant (Fig. 4). This observation suggests that there is a buffering system able to maintain a certain level of H2O2, and that such system is deregulated in the presence of catalase.
Infection of macrophages with T. cruzi induces oxidative stress, which stimulates parasite replication (Bergeron et al. Reference Bergeron, Blanchette, Rouleau and Olivier2008; Alvarez et al. Reference Alvarez, Peluffo, Piacenza and Radi2011; Paiva et al. Reference Paiva, Feijó, Dutra, Carneiro, Freitas, Alves, Mesquita, Fortes, Figueiredo, Fantappiè, Lannes-Vieira and Bozza2012). Here, we verified that expression of katE in T. cruzi increases parasitism in macrophages (Fig. 6A), and that treatment with exogenous catalase reduces MutT-expressing T. cruzi capacity to parasite these immune cells (Fig. 6B). In line with these data, it is known that treatment with antioxidants inhibits epimastigote proliferation in vitro (Nogueira et al. Reference Nogueira, de Souza, de Souza Saraiva, Sultano, Dalmau, Bruno, Gonçalves, Laranja, Leal, Coelho, Masuda, Oliveira and Paes2011), and reduces T. cruzi parasitaemia (Figueiredo et al. Reference Figueiredo, Rosa and Soares2000). Taken together, these findings corroborate the hypothesis that oxidant signalling is needed for T. cruzi proliferation in a manner prevented by the expression of catalase.
When in the intestine of R. prolixus, T. cruzi has to handle with a highly oxidative environment. In fact, blood digestion results in the release of extremely high concentrations of haeme, the prosthetic group of haemoglobin (Graça-Souza et al. Reference Graça-Souza, Maya-Monteiro, Paiva-Silva, Braz, Paes, Sorgine, Oliveira and Oliveira2006). Haeme toxicity is often related to ROS generation through Fenton reaction, promoting lipid, protein and DNA oxidations (Aft and Mueller, Reference Aft and Mueller1983; Gutteridge and Smith, Reference Gutteridge and Smith1988). Parasites expressing catalase could be more resistant to this environment by degrading H2O2. This fact, together with the finding that a group of monoxenous parasites, such as Crithidia fasciculata, Crithidia acanthocephali, Crithidia luciliae, Leptomonas pyrrhocoris, Leptomonas seymouri, Lotmaria passim, Novymonas esmeraldas (trypanosomatids which only infects insects), express catalase (Alcolea et al. Reference Alcolea, Alonso, Garcia-Tabares, Toraño and Larraga2014; Kraeva et al. Reference Kraeva, Horáková, Kostygov, Butenko, Yurchenko and Luke2016), suggest that this enzyme is very important to confer resistance to the oxidative stress in the invertebrate host. With regard to the vertebrate host, however, catalase expression might impair oxidative stress signalling triggered by H2O2 – it has been already shown that this molecule is found in reduced levels in this environment (Mueller et al. Reference Mueller, Riedel and Stremmel1997).
Phylogenetic hypotheses suggest that, during evolution, a free-living kinetoplastid ancestral may have been ingested by insects, and have been adapted itself to the intestinal habitat, originating the monoxenous trypanosomatids. When the haematophagy appeared in nature, it is possible that the parasites were inoculated into vertebrates. Thus, parasites which were adapted to this new environment started to alternate between insects and vertebrates, originating the dixenous trypanosomatids (Simpson et al. Reference Simpson, Simpson, Kidane, Livingston and Spithill1980, Reference Simpson, Stevens and Lukeš2006; Hamilton et al. Reference Hamilton, Stevens, Gaunt, Gidley and Gibson2004; Stevens, Reference Stevens2008). As postulated by Kraeva et al. (Reference Kraeva, Horáková, Kostygov, Butenko, Yurchenko and Luke2016), a group of monoxenous trypanosomatids received the catalase gene by horizontal transfer from some unknown bacterium. In this sense, it is possible that dixenous trypanosomatids, as T. cruzi, have never received this gene by horizontal transfer. Noteworthy is the fact that Leishmania does not have the catalase gene, even belonging to the catalase-positive branch phylogenetic tree (Kraeva et al. Reference Kraeva, Horáková, Kostygov, Butenko, Yurchenko and Luke2016); this finding reinforces the idea that the presence of catalase impairs oxidative signalling in dioxenous kinetoplastids (Kraeva et al. Reference Kraeva, Horáková, Kostygov, Butenko, Yurchenko and Luke2016). Catalase expression could impair the oxidative stress signal originated in an oxidant environment which is required for cellular differentiation, and, to some extent, allowed protection to the parasite against oxidative damage. In this context, our findings suggest that the absence of catalase gene could improve signalling capacity triggered by oxidative mediator molecules in T. cruzi.
ACKNOWLEDGEMENTS
We are grateful to Neuza Antunes Rodrigues for technical support. Authors have no conflict of interest to declare.
FINANCIAL SUPPORT
This work was supported by CNPq-Brazil (Universal and INCTV), PRONEX, Newton Fund/FAPEMIG and FAPESP.
