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Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease, a debilitating and sometimes fatal disease that continues to spread to new areas. Yet, Chagas disease is still only treated with two related nitro compounds that are insufficiently effective and cause severe side effects. Nucleotide metabolism is one of the known vulnerabilities of T. cruzi, as they are auxotrophic for purines, and nucleoside analogues have been shown to have genuine promise against this parasite in vitro and in vivo. Since purine antimetabolites require efficient uptake through transporters, we here report a detailed characterisation of the T. cruzi NB1 nucleobase transporter with the aim of elucidating the interactions between TcrNB1 and its substrates and finding the positions that can be altered in the design of novel antimetabolites without losing transportability. Systematically determining the inhibition constants (K i ) of purine analogues for TcrNB1 yielded their Gibbs free energy of interaction, ΔG 0 . Pairwise comparisons of substrate (hypoxanthine, guanine, adenine) and analogues allowed us to determine that optimal binding affinity by TcrNB1 requires interactions with all four nitrogen residues of the purine ring, with N1 and N9, in protonation state, functioning as presumed hydrogen bond donors and unprotonated N3 and N7 as hydrogen bond acceptors. This is the same interaction pattern as we previously described for the main nucleobase transporters of Trypanosoma brucei spp. and Leishmania major and makes it the first of the ENT-family genes that is functionally as well as genetically conserved between the three main kinetoplast pathogens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Trypanosoma cruzi is a parasite with a high capacity to adapt to the host. Animal models have already demonstrated that the tropism of this parasite occurs not only in cardiac/digestive tissues but also in adipose tissue (AT). That said, the consequences ofT. cruziinfection for AT and the implications of treatment with Benzonidazole in this tissue are under discussion. Here, we tested the hypothesis that T. cruzi infection in adipose tissue upon treatment with Benzonidazole (Bz) and the interaction of mononuclear immune cells (PBMC) influences the relative expression of ACAT1, FASN, and PNPLA2 genes. Thus, stem cells derived from adipose tissue (ADSC) after adipogenic differentiation were indirectly cultivated with PBMC after infection with the T. cruzi Y strain and treatment with Bz. We use the TcSAT-IAM system and RT-qPCR to evaluate the parasite load and the relative quantification (ΔCt) of the ACAT1, FASN, and PNPLA2 genes. Our results demonstrate that treatment with Bz did not reduce adipocyte infection in the presence (p-value: 0.5796) or absence (p-value: 0.1854) of cultivation with PBMC. In addition, even though there is no statistical difference when compared to the control group (AT), T. cruzi induces the FASN expression (Rq: 14.00). However, treatment with Bz in AT suggests the increases of PNPLA2 expression levels (Rq: 12.58), even in the absence of T. cruzi infection. During indirect cultivation with PBMC, T. cruzi smooths the expression of PNPLA2 (Rq: 0.824) and instigates the expression of ACAT1 (Rq: 1.632) and FASN (Rq: 1.394). Furthermore, the treatment with Bz during infection induces PNPLA2 expression (Rq: 1.871), maintaining FASN expression levels (Rq: 1.334). Given this, our results indicate that treatment with Benzonidazole did not decrease T. cruzi infection in adipose tissue. However, treating the adipocyte cells with Bz during the interaction with PBMC cells influences the lipid pathways scenario, inducing lipolytic metabolism through the expression of PNPLA2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Previous work has shown that the TbFUT1 and LmjFUT1 genes encode essential fucosyltransferases located inside the single mitochondria of the protozoan parasites Trypanosoma brucei and Leishmania major, respectively. However, nothing was known about the orthologous gene TcFUT1 or its gene product in Trypanosoma cruzi, aetiological agent of Chagas disease. In this study, we describe the overexpression of TcFUT1 with a C-terminal 6xMyc epitope tag in T. cruzi epimastigote cells. Overexpressed and immunoprecipitated TcFUT1-6xMyc was used to demonstrate enzymatic activity and to explore substrate specificity. This defined TcFUT1 as a GDP-Fuc : βGal α1-2 fucosyltransferase with a strict requirement for acceptor glycans with non-reducing terminal Galβ1-3GlcNAc structures. This differs from the specificity of the T. brucei orthologue TbFUT1, which can also tolerate non-reducing terminal Galβ1-4GlcNAc and Galβ1-4Glc acceptor sites. Immunofluorescence microscopy using α-Myc tag antibodies also showed a mitochondrial location for TcFUT1 in T. cruzi epimastigote cells. Collectively, these results are like those described for TbFUT1 and LmjFUT1 from T. brucei and L. major, suggesting that FUT1 gene products have conserved function for across the trypanosomatids and may share therapeutic target potential., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in glycosomes, such as β-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na 2 CO 3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite's glycosomes., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

The nuclear import of proteins in eukaryotic cells is a fundamental biological process. While it has been analysed to different extents in model eukaryotic organisms, this event has rarely been studied in the early divergent protozoa of the order Kinetoplastida. The work presented here represents an overview of nuclear import in these important species of human pathogens. Initially, an in silico study of classical nuclear localization signals within the published nuclear proteomes of Trypanosoma brucei and Trypanosoma cruzi was carried out. The basic amino acids that comprise the monopartite and bipartite classical nuclear localization signals (cNLS) in trypanosomal proteins are similar to the consensus sequences observed for the nuclear proteins of yeasts, animals and plants. In addition, a summarized description of published studies that experimentally address the NLS of nuclear proteins in trypanosomatids is presented, and the clear occurrence of non-classical NLS (NLS that lack the consensus motifs of basic amino acids) in the analysed reports indicate a complex scenario for the types of receptors in these species. In general, the information presented here agrees with the hypothetical appearance of mechanisms for the recognition of nuclear proteins in early eukaryotic evolution., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Kinetoplastid parasites such as Trypanosoma brucei, Trypanosoma cruzi, and Leishmania species rely upon their insect and vertebrate hosts to provide a plethora of nutrients throughout their life cycles. Nutrients and ions critical for parasite survival are taken up across the parasite plasma membrane by transporters and channels, polytopic membrane proteins that provide substrate-specific pores across the hydrophobic barrier. However, transporters and channels serve a wide range of biological functions beyond uptake of nutrients. This article highlights the diversity of activities that these integral membrane proteins serve and underscores the emerging complexity of their functions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The limited flexibility and time-consuming nature of the genetic manipulation procedures applicable to Trypanosoma cruzi continue to restrict the functional dissection of this parasite. We hypothesised that transformation efficiency could be enhanced if electroporation was timed to coincide with DNA replication. To test this, we generated epimastigote cultures enriched at the G1/S boundary using hydroxyurea-induced cell-cycle synchronisation, and then electroporated parasites at various time points after release from the cell-cycle block. We found a significant increase in transformation efficiency, with both episomal and integrative constructs, when cultures were electroporated 1 h after hydroxyurea removal. It was possible to generate genetically modified populations in less than 2 weeks, compared to the normal 4-6 weeks, with a 5 to 8-fold increase in the number of stably transformed clones. This straightforward optimisation step can be widely applied and should help streamline functional studies in T. cruzi., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In the present work, we evaluated the effect of mixed Trypanosoma cruzi infections, studying the biological distribution of the different parasites in blood, heart and skeletal muscle during the acute phase. Albino Swiss mice were infected with different parasite strain/isolates or with a combination of them. The parasites in the different tissues were typified through specific PCR, population variability was analyzed through RFLP studies and parasitological and histopathological parameters were evaluated. We found a predominance of TcII and TcVI in all tissues samples respect to TcV and different parasite populations were found in circulation and in the tissues from the same host. These results verify the distribution of parasites in host tissues from early stages of infection and show biological interactions among different genotypes and populations of T. cruzi., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the protozoan parasite Trypanosoma cruzi - the causative agent of Chagas disease - gene expression control is mainly post-transcriptional, where RNA-binding proteins (RBPs) play a central role, by controlling mRNA stability, distribution and translation. A large variety of RBPs are encoded in the T. cruzi genome, including the CCCH-type zinc finger (CCCH ZnF) protein family, which is characterized by the presence of the C-X 7/8 -C-X 5 -C-X 3 -H (CCCH) motif. In the related parasite T. brucei, CCCH ZnF proteins have been shown to control key differentiation steps in the parasite's life cycle. However, little is known about the CCCH ZnF proteins in T. cruzi. We have worked on the generation of T. cruzi mutants for CCCH ZnF proteins in an effort to shed light on the functions of these proteins in this parasite. Here, we characterize the expression and function of the CCCH ZnF protein TcZC3H31 of T. cruzi. TcZC3H31 is almost exclusively expressed in epimastigotes and metacyclic trypomastigotes, the parasite forms found in the invertebrate host. Importantly, we show that the epimastigote form of the T. cruzi knockout for the TcZC3H31 gene (TcZC3H31 KO) is incapable, both in vitro and in vivo (in infected triatomine insects), to differentiate into the metacyclic trypomastigote form, which is responsible for infection transmission from vectors to humans. The epimastigote forms recovered from the excreta of insects infected with TcZC3H31 KO parasites do not have the typical epimastigote morphology, suggesting that parasites are arrested in a mid-differentiation step. Also, epimastigotes overexpressing TcZC3H31 differentiate into metacyclics more efficiently than wild-type epimastigotes, in vitro. These data suggest that TcZC3H31 is an essential positive regulator of T. cruzi differentiation into the human-infective metacyclic form., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Per-ARNT-Sim (PAS) domains of proteins play important roles as modules for signalling and cellular regulation processes in widely diverse organisms such as Archaea, Bacteria, protists, plants, yeasts, insects and vertebrates. These domains are present in many proteins where they are used as sensors of stimuli and modules for protein interactions. Characteristically, they can bind a broad spectrum of molecules. Such binding causes the domain to trigger a specific cellular response or to make the protein containing the domain susceptible to responding to additional physical or chemical signals. Different PAS proteins have the ability to sense redox potential, light, oxygen, energy levels, carboxylic acids, fatty acids and several other stimuli. Such proteins have been found to be involved in cellular processes such as development, virulence, sporulation, adaptation to hypoxia, circadian cycle, metabolism and gene regulation and expression. Our analysis of the genome of different kinetoplastid species revealed the presence of PAS domains also in different predicted kinases from these protists. Open-reading frames coding for these PAS-kinases are unusually large. In addition, the products of these genes appear to contain in their structure combinations of domains uncommon in other eukaryotes. The physiological significance of PAS domains in these parasites, specifically in Trypanosoma cruzi, is discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Gene function studies in Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, have been hindered by the lack of efficient genetic manipulation protocols. In most organisms, insertion and deletion of DNA fragments in the genome are dependent on the generation of double-stranded DNA break (DSB) and repair. By inducing a site-specific DSB, zinc finger nucleases (ZFNs) have proven to be useful to enhance gene editing in many cell types. Using a pair of ZFNs targeted to the T. cruzi gp72 gene, we were able to generate gp72 knockout parasites with improved efficiency compared to the conventional gene knockout protocol. We also provide evidence that, in T. cruzi, repair of DSBs generated by ZFNs occurs primarily by the homologous recombination pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Amastins are surface glycoproteins, first identified in amastigotes of T. cruzi but later found to be expressed in several Leishmania species, as well as in T. cruzi epimastigotes. Amastins are encoded by a diverse gene family that can be grouped into four subfamilies named α, β, γ, and δ amastins. Differential expression of amastin genes results from regulatory mechanisms involving changes in mRNA stability and/or translational control. Although distinct regulatory elements were identified in the 3' UTR of T. cruzi and Leishmania amastin mRNAs, RNA binding proteins involved with amastin gene regulation have only being characterized in L. infantum where an Alba-domain protein (LiAlba20) able to bind to the 3' UTR of a δ-amastin mRNA was identified. Here we investigated the role of TcAlba30, the LiAlba20 homologue in T. cruzi, in the post transcriptional regulation of amastin genes. TcAlba30 transcripts are present in all stages of the T. cruzi life cycle. RNA immunoprecipitation assays using a transfected cell line expressing a cMyc tagged TcAlba30 revealed that TcAlba30 can interact with β-amastin mRNA. In addition, over-expression of TcAlba30 in epimastigotes resulted in 50% decreased levels of β-amastin mRNAs compared to wild type parasites. Since luciferase assays indicated the presence of regulatory elements in the 3' UTR of β-amastin mRNA and reduced levels of luciferase mRNA were found in parasites over expressing TcAlba30, we conclude that TcAlba30 acts as a T. cruzi RNA binding protein involved in the negative control of β-amastin expression through interactions with its 3'UTR., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Glucokinase and hexokinase from pathogenic protozoa Trypanosoma cruzi are potential drug targets for antiparasitic chemotherapy of Chagas' disease. These glucose kinases phosphorylate d-glucose with co-substrate ATP and yield glucose 6-phosphate and are involved in essential metabolic pathways, such as glycolysis and the pentose phosphate pathway. An inhibitor class was conceived that is selective for T. cruzi glucokinase (TcGlcK) using structure-based drug design involving glucosamine having a linker from the C2 amino that terminates with a hydrophobic group either being phenyl, p-hydroxyphenyl, or dioxobenzo[b]thiophenyl groups. The synthesis and characterization for two of the four compounds are presented while the other two compounds were commercially available. Four high-resolution X-ray crystal structures of TcGlcK inhibitor complexes are reported along with enzyme inhibition constants (Ki) for TcGlcK and Homo sapiens hexokinase IV (HsHxKIV). These glucosamine analogue inhibitors include three strongly selective TcGlcK inhibitors and a fourth inhibitor, benzoyl glucosamine (BENZ-GlcN), which is a similar variant exhibiting a shorter linker. Carboxybenzyl glucosamine (CBZ-GlcN) was found to be the strongest glucokinase inhibitor known to date, having a Ki of 0.71±0.05μM. Also reported are two biologically active inhibitors against in vitro T. cruzi culture that were BENZ-GlcN and CBZ-GlcN, with intracellular amastigote growth inhibition IC50 values of 16.08±0.16μM and 48.73±0.69μM, respectively. These compounds revealed little to no toxicity against mammalian NIH-3T3 fibroblasts and provide a key starting point for further drug development with this class of compound., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The histone H4 from Trypanosomatids diverged from other eukaryotes in the N-terminus, a region that undergoes post-translation modifications involved in the control of gene expression, DNA replication, and chromatin assembly. Nonetheless, the N-terminus of Trypanosoma cruzi histone H4 is mainly acetylated at lysine 4. The lysines 10 and 14 are also acetylated, although at less extent, increasing during the S-phase or after DNA damage, which suggests a regulatory function. Here, we investigated the roles of these acetylations by expressing non-acetylated forms of histone H4 in T. cruzi. We found that histone H4 containing arginines at positions 10 or 14, to prevent acetylation were transported to the nucleus and inserted into the chromatin. However, their presence, even at low levels, interfered with DNA replication and transcription, causing a significant growth arrest of the cells. The absence of acetylation also increased the amount of soluble endogenous histones H3 and H4 and affected the interaction with Asf1, a histone chaperone. Therefore, acetylation of lysines 10 and 14 of the histone H4 in trypanosomes could be required for chromatin assembly and/or remodeling required for transcription and replication., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, an illness that affects 6-7 million people and for which there is no effective drug therapy or vaccine. The publication of its complete genome sequence allowed a rapid advance in molecular studies including in silico screening of genes involved with pathogenicity as well as molecular targets for the development of new diagnostic methods, drug therapies and prophylactic vaccines. Alongside with in silico genomic analyses, methods to study gene function in this parasite such as gene deletion, overexpression, mutant complementation and reporter gene expression have been largely explored. More recently, the use of genome-wide strategies is producing a shift towards a global perspective on gene function studies, with the examination of the expression and biological roles of gene networks in different stages of the parasite life cycle and under different contexts of host parasite interactions. Here we describe the molecular tools and protocols currently available to perform genetic manipulation of the T. cruzi genome, with emphasis on recently described strategies of gene editing that will facilitate large-scale functional genomic analyses. These new methodologies are long overdue, since more efficient protocols for genetic manipulation in T. cruzi are urgently needed for a better understanding of the biology of this parasite and molecular processes involved with the complex and often harmful, interaction with its human host., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Flagellar calcium-binding protein (FCaBP) is a dually acylated Ca(2+) sensor in the Trypanosoma cruzi flagellar membrane that undergoes a massive conformational change upon Ca(2+) binding. It is similar to neuronal Ca(2+) sensors, like recoverin, which regulate their binding partners through a calcium acyl switch mechanism. FCaBP is washed out of permeabilized cells with buffers containing EDTA, indicating Ca(2+)-dependent flagellar membrane association. We hypothesized that, like recoverin, FCaBP projects its acyl groups in the presence of Ca(2+), permitting flagellar membrane and binding partner association and that it sequesters the acyl groups in low Ca(2+), disassociating from the membrane and releasing its binding partner to perform a presumed enzymatic function. The X-ray crystal structure of FCaBP suggests that the acyl groups are always exposed, so we set out to test our hypothesis directly. We generated T. cruzi transfectants expressing FCaBP or Ca(2+)-binding mutant FCaBP(E151Q/E188Q) and recombinant wildtype and mutant proteins as well. Both FCaBP and FCaBP(E151Q/E188Q) were found to associate with lipid rafts, indicating the Ca(2+)-independence of this association. To our initial surprise, FCaBP(E151Q/E188Q), like wildtype FCaBP, exhibited Ca(2+)-dependent flagellar membrane association, even though this protein does not bind Ca(2+) itself [16]. One possible explanation for this is that FCaBP(E151Q/E188Q), like some other Ca(2+) sensors, may form dimers and that dimerization of FCaBP(E151Q/E188Q) with endogenous wildtype FCaBP might explain its Ca(2+)-dependent localization. Indeed both proteins are able to form dimers in the presence and absence of Ca(2+). These results suggest that FCaBP possesses two distinct Ca(2+)-dependent interactions-one involving a Ca(2+)-induced change in conformation and another perhaps involving binding partner association., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In Trypanosoma cruzi three isoenzymes of phosphoglycerate kinase (PGK) are found which are simultaneously expressed: the cytosolic isoenzyme PGKB as well as two glycosomal enzymes, PGKA and PGKC. In this paper, we show that PGKA in T. cruzi epimastigotes is associated to the glycosomal membrane; it is responsible for about 23% of the glycosomal PGK activity, the fraction that remains in the pellet after osmotic shock treatment of purified organelles, in contrast to the 77% soluble activity that is mainly attributed to PGKC. Antibodies against the unique 80 amino-acid insertion of PGKA blocked almost completely the glucose consumption by epimastigotes that were partially permeabilized with digitonin. These results indicate that PGKA is the predominant isoenzyme for sustaining glycolysis through the glycosomes of these parasites., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The Trypanosoma cruzi adenylyl cyclase (AC) multigene family encodes different isoforms (around 15) sharing a variable large N-terminal domain, which is extracellular and receptor-like, followed by a transmembrane helix and a conserved C-terminal catalytic domain. It was proposed that these key enzymes in the cAMP signalling pathway allow the parasite to sense its changing extracellular milieu in order to rapidly adapt to its new environment, which is generally achieved through a differentiation process. One of the critical differentiation events the parasitic protozoan T. cruzi undergoes during its life cycle, known as metacyclogenesis, occurs in the digestive tract of the insect and corresponds to the differentiation from noninfective epimastigotes to infective metacyclic trypomastigote forms. By in vitro monitoring the activity of AC during metacyclogenesis, we showed that both the activity of AC and the intracellular cAMP content follow a similar pattern of transient stimulation in a two-step process, with a first activation peak occurring during the first hours of nutritional stress and a second peak between 6 and 48 h, corresponding to the cellular adhesion. During this differentiation process, a general mechanism of upregulation of AC expression of both mRNA and protein is triggered and in particular for a major subclass of these enzymes that are present in various gene copies commonly associated to the THT gene clusters. Although the scattered genome distribution of these gene copies is rather unusual in trypanosomatids and seems to be a recent acquisition in the evolution of the T. cruzi clade, their encoded product redistributed on the flagellum of the parasite upon differentiation could be important to sense the extracellular milieu., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Over the last years, an expanding family of small regulatory RNAs (e.g. microRNAs, siRNAs and piRNAs) was recognized as key players in novel forms of post-transcriptional gene regulation in most eukaryotes. However, the machinery associated with Ago/Dicer-dependent small RNA biogenesis was thought to be either entirely lost or extensively simplified in some unicellular organisms including Trypanosoma cruzi, Saccharomyces cerevisiae, Leishmania major and Plasmodium falciparum. Although the biogenesis of small RNAs from non-coding RNAs represent a minor fraction of the normal small RNA transcriptome in eukaryotic cells, they represent the unique small RNA pathways in Trypanosoma cruzi which produce different populations of small RNAs derived from tRNAs, rRNAs, sn/snoRNAs and mRNAs. These small RNAs are secreted included in extracellular vesicles and transferred to other parasites and susceptible mammalian cells. This process represents a novel form of cross-kingdom transfer of genetic material suggesting that secreted vesicles could represent new relevant pieces in life cycle transitions, infectivity and cell-to-cell communication. Here, we provide for the first time a detailed analysis of the small RNA cargo of extracellular vesicles from T. cruzi epimastigotes under nutritional stress conditions compared to the respective intracellular compartment using deep sequencing. Compared with the intracellular compartment, shed extracellular vesicles showed a specific extracellular signature conformed by distinctive patterns of small RNAs derived from rRNA, tRNA, sno/snRNAs and protein coding sequences which evidenced specific secretory small RNA processing pathways., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The scarcity of molecular tools for genetic manipulation is a critical obstacle for functional genomics studies on Trypanosoma cruzi. The current study adapted an inducible site-specific recombination system based on Dimerizable CRE recombinase (DiCRE). Two vectors for stable transfection were created, a first one to express inactive portions of DiCRE recombinase, and a second plasmid containing the loxP sites to test DiCRE activity. After integrating both constructs into the T. cruzi genome, it was shown that DiCRE recombinase can be efficiently used to manipulate its genome by allowing the removal of selectable markers thus generating homogeneous populations. The DiCRE recombinase success allows conditional knockout and the removal of selectable markers without prior parasite modification, which also facilitate the transferring of DiCRE recombinase to different T. cruzi strains., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cyclic AMP has been implicated as second messenger in a wide range of cellular processes. In the protozoan parasite Trypanosoma cruzi, cAMP is involved in the development of the parasite's life cycle. While cAMP effectors have been widely studied in other eukaryotic cells, little is known about cAMP's mechanism of action in T. cruzi. To date, only a cAMP-dependent protein kinase A (PKA) has been cloned and characterised in this parasite; however experimental evidence indicates the existence of cAMP-dependent, PKA-independent events. In order to identify new cAMP binding proteins as potential cAMP effectors, we carried out in silico studies using the predicted T. cruzi proteome. Using a combination of search methods 27 proteins with putative cNMP binding domains (CBDs) were identified. Phylogenetic analysis of the CBDs presented a homogeneous distribution, with sequences segregated into two main branches: one containing kinases-like proteins and the other gathering hypothetical proteins with different function or no other known. Comparative modelling of the strongest candidates provides support for the hypothesis that these proteins may give rise to structurally viable cyclic nucleotide binding domains. Pull-down and nucleotide displacement assays strongly suggest that TcCLB.508523.80 could bind cAMP and eventually be a new putative PKA-independent cAMP effector in T. cruzi., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The capacity for rapid localization of epitope-tagged or fluorescent fusion proteins in cells is an important tool for biological discovery and functional analysis. For Trypanosoma cruzi, the protozoan parasite that causes human Chagas disease, visualization of ectopically-expressed proteins in the clinically-relevant mammalian stages is hindered by the necessity to first perform transfection and lengthy selection procedures in the insect vector form of the parasite. Here, we demonstrate the ability to by-pass the insect stage with the delivery of plasmid DNA to non-dividing, tissue culture trypomastigotes such that upon host cell infection, transgenes are expressed and rapidly localized in intracellular T. cruzi amastigotes. The inclusion of a sorting step prior to host cell infection by trypomastigotes greatly enriches (>90%) the number of transgene-expressing amastigotes observed in mammalian host cells. This is a significant methodological advance that has the potential to accelerate the pace of discovery in the Chagas disease field., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A growing body of evidence in mammalian cells indicates that secreted vesicles can be used to mediate intercellular communication processes by transferring various bioactive molecules, including mRNAs and microRNAs. Based on these findings, we decided to analyze whether Trypanosoma cruzi-derived extracellular vesicles contain RNA molecules and performed a deep sequencing and genome-wide analysis of a size-fractioned cDNA library (16-40nt) from extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Our data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs. In addition, our results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Trypanosoma cruzi alternates between replicative and non-replicative stages. We analyzed the expression of components of the pre-replication machinery TcORC1/CDC6 and TcMCM7 and their interaction with DNA in all T. cruzi stages. TcORC1/CDC6 remains in the nuclear space during all stages of the life cycle and interacts with DNA in the replicative stages; however, it does not bind to DNA in the non-replicative forms. Moreover, TcMCM7 is not present in the non-replicative stages. These data suggest that the lacking of DNA replication during the T. cruzi life cycle may be a consequence of the blocking of TcORC1/CDC6-DNA interaction and of the down regulation of the TcMCM7 expression., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Benznidazole is the main drug used to treat Trypanosoma cruzi infections. However, frequent instances of treatment failure have been reported. To better understand potential resistance mechanisms, we analysed three clones isolated from a single parasite population that had undergone benznidazole-selection. These clones exhibited differing levels of benznidazole-resistance (varying between 9 and 26-fold), and displayed cross-resistance to nifurtimox (2 to 4-fold). Each clone had acquired a stop-codon-generating mutation in the gene which encodes the nitroreductase (TcNTR) that is responsible for activating nitroheterocyclic pro-drugs. In addition, one clone had lost a copy of the chromosome containing TcNTR. However, these processes alone are insufficient to account for the extent and diversity of benznidazole-resistance. It is implicit from our results that additional mechanisms must also operate and that T. cruzi has an intrinsic ability to develop drug-resistance by independent sequential steps, even within a single population. This has important implications for drug development strategies., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Phospholipase A1 (PLA1) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of protein kinase C. In the present work we determined higher levels of PLA1 expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA1 activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA1 antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA1 in the early events of parasite-host cell interaction. We also demonstrated the presence of lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA1 action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA1 with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA1 selected gene was cloned and expressed as a recombinant protein that possessed PLA1 activity. Collectively, the results presented here point out at T. cruzi PLA1 as a novel virulence factor implicated in parasite invasion., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi is expected to synthetize de novo cysteine by different routes, among which the two-step pathway involving serine acetyltransferase and cysteine synthase (CS) is comprised. Also, cystathionine β synthase (CBS) might contribute to the de novo generation of cysteine in addition to catalyze the first step of the reverse transsulfuration route producing cystathionine. However, neither the functionality of CS nor that of cystathionine γ lyase (CGL) has been assessed. Our results show that T. cruzi CS could participate notably more actively than CBS in the de novo synthesis of cysteine. Interestingly, at the protein level T. cruzi CS is more abundant in amastigotes than in epimastigotes. Unlike the mammalian homologues, T. cruzi CGL specifically cleaves cystathionine into cysteine and is unable to produce H(2)S. The expression pattern of T. cruzi CGL parallels that of CBS, which unexpectedly suggests that in addition to the de novo synthesis of cysteine, the reverse transsulfuration pathway could be operative in the mammalian and insect stages. Besides, T. cruzi CBS produces H(2)S by decomposing cysteine or via condensation of cysteine with homocysteine. The latter reaction leads to cystathionine production, and is catalyzed remarkably more efficiently than the breakdown of cysteine. In T. cruzi like in other organisms, H(2)S could exert regulatory effects on varied metabolic processes. Notably, T. cruzi seems to count on stage-specific routes involved in cysteine production, the multiple cysteine-processing alternatives could presumably reflect this parasite's high needs of reducing power for detoxification of reactive oxygen species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2Δ mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2Δ mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polβ) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolβ) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolβ showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolβ overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolβ was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolβ during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In eubacteria and eukaryotic organelles N-terminal methionine excision requires the sequential action of two activities, a peptide deformylase (PDF), which systematically removes the N-formyl group present on all nascent polypeptides and methionine aminopeptidase (MAP), which exscinds methionine specifically and depends on the previous removal of the N-formyl group. In Trypanosoma cruzi two genes encoding bacterial PDF homologues have been identified and referred to as TcPDF-1 and TcPDF-2. Here we report the biochemical characterization of a truncated soluble version of TcPDF-1 lacking the hydrophobic N-terminal domain that is active with the bacterial PDF substrate formyl-methionyl-alanyl-serine but, in contrast to other PDFs, is not inhibited by actinonin. The enzyme is strongly activated by Cu(2+) and inhibited by Ni(2+). Our results show that T. cruzi PDF exhibits unique features thus providing a new avenue for the design of potential inhibitors for use in the treatment of diseases caused by trypanosomatid parasites., (© 2012 Elsevier B.V. All rights reserved.)

In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [(3)H]dihydrosphingosine or [(3)H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B(1), a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasite's CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B(1) did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasite's CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1Δ lac1Δ double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B(1) than the parental strain., (© 2011 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi exhibits two putative isocitrate dehydrogenases (IDHs). Both idh genes were cloned and the recombinant enzymes expressed in Escherichia coli. Our results showed that T. cruzi IDHs are strictly dependent on NADP(+) and display apparent affinities towards isocitrate and the coenzyme in the low micromolar range. In T. cruzi, IDHs are cytosolic and mitochondrial enzymes, and there is no evidence for the typical Krebs cycle-related NAD-dependent IDH. Hence, like in Trypanosoma brucei, the Krebs cycle is not a canonical route in T. cruzi. However, the citrate produced in the mitochondrion could be isomerized into isocitrate in the cytosol and the mitochondrion by means of the putative aconitase, which would provide the substrate for both IDHs. The cytosolic IDH is significantly more abundant in amastigotes, cell-derived and metacyclic trypomastigotes than in epimastigotes. This observation fits in well with the expected oxidative burst this pathogen has to face when infecting the mammalian host., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Components of the DNA mismatch repair (MMR) pathway are major players in processes known to generate genetic diversity, such as mutagenesis and DNA recombination. Trypanosoma cruzi, the protozoan parasite that causes Chagas disease has a highly heterogeneous population, composed of a pool of strains with distinct characteristics. Studies with a number of molecular markers identified up to six groups in the T. cruzi population, which showed distinct levels of genetic variability. To investigate the molecular basis for such differences, we analyzed the T. cruzi MSH2 gene, which encodes a key component of MMR, and showed the existence of distinct isoforms of this protein. Here we compared cell survival rates after exposure to genotoxic agents and levels of oxidative stress-induced DNA in different parasite strains. Analyses of msh2 mutants in both T. cruzi and T. brucei were also used to investigate the role of Tcmsh2 in the response to various DNA damaging agents. The results suggest that the distinct MSH2 isoforms have differences in their activity. More importantly, they also indicate that, in addition to its role in MMR, TcMSH2 acts in the parasite response to oxidative stress through a novel mitochondrial function that may be conserved in T. brucei., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Kinetoplast DNA (kDNA) of trypanosomatid protozoa consists of an unusual arrangement of two types of circular molecules catenated into a single network: (1) a few maxicircles that encode various mitochondrial enzyme subunits and rRNA in a cryptic pattern and (2) thousands of minicircles that encode guide RNAs (gRNAs). kDNA is associated with proteins, known as kinetoplast-associated proteins (KAPs), which condense the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasite that displays different kDNA condensation patterns during its complex morphogenetic development. We cloned the T. cruzi gene encoding TcKAP3 (kinetoplast-associated protein 3). TcKAP3 is a single-copy gene that is transcribed into a 1.8-kb mRNA molecule and expressed in all stages of the parasite. Mouse antiserum raised against recombinant TcKPA3 recognized a 21.8kDa protein, which was found, by indirect immunofluorescence and immunoelectron microscopy, to be associated with the T. cruzi kinetoplast. Several features of TcKAP3, such as its small size, basic nature and similarity with KAP3 from the insect trypanosomatid Crithidia fasciculata, are consistent with a role in DNA charge neutralization and condensation. This suggests that this protein is involved in organizing the kDNA network. Gene deletion was used to investigate TcKAP3 function. Here we investigated the T. cruzi KAP3 null mutant, analyzing its fitness during proliferation, differentiation and infectivity.

Over the last years an expanding family of small RNAs (i.e. microRNAs, siRNAs and piRNAs) was recognized as key players in diverse forms of gene silencing and chromatin organization. Effectors functions of these small RNAs are achieved through ribonucleoprotein (RNP) complexes containing at their center an Argonaute/Piwi protein. Although these proteins and their small RNA-associated machinery can be traced back to the common ancestor of eukaryotes, this machinery seems to be entirely lost or extensively simplified in some unicellular organisms including Trypanosoma cruzi, which are unable to trigger RNAi related phenomena. Speculating about the presence of alternate small RNA-mediated pathways in these organisms, we constructed and analyzed a size-fractionated cDNA library (20-35 nt) from epimastigotes forms of T. cruzi. Our results showed the production of an abundant class of tRNA-derived small RNAs preferentially restricted to specific isoacceptors and whose production was more accentuated under nutritional stress. These small tRNAs derived preferentially from the 5' halves of mature tRNAs and were recruited to distinctive cytoplasmic granules. Our data favor the idea that tRNA cleavage is unlikely to be the consequence of non-specific degradation but a controlled process, whose biological significance remains to be elucidated., (Copyright 2010 Elsevier B.V. All rights reserved.)

As part of a drug discovery programme to discover new treatments for human African trypanosomiasis, recombinant trypanothione reductase from Trypanosoma brucei has been expressed, purified and characterized. The crystal structure was solved by molecular replacement to a resolution of 2.3A and found to be nearly identical to the T. cruzi enzyme (root mean square deviation 0.6A over 482 Calpha atoms). Kinetically, the K(m) for trypanothione disulphide for the T. brucei enzyme was 4.4-fold lower than for T. cruzi measured by either direct (NADPH oxidation) or DTNB-coupled assay. The K(m) for NADPH for the T. brucei enzyme was found to be 0.77microM using an NADPH-regenerating system coupled to reduction of DTNB. Both enzymes were assayed for inhibition at their respective S=K(m) values for trypanothione disulphide using a range of chemotypes, including CNS-active drugs such as clomipramine, trifluoperazine, thioridazine and citalopram. The relative IC(50) values for the two enzymes were found to vary by no more than 3-fold. Thus trypanothione reductases from these species are highly similar in all aspects, indicating that they may be used interchangeably for structure-based inhibitor design and high-throughput screening.

In animal cells, the exon junction complex (EJC) is deposited onto mRNAs during the second step of splicing, 20-24 nt upstream of the exon-exon junction. The EJC core contains four proteins: Mago, Y14, eIF4AIII and Btz. In trypanosomes, cis-splicing is very rare but all mRNAs are subject to 5'trans-splicing of a 39-nt RNA sequence. Here we show that trypanosomes have a conserved Mago and a divergent Y14 protein, but we were unable to identify a Btz orthologue. We demonstrate that Mago and Y14 form a stable heterodimer using yeast two hybrid analyses. We also show that this complex co-purifies in vivo in trypanosomes with a protein containing an NTF2 domain, typically involved in mRNA transport.

Surface adhesion proteins are essential for Trypanosoma cruzi invasion of mammalian cells. Here we show that Dispersed Gene Family-1 (DGF-1) members, previously identified as nuclear repeated sequences present in several chromosomes and comprising the third largest T. cruzi specific gene family, have conserved adhesin motifs including four segments with significant similarity to human beta 7 integrin. Flow cytometry and biotinylation assays with anti-DGF-1 antibodies indicated that, as expected, DGF-1 members are expressed on the trypomastigote surface. The DGF-1 genealogy, inferred using T. cruzi Genome Project data and network phylogeny algorithms, suggests that this gene family is separated in at least three groups with differential distribution of functional domains. To identify which members of this gene family are expressed we used a combined approach of RT-PCR and codon usage profiles, showing that expressed members have a very biased codon usage favoring GC, whereas non-expressed members have a homogeneous distribution. Shannon information entropy was used to measure sequence variability and revealed four major high entropy segments in the extracellular domain of DGF-1 overlapping with important putative functional modules of the predicted proteins. Testing for natural selection, however, indicated that these high entropy segments were not under positive selection, which contradicts the notion that positive selection is the cause of high variability in specific domains of a protein relative to other less variable regions in the same molecule. We conjectured that members of the DGF-1 family might be associated with the ability of T. cruzi to bind extracellular matrix proteins, such as fibronectin and laminin, and speculated on mechanisms that would be generating the localized diversity in these molecules in the absence of selection.

Early in the assembly of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF, consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, we identified, cloned and expressed the Trypanosoma cruzi and Trypanosoma brucei U2AF35, U2AF65 and SF1. Trypanosomatid U2AF65 strongly diverged from yeast and human homologues. On the contrary, trypanosomatid SF1 was conserved but lacked the C-terminal sequence present in the mammalian protein. Yeast two hybrid approaches were used to assess their interactions. The interaction between U2AF35 and U2AF65 was very weak or not detectable. However, as in other eukaryotes, the interaction between U2AF65 and SF1 was strong. At the cellular level, these results were confirmed by fractionation and affinity-selection experiments in which SF1 and U2AF65 were affinity-selected with TAP tagged SF1, but not with TAP tagged U2AF35. Silencing one of the three factors affected growth and trans-splicing in the first step of this reaction. Trypanosomes are the first described example of eukaryotic cells in which the interaction of two expressed U2AF factors seemed to be very weak, or not detectable.

The 195-bp satellite DNA is the most abundant Trypanosoma cruzi repetitive sequence. Here we show by RNA blotting and RT-PCR that 195 SAT is intensely transcribed. We observed a positive correlation between the level of satellite RNA and the abundance of the satellite copies in the genome of T. cruzi strains and that the satellite expression is not developmentally regulated. By analyzing CL Brener individual reads, we estimated that 195 SAT corresponds to approximately 5% of the CL Brener genome. 195 SAT elements were found in only 37 annotated contigs, indicating that a large number of satellite copies were not incorporated into the assembled data. The assembled satellite units are distributed in non-syntenic regions with Trypanosoma brucei and Leishmania major genomes, enriched with surface proteins, retroelements, RHS and hypothetical proteins. Satellite repeats were not observed in annotated subtelomeric regions. We report that 12 satellite sequences are truncated by the retroelement VIPER.

Cytochrome P450 hemoproteins (CYPs) are involved in the synthesis of endogenous compounds such as steroids, fatty acids and prostaglandins as well as in the activation and detoxification of foreign compounds including therapeutic drugs. Cytochrome P450 reductase (CPR, E.C.1.6.2.4) transfers electrons from NADPH to a number of hemoproteins such as CYPs, cytochrome c, cytochrome b5, and heme oxygenase. This work presents the complete sequences of three non-allelic CPR genes from Trypanosoma cruzi. The encoded proteins named TcCPR-A, TcCPR-B and TcCPR-C have calculated molecular masses of 68.6kDa, 78.4kDa and 71.3kDa, respectively. Deduced amino acid sequences share 11% amino acid identity, possess the conserved binding domains for FMN, FAD and NADPH and differ in the hydrophobic 27-amino acid residues of the N-terminal extension, which is absent in TcCPR-A. Every T. cruzi CPRs, TcCPR-A, TcCPR-B and TcCPR-C, were cloned and expressed in Escherichia coli. All of the recombinant enzymes reduced cytochrome c in a NADPH absolutely dependent manner with low K(m) values for this cofactor. They all were also strongly inhibited by diphenyleneiodonium, a classical flavoenzyme inhibitor. In addition, TcCPRs could support CYP activities when assayed in reconstituted systems containing rat liver microsomes. Polyclonal antiserum rose against the recombinant enzymes TcCPR-A and TcCPR-B demonstrated its presence in every T. cruzi developmental stages, with a remarkable expression of TcCPR-A in cell-cultured trypomastigotes. Overexpression of TcCPR-B in T. cruzi epimastigotes increased its resistance to the typical chemotherapeutic agents Nifurtimox and Benznidazole. We suggest a participation of TcCPR-B in the detoxification metabolism of the parasite.

Several copies of genes belonging to three multigene families present in the genome of Trypanosoma cruzi were sequenced and comparatively analyzed across six different strains of the parasite belonging to the T. cruzi I lineage (Colombiana, Silvio X10 and Dm28c), the T. cruzi II lineage (Esmeraldo and JG) and a hybrid strain (CL Brener). For all three gene families analyzed, our results support the division in T. cruzi I and II lineages. Furthermore, in agreement with its hybrid nature, sequences derived from the CL Brener clone clustered together with T. cruzi II sequences as well as with a third group of sequences. Paralogous sequences encoding Amastin, an amastigote surface glycoprotein and TcAG48, an antigenic RNA binding protein, which are clustered in the parasite genome, present higher intragenomic variability in T. cruzi II and CL Brener strains, when compared to T. cruzi I strains. Paralogous sequences derived from the TcADC gene family, which encode various isoforms of adenylyl cyclases and are dispersed throughout the T. cruzi genome, exhibit similar degree of variability in all strains, except in the CL Brener strain, in which the sequences were more divergent. Several factors including mutation rates and gene conversion mechanisms, acting differently within the T. cruzi population, may contribute to create such distinct levels of sequence diversity in multigene families that are clustered in the T. cruzi genome.

Glucokinase genes, found in the genome databases of Trypanosoma cruzi and Leishmania major, were cloned and sequenced. Their expression in Escherichia coli resulted in the synthesis of soluble and active enzymes, TcGlcK and LmjGlcK, with a molecular mass of 43 kDa and 46 kDa, respectively. The enzymes were purified, and values of their kinetic parameters determined. The K(m) values for glucose were 1.0 mM for TcGlcK and 3.3 mM for LmjGlcK. For ATP, the K(m) values were 0.36 mM (TcGlcK) and 0.35 mM (LmjGlcK). A lower K(m) value for glucose (2.55 mM) was found when the (His)(6)-tag was removed from the recombinant LmjGlcK, whereas the TcGlcK retained the same value. The V(max)'s of the T. cruzi and L. major GlcKs were 36.3 and 30.9 U/mg of protein, respectively. No inhibition was exerted by glucose-6-phosphate. Similarly, no inhibition by inorganic pyrophosphate was found in contrast to previous observations made for the T. cruzi and L. mexicana hexokinases. Both trypanosomatid enzymes were only able to phosphorylate glucose indicating that they are true glucokinases. Gel-filtration chromatography showed that the GlcK of both trypanosomatids may occur as a monomer or dimer, dependent on the protein concentration. Both GlcK sequences have a type-1 peroxisome-targeting signal. Indeed, they were shown to be present inside glycosomes using three different methods. These glucokinases present highest, albeit still a moderate 24% sequence identity with their counterpart from Trichomonas vaginalis, which has been classified into group A of the hexokinase family. This group comprises mainly eubacterial and cyanobacterial glucokinases. Indeed, multiple sequence comparisons, as well as kinetic properties, strongly support the notion that these trypanosomatid enzymes belong to group A of the hexokinases, in which they, according to a phylogenetic analysis, form a separate cluster.

Entomopathogenic nematode Heterorhabditis bacteriophora infective juveniles (IJs) employ multiple strategies to combat with insect innate immune system after invasion. We employed suppressive subtractive hybridization (SSH) technique to study the gene expression in the IJs upon contact with the insect hemolymph in vitro. We identified two clones having higher expression levels in the IJs than IJs treated with insect hemolymph. The differential expression levels were confirmed by Northern blot hybridization with reference to the constitutive expression level of Heterorhabditis bacteriophora actin2 gene. Clone HbGPS11.C1G02 encoded a phosphofructokinase (PFK) with a 2.5 kb transcript and clone HbGPS11.C4C08 corresponded to a 2.1 kb transcript encoding a protein that had weak similarity to trans-sialidase from Trypanosoma cruzi. The differential expression of PFK in H. bacteriophora IJs before and during recovery process represented a switch from active movement in search of insect hosts to a state of combating insect innate immune system. This first report of H. bacteriophora differential gene expression provides a glimpse at the gene expression profile of H. bacteriophora IJ recovery process.

Parvulins are a conserved group of peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze the cis/trans isomerization of proline-preceding peptide bonds. Parvulin-class PPIases are structurally unrelated to cyclophilins and FK506-binding proteins that are defined as receptors for immunosuppressive drugs. In Trypanosoma cruzi we identified parvulin TcPIN1 as a homolog of the human hPin1 PPIase. The 117 amino acids of the TcPIN1 display 40% identity with the catalytic core of hPin1 and exhibit prolyl cis/trans isomerase activity. TcPIN1 lacks the WW domain at the N-terminus, and is able to rescue the temperature-sensitive phenotype on a mutation in the Saccharomyces cerevisiae hPin1 homolog, ESS1/PTF1. Western blot analysis revealed that the enzyme was present both in dividing and non-dividing forms of T. cruzi. In epimastigote cells neither cell growth kinetics nor cell morphology was affected by the overexpression of the small parvulin TcPIN1. These results suggest the occurrence of a supplementary conserved level of post-translational control in trypanosomatids.

Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity.

Bioinformatic analyses have been used to identify potential downstream targets of the essential enzyme N-myristoyl transferase in the TriTryp species, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. These database searches predict approximately 60 putative N-myristoylated proteins with high confidence, including both previously characterised and novel molecules. One of the latter is an N-myristoylated protein phosphatase which has high sequence similarity to the Protein Phosphatase with EF-Hand (PPEF) proteins identified in sensory cells of higher eukaryotes. In L. major and T. brucei, the PPEF-like phosphatases are encoded by single-copy genes and are constitutively expressed in all parasite life cycle stages. The N-terminus of LmPPEF is a substrate for N-myristoyl transferase and is also palmitoylated in vivo. The wild type protein has been localised to the endocytic system by immunofluorescence. The catalytic and fused C-terminal domains of the kinetoplastid and other eukaryotic PPEFs share high sequence similarity, but unlike their higher eukaryotic relatives, the C-terminal parasite EF-hand domains are degenerate and do not bind calcium.

Histone tails provide sites for a variety of post-translational modifications implicated in the control of gene expression and chromatin assembly. As both histones and control of gene expression in trypanosomes are highly divergent compared to most eukaryotes, post-translational modifications of Trypanosoma cruzi histones were investigated. After in vivo incubation of live parasites with radiolabeled precursors, histone H4 mainly incorporates [(3)H]-acetyl, and to a lesser extent [(3)H]-methyl residues. In contrast, histone H3 preferentially incorporates [(3)H]-methyl residues. The modifications of histone H4 were further characterized by mass spectrometry. MALDI-TOF-TOF-MS analysis revealed that peptides from histone H4 amino-terminus, obtained by either endoproteinase Glu-C or endoproteinase Arg-C digestion, contain isoforms with 14 and 42Da additions, suggesting the presence of simultaneous acetylations and/or methylations. Tandem mass spectrometry analysis demonstrated that the N-terminal alanine is methylated, and lysine residues at positions 4, 10, 14 and 57 are acetylated; lysine at position 18 is mono-methylated, while arginine at position 53 is dimethylated. Immunoblotting analyses using specific antibodies raised against synthetic and acetylated peptides of T. cruzi histone H4 indicate that lysine 4 is acetylated in the majority of histone H4, while other acetylations at the N-terminus portion of histone H4 are less abundant.

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form). Every year 2-3% of these individuals develop severe clinical manifestations (cardiac and digestive forms). In this study a Trypanosoma cruzi DNA microarray was used to compare the transcript profiles of six human isolates: three from asymptomatic and three from cardiac patients. Seven signals were expressed differentially between the two classes of isolates, including tryparedoxin, surface protease GP63, cyclophilin, some hypothetical proteins and the pre-edited maxicircle gene NADH dehydrogenase subunit 7 (ND7). The approximately 30-fold greater signal in cardiac strains for ND7 was the most pronounced of the group, and differential levels of pre-edited ND7 transcript confirmed the microarray analysis. The ND7 gene from asymptomatic isolates showed a deletion of 455bp from nt 222 to nt 677 relative to ND7 of the CL Brener reference strain. The ND7 gene structure correlated with disease manifestation for 20 isolates from clinically characterised, chronic phase patients. The ND7 lesion produces a truncated product that could impair the function of mitochondrial complex I. Possible links between the integrity of the electron transport chain and symptom presentation are discussed. We propose that ND7 and other genes of the pathway constitute valuable targets for PCR assays in the differential diagnosis of the infective T. cruzi strain. While this hypothesis requires validation by the examination of additional recent parasite isolates from patients with defined pathologies, the identification of specific molecular markers represents a promising advance in the association between parasite genetics and disease pathology.