Glutathione metabolism in protozoan parasites : making the difference

Generation of reactive oxygen species (ROS) is a common feature of abiotic and biotic stresses. ROS need to be detoxified to avoid the occurrence of deleterious reactions. Parasitic diseases such as sleeping sickness, Chagas disease, and malaria, between others, are major health problems in poverty-stricken areas. Redox metabolism in parasitic protozoa is a potentially relevant target for developing specific drug inhibitors. The design of efficacious and safe drugs is possible after the occurrence of metabolic pathways that are unique and essential to the parasite, but absent in the host. In most organisms, glutathione is the most abundant low-molecular weight thiol acting in cellular redox systems. In those parasites with deficit or lacking the glutathione machinery, other active molecules are functional in mimicking the former reductive compound. Many of the known glutathione-dependent processes are directly related to specific life-style of the respective parasite. Thus, malaria parasites have a dual antioxidant system based on glutathione and thioredoxin. Proteins involved in glutathione-dependent metabolic pathways include glutaredoxins, glyoxalases, thioredoxins, glutathione reductase and glutathione S-transferases. In contrast, in trypanosomatids (Trypanosoma and Leishmania spp.), the redox network is centered around trypanothione [N1,N8-bis(glutathionyl)spermidine], instead of the ubiquitous glutathione. In these parasites, trypanothione participates in essential thiol?disulfide exchange reactions as electron donor to different trypanothione-dependent enzymes such as tryparedoxin, glutaredoxin and peroxiredoxins. Since trypanosomatids lack glutathione reductase and thioredoxin reductase, trypanothione is the central node in these microorganisms taking electrons from NADPH to achieve antioxidant enzymes. Concerning some anaerobic parasites such as Entamoeba histolytica or Giardia lamblia, they are human pathogens that lack the capacity to synthesize glutathione, and they have cysteine as the major low-molecular weight thiol. In this chapter, we review the glutathione dependent metabolism in protozoan parasitic cells, pointing out its relevance in vital functions of the parasite. We also comparatively analyze differences and similarities between glutathione and alternative metabolisms in the different protozoa. Particular attention is given to the role of glutathione in redox regulation and adaptation to stresses, highlighting the importance of enzymes belonging to redox metabolism, many of them proposed as target of antiparasitic drugs.Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Márquez, Vanina Elizabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Cabeza, Matías Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum

Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg-1 with an optimal activity at pH 8.0 and 32 °C. We determined Km values of 14 μM and 60 μM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Márquez, Vanina Elizabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Beccaria, Alejandro José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Physiological and morphological responses of green microalgae Chlorella vulgaris to silver nanoparticles

The toxic effects of silver nanoparticles (AgNPs) on the physiology and morphology of the green microalga Chlorella vulgaris were studied. AgNPs were characterized by particle size distribution, ζ potential measurement, and atomic force microscopy (AFM). Chlorella vulgaris was exposed to 90–1440 μg/L of AgNPs range in Bold's Basal Medium for 96 h. The inhibition of algae growth rate and changes in the concentrations of chlorophyll-a, chlorophyll-b, pheophytin, and carotenoids was determined at the beginning and end of the trial. Cell diameter and volume, carbohydrate, total lipids, and protein content were also determined. Our data strongly suggest that the toxic effects of the AgNPs resulted in concentration and time-dependent. AgNPs altered C. vulgaris growth kinetics and cell metabolism expressed in photosynthetic pigments and biochemical composition. Our study confirmed the cytotoxicity of AgNPs through the algal growth inhibition with an EC50 value of 110 μg/L. Also, changes of chlorophyll-a, chlorophyll-b, pheophytin, and carotenoids concentrations were observed associated with a color shift from green to pale brown of algae cultures exposed to AgNPs for 96 h. Furthermore, algae cell concentration, diameter, and volume, plus total lipid, protein, and carbohydrates contents in the presence of AgNPs, were significantly altered compared to untreated cells. In synthesis, this study highlighted AgNPs toxic effects on morphological and physiological traits of C. vulgaris and warns about possible impacts on energy flow and aquatic food web structure, and on the transfer efficiency of energy to higher trophic levels.Fil: Romero, Natalí. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias. Laboratorio de Ecotoxicologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Visentini, Flavia Fátima. Universidad Nacional del Litoral. Facultad de Ingeniería Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Márquez, Vanina Elizabet. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Santiago, Liliana G.. Universidad Nacional del Litoral. Facultad de Ingeniería Química; ArgentinaFil: Castro, Guillermo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Gagneten, Ana María. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias. Laboratorio de Ecotoxicologia; Argentin

Redox metabolism in Trypanosoma cruzi. Biochemical characterization of dithiol glutaredoxin dependent cellular pathways

In Trypanosoma cruzi, the modification of thiols by glutathionylationedeglutathionylation and its potential relation to protective, regulatory or signaling functions have been scarcely explored. Herein we characterize a dithiolic glutaredoxin (TcrGrx), a redox protein with deglutathionylating activity, having potential functionality to control intracellular homeostasis of protein and non-protein thiols. The catalytic mechanism followed by TcrGrx was found dependent on thiol concentration. Results suggest that TcrGrx operates as a dithiolic or a monothiolic Grx, depending on GSH concentration. TcrGrx functionality to mediate reduction of protein and non-protein disulfides was studied. TcrGrx showed a preference for glutathionylated substrates respect to protein disulfides. From in vivo assays involving TcrGrx overexpressing parasites, we observed the contribution of the protein to increase the general resistance against oxidative damage and intracellular replication of the amastigote stage. Also, studies performed with epimastigotes overexpressing TcrGrx strongly suggest the involvement of the protein in a cellular pathway connecting an apoptotic stimulus and apoptotic-like cell death. Novel information is presented about the participation of this glutaredoxin not only in redox metabolism but also in redox signaling pathways in T. cruzi. The influence of TcrGrx in several parasite physiological processes suggests novel insights about the protein involvement in redox signaling.Fil: Márquez, Vanina Elizabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Chiribao, Maria L.. Instituto Pasteur de Montevideo. Unidad de Biologia Molecular; Uruguay. Universidad de la República. Facultad de Medicina. Departamento de Bioquímica; UruguayFil: Faral Tello, Paula. Instituto Pasteur de Montevideo. Unidad de Biologia Molecular; UruguayFil: Robello, Carlos. Instituto Pasteur de Montevideo. Unidad de Biologia Molecular; Uruguay. Universidad de la República. Facultad de Medicina. Departamento de Bioquímica; UruguayFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; Argentin

Redox metabolism in Trypanosoma cruzi: functional characterization of tryparedoxins revisited

Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to different thiol proteins. In Trypanosoma cruzi, two genes coding for TXN like proteins have been identified: TXNI, previously characterized as a cytoplasmic protein, and TXNII, a putative tail-anchored membrane protein. In this work, we performed a comparative functional characterization of T. cruzi TXNs. Particularly, we cloned the gene region coding for the soluble version of TXNII for its heterologous expression. The truncated recombinant protein (without its 22 C-terminal transmenbrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione or dihydrolipoamide to different acceptors, including methionine sulfoxide reductases and peroxiredoxins. Results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in redox scenario in T. cruzi.Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Márquez, Vanina Elizabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Chiribao, Maria L.. Universidad de la República; UruguayFil: Gadhela, Fernanda R.. Universidade Estadual Do Campinas. Instituto de Biología; BrasilFil: Robello, Carlos. Universidad de la República; UruguayFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Toxicity of "Bacillus thuringiensis" var. "israelensis" in aqueous suspension on the South American common frog "Leptodactylus latrans" (Anura: Leptodactylidae) tadpoles

The effects of commercial formulations of Bacillus thuringiensis var. israelensis (Bti) on non-target organisms are still a matter of debate; in amphibians, the risks of Bti are little known. To evaluate the toxicity of a commercial liquid (aqueous suspension, AS) formulation of Bti (Introban®) on Leptodactylus latrans tadpoles, including median lethal concentration (LC50) and no-and lowest-observed-effect concentrations (NOEC and LOEC, respectively), as well as the possible effects of Bti on oxidative responses, erythrocytes genotoxicity, and histology of the intestines. In the laboratory, tadpoles were exposed to nominal concentrations of 0 (control), 2.5, 5, 10, 20 and 40mg/L of formulated Bti-AS. Glutathione S-transferase (GST) and catalase (CAT) activities, as well as formation of erythrocyte nuclear abnormalities (ENAs), and histological effect were measured in tadpoles displaying survival rates >85%. L. latrans tadpoles were sensitive to exposure to Bti-AS, reaching 100% mortality after 48h of exposure at the highest concentration. Bti-AS induced GST and CAT enzymes and genotoxicity (erythrocyte's nuclear abnormalities), and caused intestine's histopathology. Our results demonstrate that toxicity of Bti-AS is dose-dependent for L. latrans tadpoles and that sublethal exposure alters enzymes of oxidative stress, induces genotoxicity, and causes intestine damage. Further research is needed to evaluate the ecotoxicological risk of the massive use of Bti formulations on amphibian populations that commonly used suburban wastewater or urban waterbodies to reproduce and where this biopesticide is frequently applied.Fil: Lajmanovich, Rafael Carlos. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Saneamiento Ambiental. Cátedra de Ecotoxicología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Junges, Celina Maria. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Saneamiento Ambiental. Cátedra de Ecotoxicología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Cabagna Zenklusen, Mariana Cristina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Attademo, Andres Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Saneamiento Ambiental. Cátedra de Ecotoxicología; ArgentinaFil: Peltzer, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Saneamiento Ambiental. Cátedra de Ecotoxicología; ArgentinaFil: Maglianese, Mariana. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Márquez, Vanina Elizabet. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Beccaria, Alejandro José. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin