Carlos Robello

A PTERIDINE REDUCTASE GENE PTR1 CONTIGUOUS TO A P-GLYCOPROTEIN CONFERS RESISTANCE TO ANTIFOLATES IN TRYPANOSOMA CRUZI

We have isolated the pteridine reductase-1 gene (ptr1), from Trypanosoma cruzi (Y strain), located contiguous to the Trypanosoma cruzi P-glycoprotein-2 (tcpgp2). The gene encodes a member of the family of short-chain dehydrogenases, enzymes that are involved in several oxidoreduction reactions. One member of the family, pteridine reductase-1 (PTR1) has been previously described in Leishmania as being involved in antifolate resistance. The ptr1 gene from T. cruzi presents an 828 bp open reading frame, coding for a 276 amino acid protein with a predicted molecular mass of 30 kDa. The deduced amino acid sequence exhibited a remarkable homology with the ptr1 genes of Leishmania major and Leishmania tarentolae. Southern blot analysis using a specific probe indicated that T. cruzi PTR1 is encoded by a single copy gene located in two chromosomes of about 0.9 and 1.2 Mb. Western blot analysis using a polyclonal antiserum against recombinant PTR1 revealed that the protein is only expressed in the epimastigote forms of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. Purified recombinant PTR1 exhibits a NADPH-dependent pteridine reductase activity comparable with those described in Leishmania. Gene transfection experiments using the pTEX expression vector show that, under the conditions tested, T. cruzi PTR1 is involved in resistance to the methotrexate, aminopterin and trimethoprim antifolates.

Evolutionary relationships in Trypanosoma cruzi: Molecular phylogenetics supports the existence of a new major lineage of strains

For the purpose of investigating the evolutionary relationships among strains of the human parasite Trypanosoma cruzi, we have determined the nucleotide sequence, in 16 T. cruzi stocks, of a DNA fragment having approximately 1030 nucleotides in length. Phylogenetic analyses show the presence of at least three major groups of T. cruzi strains, a result that contradicts previous phylogenetic inferences based on polymorphism data. We also performed an analysis of the relative extent of nucleotide divergence among T. cruzi strains compared to the divergence between Leishmania species, using the gene encoding pteridine reductase. The results presented in this work show that the divergence among the most distant T. cruzi strains is at least as high as the divergence between two different species complexes of Leishmania, those containing L. major and L. mexicana.

Redox metabolism in Trypanosoma cruzi: Functional characterization of tryparedoxins revisited

Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to various 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 transmembrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione, or dihydrolipoamide to various acceptors, including methionine sulfoxide reductases and peroxiredoxins. The results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in the redox scenario for T. cruzi.

Tryparedoxin peroxidases and superoxide dismutases expression as well as ROS release are related to Trypanosoma cruzi epimastigotes growth phases

Trypanosoma cruzis antioxidant system is unique and relevant to the parasite. In this study, quantitative assays were performed to determine cytosolic and mitochondrial tryparedoxin peroxidases and superoxide dismutases expression (TcCPx, TcMPx, SODB and SODA) in correlation to H(2)O(2) release and O(2)(-) production. Differences were observed regarding H(2)O(2) release and O(2)(-) production between strains and along the growth curve. All of the enzymes studied exhibited varied expression as a function of time in culture. Although at lower levels, the Y strain exhibited the same pattern of Tulahuen 2 enzyme expression for all of the proteins studied, except SODA. In the stationary phase, the degree of expression of all of the enzymes in the Y strain returned to similar levels as those detected in the log phase with the exception of TcCPx and SODA. In Tulahuen 2, a higher expression of TcMPx, SODA and SODB was detected in the early stationary phase, and a slight decrease was observed in the late stationary phase for each enzyme, excluding TcMPx, which exhibited a marked decrease, and TcCPx, which increased its level. Because of the significance of ROS in redox signaling, these differences in enzyme expression underscore the importance of these parameters for epimastigote proliferation.

Role of Trypanosoma cruzi peroxiredoxins in mitochondrial bioenergetics.

Trypanosoma cruzi cytosolic (TcCPx) and mitochondrial tryparedoxin peroxidase (TcMPx) play a fundamental role in H2O2 detoxification. Herein, mitochondrial bioenergetics was evaluated in cells that overexpressed TcCPx (CPx) and TcMPx (MPx) and in pTEX. In MPx, a higher expression was observed for TcCPx, and the same correlation was true for CPx. Differences in H2O2 release among the overexpressing cells were detected when the mitochondrial respiratory chain was inhibited using antimycin A or thenoyltrifluoroacetone. MPx had higher O2 consumption rates than pTEX and CPx, especially in the presence of oligomycin. In all of the cells, the mitochondrial membrane potential and the ATP levels were similar. Because of the mild uncoupling that was observed in MPx, the presence or induction of a proton transporter in the mitochondrial membrane is suggested when TcMPx is expressed at higher levels. Our results show a possible interplay between the cytosolic and mitochondrial antioxidant systems in a trypanosomatid.

Two casein kinase 1 isoforms are differentially expressed in Trypanosoma cruzi

The cDNAs for two casein kinase 1 (CK1) homologues, TcCK1.1 and TcCK1.2, have been isolated from Trypanosoma cruzi. Both isoforms showed strong identity with other known CK1s. Their corresponding genes encode proteins of 312- and 330-amino acid residues with apparent molecular weights of 16 and 37 kDa, respectively. TcCK1.1 is a two-copy gene while TcCK1.2 is tandemly repeated, an arrangement not yet found in any other CK1. TcCK1.1 has been overexpressed in Escherichia coli and the recombinant protein exhibited properties characteristic of the CK1 family. Northern blot indicated that both TcCK1s are expressed differentially during the life stages of the parasite: the isoform TcCK1.1 shows low levels of mRNA expression in epimastigotes and increased expression in trypomastigotes while TcCK1.2 presents an augmented expression in amastigotes as compared with the other two life stages of the parasite. The CK1-like activity of amastigotes and trypomastigotes is significantly higher than that of epimastigotes and, independent of the life stage of the parasite, a constitutive activity is observed which, in the epimastigote forms, is found predominantly in the microsomal fraction. Also in the epimastigote forms, the CK1-like activity increases in the log phase of growth of the parasites, and, through synchronization studies, this activity has been most conspicuously circumscribed to the S and M phases of the cell cycle.

A NEW MEMBER OF YER057C FAMILY IN TRYPANOSOMA CRUZI IS ADJACENT TO AN ABC-TRANSPORTER

Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2. The protein contains 160 amino acid residues with a predicted molecular mass of 16.5kDa. Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized. Here, we propose a new signature of this family considering the N-terminal: [IV]-X(4)-[AV]-[AP]-X-[AP]-X(3)-Y-X(9)-[LIVF]-X(2)-[SA]-G-[QS], and the C-terminal: [AT]-R-X(2)-[IVFY]-X-[VC]-X(2)-L-P-X(4)-[LIVM]-E-[IVM] -[DE] motifs. Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles. Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.

Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells

Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response), a great number of transcription factors (including the majority of NFκB family members), and host metabolism (cholesterol, fatty acids, and phospholipids). These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination.

Old yellow enzyme from Trypanosoma cruzi exhibits in vivo prostaglandin F-2 alpha synthase activity and has a key role in parasite infection and drug susceptibility

The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F2α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF2α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host–parasite interaction.

Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi

Background: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione‐spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol‐redox metabolite in trypanosomatids. Methods and results: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti‐protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36–71% higher resistance to H2O2 (50–1000 &mgr;M) and heavy metal (1–500 &mgr;M) toxicity. Treatment with TryS inhibitors (5–30 &mgr;M) abolished the proliferation and survival advantages against H2O2 pressure in a dose‐dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. Conclusions: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti‐trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease. Graphical abstract Figure. No Caption available. HighlightsTrypanothione synthetase (TryS) is an essential enzyme for T. cruzi.TryS‐overexpression confers higher replication rate to T. cruzi epimastigotes.TryS provides protection against oxidative stress to different parasite stages.TryS confers resistance to current treatment drugs, benznidazole and nifurtimox.