Elsevier

Journal of Proteomics

Volume 74, Issue 9, 24 August 2011, Pages 1683-1692
Journal of Proteomics

Molecular characterization and interactome analysis of Trypanosoma cruzi Tryparedoxin 1

https://doi.org/10.1016/j.jprot.2011.04.006Get rights and content

Abstract

Trypanosoma cruzi tryparedoxin 1 (TcTXN1) is an oxidoreductase belonging to the thioredoxin superfamily, which mediates electron transfer between trypanothione and peroxiredoxins. In trypanosomes TXNs, and not thioredoxins, constitute the oxido-reductases of peroxiredoxins. Since, to date, there is no information concerning TcTXN1 substrates in T. cruzi, the aim of this work was to characterize TcTXN1 in two aspects: expression throughout T. cruzi life cycle and subcellular localization; and the study of TcTXN1 interacting-proteins. We demonstrate that TcTXN1 is a cytosolic and constitutively expressed protein in T. cruzi. In order to start to unravel the redox interactome of T. cruzi we designed an active site mutant protein lacking the resolving cysteine, and validated the complex formation in vitro between the mutated TcTXN1 and a known partner, the cytosolic peroxiredoxin. Through the expression of this mutant protein in parasites with an additional 6xHis-tag, heterodisulfide complexes were isolated by affinity chromatography and identified by 2-DE/MS. This allowed us to identify fifteen TcTXN1 proteins which are involved in two main processes: oxidative metabolism and protein synthesis and degradation. Our approach led us to the discovery of several putatively TcTXN1-interacting proteins thereby contributing to our understanding of the redox interactome of T. cruzi.

Introduction

Trypanosoma cruzi, the causative agent of Chagas disease, constitutes a major sanitary problem in Latin America. This illness mainly affects the poor rural areas of Latin America, where the number of estimated infected persons is around 10 million, 28 million people are at risk of infection and ~ 20,000 deaths occur per year in endemic regions [1]. Remote location of infected people makes Chagas disease neglected and unattractive for search of new drugs. No vaccines are available at present, and drugs currently in use, nifurtimox and benznidazol, were developed decades ago and are effective only during the symptomatic stage of Chagas disease and have undesirable side effects. Programs have focused on disease prevention by eliminating the vector, and prevention of other means of transmission, such as infection by blood donors or maternal transmission [2]. However, new means of transmission have been described, such as oral infection by contaminated food and accidental contamination during laboratory work and via organ transplantation [3]. Due to human migration, there are estimations that 100,000 chronically infected people from endemic countries are now living in non-endemic countries which do not screen donors serologically for Chagas disease [4]. There has been reported more number of cases in urban locations, novel means of transmission, vector resistance, new carriers and disease reactivation in AIDS individuals. Chagas disease can become an international threat. New effective treatments are a priority so novel specific targets for drug development against T. cruzi need to be discovered.

The protozoan parasite T. cruzi has a complex life cycle which alternates between two intermediate hosts (the first, a triatomine insect and the second, a mammalian vertebrate which could be a human), where it invades different cell types [5]. During macrophage invasion, parasites are temporarily located in phagocytic vacuoles, where reactive oxygen and nitrogen species are synthesized [6]. Oxidizing species such as peroxynitrite and hydrogen peroxide (H2O2) can be toxic to parasites and therefore, their ability to circumvent such an oxidative environment will determine the success and persistence of the infectious process. Therefore, T. cruzi antioxidant mechanisms constitute an active field of investigation, since they could provide the basis for a rational drug development.

As revealed by its genome sequencing [7], T. cruzi lacks genes for glutathione reductase, thioredoxin reductase, catalase and selenium-dependent glutathione peroxidases. Instead, the trypanosomes redox metabolism is based on the low molecular mass dithiol trypanothione (N(1), N(8)-bis (glutathionyl) spermidine). Thiol redox homeostasis in trypanosomes is efficiently maintained through the participation of different peroxidases which are part of a complex system of enzymes that includes trypanothione synthetase trypanothione reductase and tryparedoxins (for a review see [8]). Several of these enzymes are found exclusively in trypanosomes. This is the case of tryparedoxins (TXNs), oxidoreductases belonging to the thioredoxin superfamily which transfer reducing equivalents from trypanothione to peroxidases such as peroxiredoxins and glutathione peroxidases [9], [10], [11].

The regulation of a number of phenomena in the cell has been linked to the reversible conversion of disulfides to dithiols thereby modulating the activities of the respective proteins [12]. This conversion activity is mainly carried out by thioredoxins, another type of oxidoreductase. Several recent articles have described the identification of thioredoxin-interacting proteins in eukaryotes [13], [14], [15], [16], [17]. The putative target proteins are involved in many processes, including oxidative stress response (e.g. peroxiredoxins), nucleotide metabolism (e.g. ribonucleotide reductase) and protein synthesis (e.g. several elongation factors), among others.

However this does not seem to be the case for trypanosomes since there is no thioredoxin reductase, and thioredoxins are expressed at very low concentrations [18]. The predominant low molecular mass dithiol proteins in trypanosomes are tryparedoxins (TXNs), which are uniquely expressed in these parasites, and despite belonging to the thioredoxin superfamily they are quite different to thioredoxins. TXNs have a different active site motif (WCPPCR instead of WCG(A)PK in most thioredoxins), and are also considerably larger than thioredoxins due to several insertions summing up to about 5 kDa [19]. As in thioredoxins, the reduction of protein disulfides by TXNs is based on a dithiol exchange mechanism [19] in which the N-terminal cysteine residue of the CXXC motif (Cys40 in T. cruzi) initiates a nucleophilic attack on the disulfide target resulting in the formation of a mixed disulfide. The intermolecular disulfide bond is subsequently cleaved by the C-terminal resolving cysteine residue of the active site motif (Cys43 in T. cruzi), yielding the reduced substrate and the oxidized TXN. In trypanosomes TXNs, and not thioredoxins, constitute the oxidoreductases of peroxiredoxins (known as tryparedoxin peroxidases) and ribonucleotide reductase, while thioredoxins seem to be less relevant, as can be inferred from gene knock out and dsRNAi experiments, showing no phenotypic changes [20].

In Trypanosoma brucei and Leishmania infantum there are two TXN isoforms, one cytosolic (TXN1) and one mitochondrial (TXN2) [21], [22]. The importance of cytosolic TXNs in trypanosomatids was evidenced by experiments demonstrating that they are essential for parasite survival. In T. brucei the depletion of TbTXN1 showed that it plays a pivotal role in the hydroperoxide metabolism, and parasites become significantly more sensitive to oxidative damage [23]. In L. infantum the replacement of both chromosomal LiTXN1 alleles was only possible upon parasite complementation with an episomal copy of the gene. Furthermore, ex vivo infection assays suggest that wild-type levels of LiTXN1 are required for optimal L. infantum virulence [24]. The role of TXN1 in immunopathological processes has also been described in L. infantum by targeting B-cell effector functions, leading to IL-10 secretion and production of specific antibodies [25]. These results must not only be considered in the context of oxidative metabolism; since in trypanosomes TXNs may substitute thioredoxins in some functions, we can expect the presence of protein targets from different cellular processes.

In T. cruzi two genes that code for TXN have been described: TcTXN1 and TcTXN2. It has been shown that TcTXN1 is capable of reducing T. cruzi glutathione peroxidase [26]. However, up to date, no molecular studies have been performed in T. cruzi's TXN1 (TcTXN1). In the first part of this work we present our findings upon cloning the gene and conducting studies to determine its subcellular localization and expression profile. In order to begin the unraveling of the TXN1 interactome, we developed an in vivo approach, by expressing a mutated form of TcTXN1 in T. cruzi. This mutated TcTXN1 contains a substitution in its resolving cysteine as well as an additional 6xHis tag (TcTXN1C43S). Complexes between mutant TXN1 and targets were affinity purified and analyzed by two dimensional electrophoresis and mass spectrometry (MALDI-TOF-TOF). Our approach led us to the identification of some potential TXN1 binding partners. The advantage of this in vivo approach is that it was carried out maintaining intracellular conditions, and thus allowing a better appreciation of physiological roles of disulfide oxidoreductases.

Section snippets

Parasites

T. cruzi parasites of the Dm28c strain were used throughout this work [27]. Epimastigotes were grown in liver infusion tryptose (LIT) medium supplemented with 10% heat inactivated fetal bovine serum (FBS) at 28 °C. Vero cell derived trypomastigotes, amastigotes and metacyclic trypomastigotes were obtained as described in [28].

Production of polyclonal antiserum against TcTXN1 and western blot

Rabbits were immunized with 200 μg of TcTXN1 protein (purified as described below) in Freund's Complete Adjuvant (Sigma), for the first immunization. They were boosted three

Tryparedoxin 1 is a cytosolic and constitutively expressed enzyme in T. cruzi

Based on the GenBank sequence (Accession number AJ313314.1), we designed specific oligonucleotides to PCR amplify the gene coding for TcTXN1 from T. cruzi Dm28c strain genomic DNA. The amplified product was cloned and analyzed by sequencing, showing to be identical to the reference gene (not shown). The predicted gene product is a 144 amino acid protein with a theoretical molecular mass of 16 kDa and a calculated isoelectric point of 5.27. The gene was cloned and expressed in E. coli as

Conclusions

This proteomic approach reports the identification of putative partners of tryparedoxin 1 from T. cruzi. We demonstrate that TcTXN1 is a protein expressed along the life cycle of T. cruzi, without significant changes on its expression, and it is located in the cytosol. In order to start unravelling the redox interactome of T. cruzi, and based on the mechanism of action of tryparedoxins, we propose an improved approach for capture of partners. The pipeline for this method is outlined in Fig. 5.

Acknowledgements

We thank Monica Gardner (Institut Pasteur de Montevideo) for revision of the manuscript and Rosario Durán for helpful experimental suggestions. This work was supported partially by the Institut Pasteur de Montevideo and from the CSIC-Universidad de la República (Montevideo, Uruguay). M.D.P, A.P and C.R are researchers from the Sistema Nacional Investigadores (ANII), Montevideo Uruguay.

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