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Chagas Disease, African sleeping sickness, and leishmaniasis are neglected diseases caused by pathogenic trypanosomatid parasites, which have a considerable impact on morbidity and mortality in poor countries. The available drugs used as treatment have high toxicity, limited access, and can cause parasite drug resistance. Long-term treatments, added to their high toxicity, result in patients that give up therapy. Trypanosomatids presents a unique trypanothione based redox system, which is responsible for maintaining the redox balance. Therefore, inhibition of these essential and exclusive parasite’s metabolic pathways, absent from the mammalian host, could lead to the development of more efficient and safe drugs. The system contains different redox cascades, where trypanothione and tryparedoxins play together a central role in transferring reduced power to different enzymes, such as 2-Cys peroxiredoxins, non-selenium glutathione peroxidases, ascorbate peroxidases, glutaredoxins and methionine sulfoxide reductases, through NADPH as a source of electrons. There is sufficient evidence that this complex system is essential for parasite survival and infection. In this review, we explore what is known in terms of essentiality, kinetic and structural data, and the development of inhibitors of enzymes from this trypanothione-based redox system. The recent advances and limitations in the development of lead inhibitory compounds targeting these enzymes have been discussed. The combination of molecular biology, bioinformatics, genomics, and structural biology is fundamental since the knowledge of unique features of the trypanothione-dependent system will provide tools for rational drug design in order to develop better treatments for these diseases.

Background: Trypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin that plays an important role in coping with host cell oxidative response during the infection process, for which it has been described as a virulence factor. Methods: Four residues corresponding to c-TXNPx catalytic and solvent-exposed cysteines were individually mutated to serine by site-specific mutagenesis. Susceptibility to redox treatments and oligomeric dynamics were investigated by western-blot and gel filtration chromatography. Chaperone and peroxidase activities were determined. Results: In this study we demonstrated that c-TXNPx exists as different oligomeric forms, from decameric to high molecular mass aggregates. Moreover, c-TXNPx functions as a dual-function protein acting both as a peroxidase and as a molecular chaperone. Its chaperone function was shown to be independent of the presence of catalytic cysteines, even in the reduced and decameric forms, although it is enhanced when the protein is overoxidized leading to the formation of high molecular mass aggregates. Conclusions: c-TXNPx has chaperone activity which does not depend on the redox state. c-TXNPx does not undergo the dimer-decamer transition in the oxidized state described for other peroxiredoxins. Overoxidized c-TXNPx exists as different oligomeric forms from decamer to high molecular mass aggregates which are in a very slow dynamic equilibrium. The non-catalytic C57 residue may have a role in the maintenance of the decameric form, but seems not to have an alternative CP and CR role. General significance: This study provides novel insights into some key aspects of the oligomerization dynamics and function of c-TXNPx. Fil: Piñeyro, María Dolores. Universidad de la Republica Facultad de Medicina; Uruguay. Instituto Pasteur de Montevideo; Uruguay Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Ricciardi, Guillermo Alejandro. Instituto Pasteur de Montevideo; Uruguay Fil: Robello, Carlos. Universidad de la Republica Facultad de Medicina; Uruguay. Instituto Pasteur de Montevideo; Uruguay Fil: Parodi-Talice, Adriana. Universidad de la Republica; Uruguay. Instituto Pasteur de Montevideo; Uruguay

Background: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. Methods: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications. Findings: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. Interpretation: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection. Fil: Niborski, Leticia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Potenza, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Chirivi, Renato G.S.. No especifíca; Fil: Simonetti, Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Ossowski, Micaela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Grippo, Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: May, Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Staquicini, Daniela I.. Universidade Federal de Sao Paulo; Brasil Fil: Parodi Talice, Adriana. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; Uruguay Fil: Robello, Carlos. Universidad de la República; Uruguay Fil: Comini, Marcelo. Universidad de la Republica; Uruguay Fil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Raats, Jos M.H.. Universidad de la República; Uruguay Fil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Trypanosoma cruzi is a genetically heterogeneous group of organisms that cause Chagas disease. It has been long suspected that the clinical outcome of the disease and response to therapeutic agents are, at least in part, related to the genetic characteristics of the parasite. Herein, we sought to validate the significance of the genotype of T. cruzi isolates recovered from patients with different clinical forms of Chagas disease living in Argentina on their biological behaviour and susceptibility to drugs. Genotype identification of the newly established isolates confirmed the reported predominance of TcV, with a minor frequency of TcI. Epimastigote sensitivity assays demonstrated marked dissimilar responses to benznidazole, nifurtimox, pentamidine and dihydroartemisinin in vitro. Two TcV isolates exhibiting divergent response to benznidazole in epimastigote assays were further tested for the expression of anti-oxidant proteins. Benznidazole-resistant BOL-FC10A epimastigotes had decreased expression of Old Yellow Enzyme and cytosolic superoxide dismutase, and over-expression of mitochondrial superoxide dismutase and tryparedoxin- 1, compared to benznidazole-susceptible AR-SE23C parasites. Drug sensitivity assays on intracellular amastigotes and trypomastigotes reproduced the higher susceptibility of AR-SE23C over BOL-FC10A parasites to benznidazole observed in epimastigotes assays. However, the susceptibility/resistance profile of amastigotes and trypomastigotes to nifurtimox, pentamidine and dihydroartemisinin varied markedly with respect to that of epimastigotes. C3H/He mice infected with AR-SE23C trypomastigotes had higher levels of parasitemia and mortality rate during the acute phase of infection compared to mice infected with BOL-FC10A trypomastigotes. Treatment of infected mice with benznidazole or nifurtimox was efficient to reduce patent parasitemia induced by either isolate. Nevertheless, qPCR performed at 70 dpi revealed parasite DNA in the blood of mice infected with AR-SE23C but not in BOL-FC10A infected mice. These results demonstrate high level of intra-type diversity which may represent an important obstacle for the testing of chemotherapeutic agents. Fil: Quebrada Palacio, Luz Piedad. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: González, Mariela Natacha. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hernandez Vasquez, Yolanda Maria. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina Fil: Perrone, Alina Elizabeth. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Parodi Talice, Adriana. Universidad de la República; Uruguay Fil: Bua, Jacqueline Elena. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Postan, Miriam. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus that causes one of the most persistent respiratory diseases in poultry. The virus is classified in genotypes and lineages with different epidemiological relevance. Two lineages of the GI genotype (11 and 16) have been widely circulating for decades in South America. GI-11 is an exclusive South American lineage while the GI-16 lineage is distributed in Asia, Europe and South America. Here, we obtained the whole genome of two Uruguayan strains of the GI-11 and GI-16 lineages using Illumina high-throughput sequencing. The strains here sequenced are the first obtained in South America for the infectious bronchitis virus and provide new insights into the origin, spreading and evolution of viral variants. The complete genome of the GI-11 and GI-16 strains have 27,621 and 27,638 nucleotides, respectively, and possess the same genomic organization. Phylogenetic incongruence analysis reveals that both strains have a mosaic genome that arose by recombination between Euro Asiatic strains of the GI-16 lineage and ancestral South American GI-11 viruses. The recombination occurred in South America and produced two viral variants that have retained the full-length S1 sequences of the parental lineages but are extremely similar in the rest of their genomes. These recombinant virus have been extraordinary successful, persisting in the continent for several years with a notorious wide geographic distribution. Our findings reveal a singular viral dynamics and emphasize the importance of complete genomic characterization to understand the emergence and evolutionary history of viral variants., Highlights • Genomic analysis was performed in two main lineages of Infectious bronchitis virus. • Lineages differ in their S1 sequences but are similar in the rest of the genome. • Genomic similarity between both lineages arise by inter-lineage recombination. • Inter-lineage recombination occurred in South America between European/Asiatic and local strain. • Recombinant forms have persisted in the continent for several years with wide geographic distribution.

Trypanosoma cruzi species is categorized into six discrete typing units (TcI to TcVI) of which TcI is most abundantly noted in the sylvatic transmission cycle and considered the major cause of human disease. In our study, the TcI strains Colombiana (COL), SylvioX10/4 (SYL), and a cultured clone (TCC) exhibited different biological behavior in a murine model, ranging from high parasitemia and symptomatic cardiomyopathy (SYL), mild parasitemia and high tissue tropism (COL), to no pathogenicity (TCC). Proteomic profiling of the insect (epimastigote) and infective (trypomastigote) forms by two-dimensional gel electrophoresis/ matrix-assisted laser desorption ionization-time of flight mass spectrometry, followed by functional annotation of the differential proteome data sets (≥2-fold change, P

International audience; The surface of Trypanosoma cruzi is covered by a dense glycocalix which is characteristic of each stage of the life cycle. Its composition and complexity depend mainly on mucin-like proteins. A remarkable feature of O-glycan biosynthesis in trypanosomes is that it initiates with the addition of a GlcNAc instead of the GalNAc residue that is commonly used in vertebrate mucins. The fact that the interplay between trans-sialidase and mucin is crucial for pathogenesis, and both families have stage-specific members is also remarkable. Recently the enzyme that transfers the first GlcNAc from UDP-GlcNAc to a serine or threonine residue was kinetically characterized. The relevance of this enzyme is evidenced by its role as catalyzer of the first step in O-glycosylation. In this paper we describe how this gene is expressed differentially along the life cycle with a pattern that is very similar to that of trans-sialidases. Its localization was determined, showing that the protein predicted to be in the Golgi apparatus is also present in reservosomes. Finally our results indicate that this enzyme, when overexpressed, enhances T. cruzi infectivity.

International audience; 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.

Trypanosoma cruzi, the causative agent of the Chagas disease, has a complex life cycle alternating between replicative and noninfective forms with nonreplicative and infective forms of the parasite. Metacyclogenesis is a process that takes place in the invertebrate host, comprising morphogenetic transformation from a noninfective form to an infective form, such that parasites acquire the ability to invade human cells. We analyze here the metacyclogenesis process by 2D electrophoresis coupled to MALDI-TOF MS. A large proportion of unique proteins expressed during metacyclogenesis were observed. Interestingly, 50% of the spots were found to differ between epimastigotes and trypomastigotes. We provide a 2D map of the infective metacyclic trypomastigotes. Sixty six protein spots were successfully identified corresponding to 43 different proteins. We analyzed the expression profiles for the identified proteins along metacyclogenesis and classified them into three groups according to their maximal level of expression. We detected several isoforms for a number of proteins, some displaying differential expression during metacyclogenesis. These results suggest that posttranslational modifications may be a fundamental part of the parasite's strategy for regulating gene expression during differentiation. This study contributes to the identification of relevant proteins involved in the metacyclogenesis process. The identification and molecular characterization of these proteins will render vital information about the steps of the parasite differentiation into the infective form.

Protozoan parasites are responsible of important healthy problems, among others malaria, leishmaniasis and trypanosomiasis. The present work reports the characterization of the first mammalian ATP-binding cassette transporter, subfamily A (ABCA)-like in Trypanosoma cruzi. TcABC1 is a single copy gene differentially expressed along the life cycle of the parasite, being absent in its infective form. TcABC1 localizes to the plasma membrane, flagellar pocket and intracellular vesicles. Functional studies of TcABC1 in transfected parasites suggest that the protein is implicated in intracellular trafficking, as determined by the analysis of endocytosis and exocytosis events. The accumulation of the endocytic markers FM4-64 and NBD-SM is increased in transfected parasites. Similarly, ectophosphatase and ectoATPase activities are increased in TcABC1 overproducers. Indeed, transmission electronic microscopy analysis showed a higher number of intracellular vesicles in TcABC1 transfectants. Taken together, these results suggest that the protein is involved in the endocytic and exocytic pathways of T. cruzi.

Trypanosoma cruzi, the causative agent of Chagas disease, has evolved particular mechanisms of gene regulation. Gene expression is regulated firstly at post-transcriptional level. This feature makes proteomic methods a promising tool for studying adaptative changes in these parasites. In this work we generated a reproducible method for protein analysis by two-dimensional electrophoresis coupled to mass spectrometry, and a protein map for T. cruzi. Western-blot analysis supported the identity of some of the proteins. This work points to proteomic approach as a powerful tool to study differential expression, stress response or drug resistance in T. cruzi.

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 theT. cruziinvasion 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 ofT. cruziin 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 byT. 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.

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. Fil: Piñeyro, María Dolores. Universidad de la Republica Facultad de Medicina; Uruguay. Instituto Pasteur de Montevideo; Uruguay Fil: Parodi Talice, Adriana. Universidad de la Republica; Uruguay. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; Uruguay Fil: Portela, Magdalena. Universidad de la República; Uruguay. Universidad de la Republica; Uruguay Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Robello, Carlos. Instituto Pasteur de Montevideo; Uruguay. Universidad de la Republica Facultad de Medicina; Uruguay

Cytosolic and mitochondrial Trypanosoma cruzi tryparedoxin peroxidases belong to the family of 2-Cys peroxiredoxins. These enzymes play an essential role as antioxidants by their peroxidase and peroxynitrite reductase activities. TXNPx are key components of the trypanosomatid peroxide detoxification pathways. The aim of this work was to determine the role of TXNPx as virulence factors in the parasite, and whether these enzymes are good candidates for drug design. We observed that peroxiredoxins are not highly abundant proteins expressed at similar levels throughout the T. cruzi life cycle. In order to study the role of c-TXNPx and m-TXNPx in invasion and infectivity, parasites overexpressing TXNPx were produced, and infection experiments were carried out using phagocytic and non-phagocytic cells. Parasites overexpressing peroxiredoxins showed a significant increase in infectivity with respect to the control ones. The results presented in this work point out that the T. cruzi peroxiredoxins are important in survival, replication and differentiation of T. cruzi and could constitute virulence factors. Moreover, their expression in the infective forms of the life cycle and their low intracellular concentration make them good candidates to become targets for drug design.

Miltefosine (hexadecylphosphocholine) is the first orally active drug approved for the treatment of leishmaniasis. We have previously shown the involvement of LtrMDR1, a P-glycoprotein-like transporter belonging to the ATP-binding cassette superfamily, in miltefosine resistance in Leishmania. Here we show that overexpression of LtrMDR1 increases miltefosine efflux, leading to a decrease in drug accumulation in the parasites. Although LtrMDR1 modulation might be an efficient way to overcome this resistance, a main drawback associated with the use of P-glycoprotein inhibitors is related to their intrinsic toxicity. In order to diminish possible side effects, we have combined suboptimal doses of modulators targeting both the cytosolic and transmembrane domains of LtrMDRl. Preliminary structure-activity relationships have allowed us to design a new and potent flavonoid derivative with high affinity for the cytosolic nucleotide-binding domains. As modulators directed to the transmembrane domains, we have selected one of the most potent dihydro-β-agarofuran sesquiterpenes described, and we have also studied the effects of two of the most promising, latest-developed modulators of human P-glycoprotein, zosuquidar (LY335979) and elacridar (GF120918). The results show that this combinatorial strategy efficiently overcomes P-glycoprotein-mediated parasite miltefosine resistance by increasing intracellular miltefosine accumulation without any side effect in the parental, sensitive, Leishmania line and in different mammalian cell lines. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Here we report the characterization of LtrABCA2, a new ABC transporter of the ABCA subfamily in the protozoan parasite Leishmania tropica, localized at the flagellar pocket region and in internal vesicles. The overexpression of this transporter reduced the accumulation of fluorescent glycerophospholipid analogs, increased the exocytic activity, and decreased infectivity of macrophage, but did not confer resistance to drugs. Together, these results suggest that this new ABC transporter plays a role in phospholipid trafficking, which may be modifying the vesicular trafficking and the infectivity of the parasite. © 2005 Elsevier Inc. All rights reserved.

Protozoan parasites are responsible of important healthy problems, among others malaria, leishmaniasis and trypanosomiasis. The present work reports the characterization of the first mammalian ATP-binding cassette transporter, subfamily A (ABCA)-like in Trypanosoma cruzi. TcABC1 is a single copy gene differentially expressed along the life cycle of the parasite, being absent in its infective form. TcABC1 localizes to the plasma membrane, flagellar pocket and intracellular vesicles. Functional studies of TcABC1 in transfected parasites suggest that the protein is implicated in intracellular trafficking, as determined by the analysis of endocytosis and exocytosis events. The accumulation of the endocytic markers FM4-64 and NBD-SM is increased in transfected parasites. Similarly, ectophosphatase and ectoATPase activities are increased in TcABC1 overproducers. Indeed, transmission electronic microscopy analysis showed a higher number of intracellular vesicles in TcABC1 transfectants. Taken together, these results suggest that the protein is involved in the endocytic and exocytic pathways of T. cruzi.

This paper reports the characterization of a new ABC transporter (LtrABC1.1), related to the human ABCA subfamily, in the protozoan parasite Leishmania tropica. LtrABC1.1 is a tandem duplicated gene flanked by inverted repeats. LtrABC1.1 is expressed mainly in the flagellar pocket of the parasite. Drug resistance studies in Leishmania overexpressing LtrABC1.1 showed the transporter not to confer resistance to a range of unrelated drugs. LtrABC1.1 appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. The activity of this protein may also affect membrane movement processes since secreted acid phosphatase (SAP) activity was significantly lower in promastigotes overexpressing LtrABC1.1. In vitro infection experiments with macrophages indicated LtrABC1.1-transfected parasites to be significantly less infective. Together, these results suggest that this new ABC transporter could play a role in lipid movements across the plasma membrane, and that its activity might influence vesicle trafficking. This is the first ABCA-like transporter described in unicellular eukaryotes.

ATP-binding cassette (ABC) transporters constitute one of the biggest and most conserved protein families in the evolutionary scale. Many of them are of enormous clinical relevance, due to their relationship with genetic diseases and drug resistance during the treatment of cancer and infectious diseases. Leishmaniasis is a major and globally widespread group of parasitic diseases, whose treatment has been complicated by the expansion of resistance to conventional drugs. Here, we review the current knowledge about ABC transporters in Leishmania spp, with special attention to their relationship with the drug-resistance phenotype. © Springer-Verlag and SEM 2001.

Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C 5 -PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.