Parasitol Res DOI 10.1007/s00436-014-3950-5
ORIGINAL PAPER
Biochemical characterization and role of the proteasome in the oxidative stress response of adult Schistosoma mansoni worms Renato Graciano de Paula & Alice Maria de Magalhães Ornelas & Enyara Rezende Morais & William de Castro Borges & Massimo Natale & Lizandra Guidi Magalhães & Vanderlei Rodrigues
Received: 26 February 2014 / Accepted: 6 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The trematode Schistosoma mansoni, an important parasite of humans, is the principle agent of the disease schistosomiasis. In the human host, one of the most important stress factors of this parasite is the oxidative stress generated by both the metabolism of the worm and the immune system of the host. The proteasomal system is responsible for protein homeostasis during oxidative stress. The 26S proteasome is a multicatalytic protease formed by two compartments, a 20S core and regulatory particle 19S, and controls the degradation of intracellular proteins, hence regulating many cellular Electronic supplementary material The online version of this article (doi:10.1007/s00436-014-3950-5) contains supplementary material, which is available to authorized users. R. G. de Paula (*) : A. M. de Magalhães Ornelas : V. Rodrigues Faculdade de Medicina de Ribeirão Preto, Departamento de Bioquímica e Imunologia, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brazil e-mail: [email protected] E. R. Morais Instituto de Genética e Bioquímica, Campus Avançado Patos de Minas, Universidade Federal de Uberlândia, Avenida Getúlio Vargas, 230, Patos de Minas, Minas Gerais, Brazil W. de Castro Borges Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, s/n, Ouro Preto 35400-000, Minas Gerais, Brazil M. Natale Department of Control and Computer Engineering, Politecnico di Torino, Turin, Italy L. G. Magalhães Grupo de Pesquisa em Produtos Naturais, Centro de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Avenida Doutor Armando S. Oliveira, 201, Parque Universitário, Franca 14404-600, São Paulo, Brazil
processes. In the present report, we describe the biochemical characterization and role of the 20S proteasome in the response of adult S. mansoni worms exposed to hydrogen peroxide. Characterization of the response to the oxidative stress included the evaluation of viability, egg production, mortality, tegument integrity, and both expression and activity of proteasome. We observed decreases in viability, egg production as well as 100 % mortality at the higher concentrations of hydrogen peroxide tested. The main changes observed in the tegument of adult worms were peeling as well as the appearance of bubbles and a decrease of spines on the tubercles. Furthermore, there were increases in 26S activity to the same extent as 20S proteasome activity, although there was increase of 20S proteasome content, suggesting that degradation of protein oxidized in adult worms is due to the 20S proteasome. It was demonstrated that adult S. mansoni worms are sensitive to oxidative stress, and that a variety of processes in this parasite are altered under this condition. The work contributes to a better understanding of the mechanisms employed by S. mansoni to survive under oxidative stress. Keywords S. mansoni . Proteasome . Oxidative stress
Introduction Schistosoma mansoni is one of the etiologic agents of schistosomiasis, a severe disease that affects almost 240 million people worldwide, mainly in the tropical and subtropical areas, and more than 700 million people live under conditions where they are exposed to the risk of infection (WHO, 2013; Mcdonald et al. 2010). Praziquantel is currently the only drug available to treat the disease (Caffrey 2007). However, resistance of the parasite has been reported, and juvenile worms are
Parasitol Res
able to evade the action of the drug (Ismail et al. 1999). An understanding of the biology of the parasite is therefore vital in order to develop new therapeutic techniques (Soliman and Ibrahim 2005). In the course of its lifecycle, the parasite is exposed to a variety of stresses, which can be either physical or chemical in nature. The free-living stages are subjected to threats that include chemical pollutants present in the aquatic environment, variations in temperature, and solar radiation (Prah and James 1977; Coelho and Bezerra 2006; Ruelas et al. 2007). In adult worms, the most important stress factor is probably the reactive oxygen species (ROS) released by cells of the host immune system during aerobic metabolism. Consistent with this hypothesis, in infection-resistant snails, the miracidium is rapidly eliminated by a process that results in an increased rate of oxygen consumption, which is indicative of a role of oxidative stress in the defense mechanism of the snails (Hahn et al. 2001). Further evidence is provided by high levels of antioxidant enzyme transcription in adult worms (Mei and Loverde 1997), suggesting the need to adapt to an intense exposure to ROS during this stage of the lifecycle. Adult worms can live for up to 30 years in the definitive host organism, and must therefore possess antioxidant mechanisms in order to preserve the integrity of their cells and survive bursts of oxidative activity (Vieira et al. 2007). ROS are released from S. mansoni and its hosts during the course of cellular processes including aerobic respiration, immune defense, and other oxidative pathways (Kazura et al. 1981). Hydrogen peroxide and its subproducts are probably the most important ROS in biological systems, and directly affect different groups of biomolecules found in cells, such as lipids, proteins, and nucleic acids (Kastle and Grune 2012; Corcoran and Cotter 2013). Many studies have demonstrated that the susceptibility to oxidative stress depends on the stage in the lifecycle, with the schistosomulae being more sensitive than adult worms (Hong et al. 1992). Oxidatively-modified proteins are continuously produced in cells due to the action of reactive oxygen species generated as a consequence of aerobic metabolism. The main changes that occur during oxidative stress are the formation of protein carbonyls and the breaking of disulfide bonds, which renders impossible the maintenance of the native protein conformation (Kastle and Grune 2011). The proteasome represents the main cytoplasmic proteolytic machinery for the preferential degradation of damaged rather than normal proteins (Dudek et al. 2005; Friguet 2006), and is responsible for the maintenance of protein homeostasis during oxidative stress (Mayer 2010; Toivola et al. 2010). The 20S proteasome particle is the main agent responsible for oxidized protein degradation (Grune et al. 2011), and the evidence suggests that the process is ubiquitin-free (Shringarpure et al. 2003). Other studies have demonstrated that oxidized proteins are degraded by the 20S proteasome, independent of the 19S regulator or ubiquitin (Yamanaka et al.
2003; Pickering et al. 2010). Shringarpure et al. (2003) demonstrated that disruption of the ubiquitination system did not impair the degradation of oxidized proteins, while Wang et al. (2010) and Kriegenburg et al. (2011) reported that the components of the ubiquitin-proteasome system are highly sensitive to oxidative stress, which could result in inhibition of the ubiquitination protein or impairment of the 26S complex. Nevertheless, the role of the proteasome in oxidized protein degradation in S. mansoni remains unclear. In order to improve understanding of the different pathways involved in the response to oxidative damage in this parasite, the aim of the present work was to identify the main changes that occur in adult S. mansoni worms subjected to oxidative stress. The effects included changes in viability, egg production, and tegument morphology. In addition, increases in both the content and activity of the 20S proteasome were observed. Materials and methods Ethics statement All experiments involving animals were authorized by the Animal Research Ethics Commission (CETEA) of the University of São Paulo (protocol number 182/2011) and abided by the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation (COBEA). These were in accordance with accepted national and international norms for laboratory animal welfare. Parasites and oxidative stress treatment The Luis Evangelista (LE) strain of S. mansoni was maintained by successive passages through Biomphalaria glabrata snails and BALB/c mice. After 56 days, adult S. mansoni worms were recovered under aseptic conditions from mice that had been previously infected with 200 cercariae by perfusion of the livers and mesenteric veins (Smithers and Terry 1965). The paired adult worms were maintained in RPMI 1640 medium (Invitrogen) supplemented with penicillin (100 UI/mL), streptomycin (100 μg/mL), and 10 % bovine fetal serum (Gibco), at 37 ºC with 5 % CO2, under dark conditions. After 24 h of incubation, oxidative stress was induced by the addition of H2O2 to final concentrations in the range 100–5,000 μM, and the worms were incubated for 0 (non-exposed control), 0.5, 1, 4, and 24, and 120 h, at 37 ºC with 5 % CO2. After incubation, either subsequent experiments were performed or the worms were frozen in liquid nitrogen. In vitro studies with S. mansoni After the oxidative stress treatment, the parasites were monitored at 24 and 120 h to evaluate their general condition, including alterations in the tegument, couples separated,
Parasitol Res
tegumental changes, and mortality rate (Xiao et al. 2007). At the same time, changes in pairing and egg production were examined using an inverted microscope (Leitz) (Magalhães et al. 2009; Manneck et al. 2010). The worms were considered dead when no movement was observed over at least 2 min. All experiments were performed in triplicate, and parasites maintained in RPMI 1640 medium were used as negative control groups. Viability assays Pairs of adult worms were incubated with H2O2 (100– 5,000 μM) for 24 or 120 h, and the viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test (Comley et al. 1989; Fernandes et al. 2013). After incubation, each pair of worms was placed individually into wells (96-well plates) containing phosphatebuffered saline (PBS) (100 μL) with 1 mg mL−1 of MTT, at 37 °C. After 30 min, the solution was carefully removed and replaced with DMSO (200 μL), in which the worms were allowed to stand for 1 h at room temperature. The absorbance was then measured at 550 nm using a microplate reader (PowerWave X340, Bio-Tek Instruments, Winooski, USA). Parasites kept in RPMI 1640 medium were used as negative control groups, and worms killed by heating at 56 °C for 15 min were used as positive control groups. All experiments were performed in triplicate.
pH 7.5, 0.5 % glycerol, 1 mM dithiothreitol, and 1x protease inhibitor cocktail) using a sonicator (five applications of 10s), and then centrifuged for 60 min at 4 ºC and 20,800×g. The supernatant was subsequently centrifuged at 4 ºC and 15,000×g for 30 min. The final supernatant was collected, and the protein content was determined according to the Bradford method using Coomassie Plus Protein Assay Reagent (Thermo Scientific). Approximately 25 μg portions of protein from the crude soluble extracts of the worms were loaded onto 12 % SDS polyacrylamide gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Amersham Biosciences). A blocking step was performed for 1 h at 4 ºC in tris-buffered saline (TBS) containing 5 % skimmed milk and 0.1 % Tween 20. The membranes were then incubated overnight with primary antibodies: human anti-20S IgG, 1:2,000 (PW8195, Biomol); human anti-19S (RPN10) IgG, 1:2,000 (04-259, Millipore); and human antiactin IgG, 1:3,000 (MAB1501, Millipore). The membranes were then washed four times with TBS-Tween and incubated for 1 h with the corresponding secondary antibody (chicken anti-mouse IgG, 1:5,000 (sc2958, Santa Cruz Biotechnology) conjugated to alkaline phosphatase for 1 h at room temperature. The membranes were revealed using the alkaline phosphatase substrates NBT/BCIP (Sigma). PageRuler TM Prestained Protein Ladder (Thermo Scientific) was used as a weight standard. Crude protein extracts from S. mansoni worms treated with 50 μM of MG132 for 24 h were used as positive controls.
Scanning electron microscopy Proteasome activity The tegument morphology of parasites submitted to oxidative stress was investigated by scanning electron microscopy. Paired adult worms were exposed to 10 μM of praziquantel (positive control) or oxidative stress at 800 μM of H2O2, being in all experiments worms incubated during 24 h. The parasites were then washed in phosphate-buffered saline (PBS) at 37 °C, fixed in 3 % glutaraldehyde for 60 min at 37 °C and 60 min at room temperature, washed twice in PBS, and maintained in PBS at 4 °C. Afterwards, the parasites were postfixed using 1 % OsO4 in 100 mM sodium phosphate buffer (pH 7.2) for 2 h at 4 °C. After postfixing, the material was washed three times in 100 mM PBS, dehydrated using an increasing ethanol series, and then maintained at the critical point (Model 030 Critical Point Dryer, Bal-Tec). The dried specimens were mounted on aluminum stubs and observed by scanning electron microscopy using a JSM 6610LV electron microscope (JEOL) operated at 25 kV. Protein extraction and Western blotting analysis S. mansoni worms submitted to oxidative stress were employed for protein extraction. Approximately 100 mg of worms were homogenized in extraction buffer (25 mM Tris,
Peptidase activity was assayed using 50 μg portions of crude extracts of worms submitted to oxidative stress. This assay was performed as described in Guerra-Sá et al. (2005). The chymotrypsin-like activity was determined by fluorimetric quantification of the Suc-LLVY-AMC substrate using a Quantech fluorometer (Barnstead Thermolyne, USA), with excitation and emission wavelengths set at 380 and 460 nm, respectively. Gene expression analyses Total RNA from adult S. mansoni worms submitted to oxidative stress and control was extracted and isolated using Trizol® reagent (Invitrogen, Carlsbad, CA). The isolated RNA was resuspended in diethylpyrocarbonate (DEPC)treated water and subjected to DNAse treatment using the DNAse I (Promega, Madison, WI) to eliminate genomic DNA contamination. RNA samples were quantified and their purity assessed on Nanodrop (Spectometer ND-1000, Thermo Scientific). One microgram of DNAse-treated RNA was used as template to synthesize cDNA using the ThermoScript™ RT-PCR System (Invitrogen, Carlsbad,
Parasitol Res
CA) following the manufacturer’s protocol. S. mansoni-specific primers were designed using the program Primer3 (Rozen and Skaletsky 2000). The sequence accession numbers and their pair of primers used in this study were: SmF10 (Smp_131110.1): Forward: 5’-GATATGGTGGAGGCTGTACT-3’; Reverse: 5’-CATA ATCACCGCCATAGG-3’ and SmRPN10 (Smp_ 000740.2): Forward: 5’-TGTCGATATTATTAACTTTGGTGAG-3’, Reverse: 5’-GAACCATCCTCACCAGCAAC-3’. Reversetranscribed cDNA samples were used as templates for PCR amplification using SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7500 System (Applied Biosystems, Rio de Janeiro, Brazil). Specific primers for S. mansoni Glyceraldehyde 3-phosphate dehydrogenase ( G A P D H ) w e r e u s ed as a n e n d o ge n o u s c on t r o l (Smp_056970.1: Forward: 5′-TCGTTGAGTCTACTGGAG TCTTTACG-3′, Reverse: 5′-AATATGAGCCTGAGCTTT ATCAATGG-3′) (Mourão et al., 2009). The efficiency of each pair of primers was evaluated according to the protocol developed by the Applied Biosystems application (cDNA dilutions were 1:10, 1:100, and 1:1,000). For all investigated transcripts, three biological replicates were performed. The gene expression of SmF10 and SmRPN10 was normalized to the GAPDH transcript using the Applied Biosystems 7500 software according to the 2-ΔΔCt method (Livak and Schmittgen 2001), being corrected to the control group (untreated worms).
Table 1 In vitro oxidative stress effects in adult S. mansoni worms Treatment
Controla
24 h
120 h Hydrogen peroxide 100 μM 24 h 120 h 200 μM 24 h 120 h 300 μM 24 h 120 h 400 μM 24 h 120 h 500 μM 24 h 120 h 800 μM 24 h 120 h 1 mM 24 h 120 h 2 mM 24 h 120 h 3 mM 4 mM
Statistical analysis 5 mM
Statistical tests were performed using GraphPad Prism v. 5.0 software (GraphPad, San Diego, CA). Significant differences (p
ORIGINAL PAPER
Biochemical characterization and role of the proteasome in the oxidative stress response of adult Schistosoma mansoni worms Renato Graciano de Paula & Alice Maria de Magalhães Ornelas & Enyara Rezende Morais & William de Castro Borges & Massimo Natale & Lizandra Guidi Magalhães & Vanderlei Rodrigues
Received: 26 February 2014 / Accepted: 6 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The trematode Schistosoma mansoni, an important parasite of humans, is the principle agent of the disease schistosomiasis. In the human host, one of the most important stress factors of this parasite is the oxidative stress generated by both the metabolism of the worm and the immune system of the host. The proteasomal system is responsible for protein homeostasis during oxidative stress. The 26S proteasome is a multicatalytic protease formed by two compartments, a 20S core and regulatory particle 19S, and controls the degradation of intracellular proteins, hence regulating many cellular Electronic supplementary material The online version of this article (doi:10.1007/s00436-014-3950-5) contains supplementary material, which is available to authorized users. R. G. de Paula (*) : A. M. de Magalhães Ornelas : V. Rodrigues Faculdade de Medicina de Ribeirão Preto, Departamento de Bioquímica e Imunologia, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brazil e-mail: [email protected] E. R. Morais Instituto de Genética e Bioquímica, Campus Avançado Patos de Minas, Universidade Federal de Uberlândia, Avenida Getúlio Vargas, 230, Patos de Minas, Minas Gerais, Brazil W. de Castro Borges Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, s/n, Ouro Preto 35400-000, Minas Gerais, Brazil M. Natale Department of Control and Computer Engineering, Politecnico di Torino, Turin, Italy L. G. Magalhães Grupo de Pesquisa em Produtos Naturais, Centro de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Avenida Doutor Armando S. Oliveira, 201, Parque Universitário, Franca 14404-600, São Paulo, Brazil
processes. In the present report, we describe the biochemical characterization and role of the 20S proteasome in the response of adult S. mansoni worms exposed to hydrogen peroxide. Characterization of the response to the oxidative stress included the evaluation of viability, egg production, mortality, tegument integrity, and both expression and activity of proteasome. We observed decreases in viability, egg production as well as 100 % mortality at the higher concentrations of hydrogen peroxide tested. The main changes observed in the tegument of adult worms were peeling as well as the appearance of bubbles and a decrease of spines on the tubercles. Furthermore, there were increases in 26S activity to the same extent as 20S proteasome activity, although there was increase of 20S proteasome content, suggesting that degradation of protein oxidized in adult worms is due to the 20S proteasome. It was demonstrated that adult S. mansoni worms are sensitive to oxidative stress, and that a variety of processes in this parasite are altered under this condition. The work contributes to a better understanding of the mechanisms employed by S. mansoni to survive under oxidative stress. Keywords S. mansoni . Proteasome . Oxidative stress
Introduction Schistosoma mansoni is one of the etiologic agents of schistosomiasis, a severe disease that affects almost 240 million people worldwide, mainly in the tropical and subtropical areas, and more than 700 million people live under conditions where they are exposed to the risk of infection (WHO, 2013; Mcdonald et al. 2010). Praziquantel is currently the only drug available to treat the disease (Caffrey 2007). However, resistance of the parasite has been reported, and juvenile worms are
Parasitol Res
able to evade the action of the drug (Ismail et al. 1999). An understanding of the biology of the parasite is therefore vital in order to develop new therapeutic techniques (Soliman and Ibrahim 2005). In the course of its lifecycle, the parasite is exposed to a variety of stresses, which can be either physical or chemical in nature. The free-living stages are subjected to threats that include chemical pollutants present in the aquatic environment, variations in temperature, and solar radiation (Prah and James 1977; Coelho and Bezerra 2006; Ruelas et al. 2007). In adult worms, the most important stress factor is probably the reactive oxygen species (ROS) released by cells of the host immune system during aerobic metabolism. Consistent with this hypothesis, in infection-resistant snails, the miracidium is rapidly eliminated by a process that results in an increased rate of oxygen consumption, which is indicative of a role of oxidative stress in the defense mechanism of the snails (Hahn et al. 2001). Further evidence is provided by high levels of antioxidant enzyme transcription in adult worms (Mei and Loverde 1997), suggesting the need to adapt to an intense exposure to ROS during this stage of the lifecycle. Adult worms can live for up to 30 years in the definitive host organism, and must therefore possess antioxidant mechanisms in order to preserve the integrity of their cells and survive bursts of oxidative activity (Vieira et al. 2007). ROS are released from S. mansoni and its hosts during the course of cellular processes including aerobic respiration, immune defense, and other oxidative pathways (Kazura et al. 1981). Hydrogen peroxide and its subproducts are probably the most important ROS in biological systems, and directly affect different groups of biomolecules found in cells, such as lipids, proteins, and nucleic acids (Kastle and Grune 2012; Corcoran and Cotter 2013). Many studies have demonstrated that the susceptibility to oxidative stress depends on the stage in the lifecycle, with the schistosomulae being more sensitive than adult worms (Hong et al. 1992). Oxidatively-modified proteins are continuously produced in cells due to the action of reactive oxygen species generated as a consequence of aerobic metabolism. The main changes that occur during oxidative stress are the formation of protein carbonyls and the breaking of disulfide bonds, which renders impossible the maintenance of the native protein conformation (Kastle and Grune 2011). The proteasome represents the main cytoplasmic proteolytic machinery for the preferential degradation of damaged rather than normal proteins (Dudek et al. 2005; Friguet 2006), and is responsible for the maintenance of protein homeostasis during oxidative stress (Mayer 2010; Toivola et al. 2010). The 20S proteasome particle is the main agent responsible for oxidized protein degradation (Grune et al. 2011), and the evidence suggests that the process is ubiquitin-free (Shringarpure et al. 2003). Other studies have demonstrated that oxidized proteins are degraded by the 20S proteasome, independent of the 19S regulator or ubiquitin (Yamanaka et al.
2003; Pickering et al. 2010). Shringarpure et al. (2003) demonstrated that disruption of the ubiquitination system did not impair the degradation of oxidized proteins, while Wang et al. (2010) and Kriegenburg et al. (2011) reported that the components of the ubiquitin-proteasome system are highly sensitive to oxidative stress, which could result in inhibition of the ubiquitination protein or impairment of the 26S complex. Nevertheless, the role of the proteasome in oxidized protein degradation in S. mansoni remains unclear. In order to improve understanding of the different pathways involved in the response to oxidative damage in this parasite, the aim of the present work was to identify the main changes that occur in adult S. mansoni worms subjected to oxidative stress. The effects included changes in viability, egg production, and tegument morphology. In addition, increases in both the content and activity of the 20S proteasome were observed. Materials and methods Ethics statement All experiments involving animals were authorized by the Animal Research Ethics Commission (CETEA) of the University of São Paulo (protocol number 182/2011) and abided by the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation (COBEA). These were in accordance with accepted national and international norms for laboratory animal welfare. Parasites and oxidative stress treatment The Luis Evangelista (LE) strain of S. mansoni was maintained by successive passages through Biomphalaria glabrata snails and BALB/c mice. After 56 days, adult S. mansoni worms were recovered under aseptic conditions from mice that had been previously infected with 200 cercariae by perfusion of the livers and mesenteric veins (Smithers and Terry 1965). The paired adult worms were maintained in RPMI 1640 medium (Invitrogen) supplemented with penicillin (100 UI/mL), streptomycin (100 μg/mL), and 10 % bovine fetal serum (Gibco), at 37 ºC with 5 % CO2, under dark conditions. After 24 h of incubation, oxidative stress was induced by the addition of H2O2 to final concentrations in the range 100–5,000 μM, and the worms were incubated for 0 (non-exposed control), 0.5, 1, 4, and 24, and 120 h, at 37 ºC with 5 % CO2. After incubation, either subsequent experiments were performed or the worms were frozen in liquid nitrogen. In vitro studies with S. mansoni After the oxidative stress treatment, the parasites were monitored at 24 and 120 h to evaluate their general condition, including alterations in the tegument, couples separated,
Parasitol Res
tegumental changes, and mortality rate (Xiao et al. 2007). At the same time, changes in pairing and egg production were examined using an inverted microscope (Leitz) (Magalhães et al. 2009; Manneck et al. 2010). The worms were considered dead when no movement was observed over at least 2 min. All experiments were performed in triplicate, and parasites maintained in RPMI 1640 medium were used as negative control groups. Viability assays Pairs of adult worms were incubated with H2O2 (100– 5,000 μM) for 24 or 120 h, and the viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test (Comley et al. 1989; Fernandes et al. 2013). After incubation, each pair of worms was placed individually into wells (96-well plates) containing phosphatebuffered saline (PBS) (100 μL) with 1 mg mL−1 of MTT, at 37 °C. After 30 min, the solution was carefully removed and replaced with DMSO (200 μL), in which the worms were allowed to stand for 1 h at room temperature. The absorbance was then measured at 550 nm using a microplate reader (PowerWave X340, Bio-Tek Instruments, Winooski, USA). Parasites kept in RPMI 1640 medium were used as negative control groups, and worms killed by heating at 56 °C for 15 min were used as positive control groups. All experiments were performed in triplicate.
pH 7.5, 0.5 % glycerol, 1 mM dithiothreitol, and 1x protease inhibitor cocktail) using a sonicator (five applications of 10s), and then centrifuged for 60 min at 4 ºC and 20,800×g. The supernatant was subsequently centrifuged at 4 ºC and 15,000×g for 30 min. The final supernatant was collected, and the protein content was determined according to the Bradford method using Coomassie Plus Protein Assay Reagent (Thermo Scientific). Approximately 25 μg portions of protein from the crude soluble extracts of the worms were loaded onto 12 % SDS polyacrylamide gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Amersham Biosciences). A blocking step was performed for 1 h at 4 ºC in tris-buffered saline (TBS) containing 5 % skimmed milk and 0.1 % Tween 20. The membranes were then incubated overnight with primary antibodies: human anti-20S IgG, 1:2,000 (PW8195, Biomol); human anti-19S (RPN10) IgG, 1:2,000 (04-259, Millipore); and human antiactin IgG, 1:3,000 (MAB1501, Millipore). The membranes were then washed four times with TBS-Tween and incubated for 1 h with the corresponding secondary antibody (chicken anti-mouse IgG, 1:5,000 (sc2958, Santa Cruz Biotechnology) conjugated to alkaline phosphatase for 1 h at room temperature. The membranes were revealed using the alkaline phosphatase substrates NBT/BCIP (Sigma). PageRuler TM Prestained Protein Ladder (Thermo Scientific) was used as a weight standard. Crude protein extracts from S. mansoni worms treated with 50 μM of MG132 for 24 h were used as positive controls.
Scanning electron microscopy Proteasome activity The tegument morphology of parasites submitted to oxidative stress was investigated by scanning electron microscopy. Paired adult worms were exposed to 10 μM of praziquantel (positive control) or oxidative stress at 800 μM of H2O2, being in all experiments worms incubated during 24 h. The parasites were then washed in phosphate-buffered saline (PBS) at 37 °C, fixed in 3 % glutaraldehyde for 60 min at 37 °C and 60 min at room temperature, washed twice in PBS, and maintained in PBS at 4 °C. Afterwards, the parasites were postfixed using 1 % OsO4 in 100 mM sodium phosphate buffer (pH 7.2) for 2 h at 4 °C. After postfixing, the material was washed three times in 100 mM PBS, dehydrated using an increasing ethanol series, and then maintained at the critical point (Model 030 Critical Point Dryer, Bal-Tec). The dried specimens were mounted on aluminum stubs and observed by scanning electron microscopy using a JSM 6610LV electron microscope (JEOL) operated at 25 kV. Protein extraction and Western blotting analysis S. mansoni worms submitted to oxidative stress were employed for protein extraction. Approximately 100 mg of worms were homogenized in extraction buffer (25 mM Tris,
Peptidase activity was assayed using 50 μg portions of crude extracts of worms submitted to oxidative stress. This assay was performed as described in Guerra-Sá et al. (2005). The chymotrypsin-like activity was determined by fluorimetric quantification of the Suc-LLVY-AMC substrate using a Quantech fluorometer (Barnstead Thermolyne, USA), with excitation and emission wavelengths set at 380 and 460 nm, respectively. Gene expression analyses Total RNA from adult S. mansoni worms submitted to oxidative stress and control was extracted and isolated using Trizol® reagent (Invitrogen, Carlsbad, CA). The isolated RNA was resuspended in diethylpyrocarbonate (DEPC)treated water and subjected to DNAse treatment using the DNAse I (Promega, Madison, WI) to eliminate genomic DNA contamination. RNA samples were quantified and their purity assessed on Nanodrop (Spectometer ND-1000, Thermo Scientific). One microgram of DNAse-treated RNA was used as template to synthesize cDNA using the ThermoScript™ RT-PCR System (Invitrogen, Carlsbad,
Parasitol Res
CA) following the manufacturer’s protocol. S. mansoni-specific primers were designed using the program Primer3 (Rozen and Skaletsky 2000). The sequence accession numbers and their pair of primers used in this study were: SmF10 (Smp_131110.1): Forward: 5’-GATATGGTGGAGGCTGTACT-3’; Reverse: 5’-CATA ATCACCGCCATAGG-3’ and SmRPN10 (Smp_ 000740.2): Forward: 5’-TGTCGATATTATTAACTTTGGTGAG-3’, Reverse: 5’-GAACCATCCTCACCAGCAAC-3’. Reversetranscribed cDNA samples were used as templates for PCR amplification using SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7500 System (Applied Biosystems, Rio de Janeiro, Brazil). Specific primers for S. mansoni Glyceraldehyde 3-phosphate dehydrogenase ( G A P D H ) w e r e u s ed as a n e n d o ge n o u s c on t r o l (Smp_056970.1: Forward: 5′-TCGTTGAGTCTACTGGAG TCTTTACG-3′, Reverse: 5′-AATATGAGCCTGAGCTTT ATCAATGG-3′) (Mourão et al., 2009). The efficiency of each pair of primers was evaluated according to the protocol developed by the Applied Biosystems application (cDNA dilutions were 1:10, 1:100, and 1:1,000). For all investigated transcripts, three biological replicates were performed. The gene expression of SmF10 and SmRPN10 was normalized to the GAPDH transcript using the Applied Biosystems 7500 software according to the 2-ΔΔCt method (Livak and Schmittgen 2001), being corrected to the control group (untreated worms).
Table 1 In vitro oxidative stress effects in adult S. mansoni worms Treatment
Controla
24 h
120 h Hydrogen peroxide 100 μM 24 h 120 h 200 μM 24 h 120 h 300 μM 24 h 120 h 400 μM 24 h 120 h 500 μM 24 h 120 h 800 μM 24 h 120 h 1 mM 24 h 120 h 2 mM 24 h 120 h 3 mM 4 mM
Statistical analysis 5 mM
Statistical tests were performed using GraphPad Prism v. 5.0 software (GraphPad, San Diego, CA). Significant differences (p
