An active Trypanosoma cruzi transmission cycle maintained by wild rodents in the Andean valleys of Cochabamba Bolivia is described. Wild and domestic Triatoma infestans with 60% infection with T. cruzi were found and was evidenced in... more
An active Trypanosoma cruzi transmission cycle maintained by wild rodents in the Andean valleys of Cochabamba Bolivia is described. Wild and domestic Triatoma infestans with 60% infection with T. cruzi were found and was evidenced in 47.5% (rodents) and 26.7% (marsupial) by parasitological and/or serologycal methods. Phyllotis ocilae and the marsupial species Thylamys elegans, are the most important reservoirs followed by Bolomys lactens and Akodon boliviensis. In spite of both genotypes (TCI and TCII) being prevalent in Bolivia, in our study area only T. cruzi I is being transmitted. Our data suggest that wild T. infestans and wild small mammals play an important role in the maintenance of the transmission cycle of T. cruzi. Furthermore, the finding of high prevalence of T. cruzi infection in wild T. infestans point to the risk of the dispersion of Chagas' disease.
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Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum,... more
Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites.
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Iron is an essential element for the survival of trichomonads during host-parasite interaction. The availability of this metal modulates several metabolic pathways of the parasites and regulates the expression of virulence factors such as... more
Iron is an essential element for the survival of trichomonads during host-parasite interaction. The availability of this metal modulates several metabolic pathways of the parasites and regulates the expression of virulence factors such as adhesins and proteolytic enzymes. In this study, we investigated the effect of iron depletion on the morphology and life cycle of Tritrichomonas foetus. Scanning and transmission electron microscopy analyses revealed that depletion of iron from the culture medium (named TYM-DIP inducer medium) induces morphological transformation of typical pear-shaped trophozoites into spherical and non-motile pseudocysts. Remarkably, inoculation of pseudocysts into an iron-rich medium (standard TYM medium), or addition of FeSO4 to a TYM-DIP inducer medium reverted the morphological transformation process and typical trophozoites were recovered. These results show that pseudocysts are viable forms of the parasite and highlight the role of iron as a modulator of the parasite phenotype. Although iron is required for the survival of T. foetus, iron depletion does not cause a cellular collapse of pseudocysts, but instead induces phenotypic alterations, probably in order to allow the parasite to survive conditions of nutritional stress. Together, these findings support previous studies that suggest pseudocysts are a resistance form in the life cycle of T. foetus and enable new approaches to understanding the multifactorial role of iron in the cell biology of this protozoan parasite.
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Anopheles (Nyssorhynchus) aquasalis is a malaria vector mainly distributed along the coastal regions of South and Central America. In the absence of an effective vaccine against malaria, strategies for controlling the vector are the main... more
Anopheles (Nyssorhynchus) aquasalis is a malaria vector mainly distributed along the coastal regions of South and Central America. In the absence of an effective vaccine against malaria, strategies for controlling the vector are the main tool for interrupting parasite transmission. Mechanisms of oogenesis and embryogenesis in anautogenous mosquitoes are mainly modulated by blood feeding. However, the expression, at the protein level, of genes involved in such mechanisms in sugar-fed females is unknown. In this work, total protein extracts of the reproductive tract of female An. aquasalis that were fed sugar were analyzed using liquid chromatography followed by mass spectrometry for protein identification and bioinformatic tools for data mining. We identified 922 proteins expressed in the organ, and using several databases, we attributed biological meaning for several of them. Remarkably, nine proteins involved in oogenesis were identified in females fed sugar. Putative vitellogenins...
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Leishmania spp. are digenetic parasites which cause a broad spectrum of fatal diseases in humans. These parasites, as well as the other trypanosomatid, regulate gene expression at the post-transcriptional and post-translational levels, so... more
Leishmania spp. are digenetic parasites which cause a broad spectrum of fatal diseases in humans. These parasites, as well as the other trypanosomatid, regulate gene expression at the post-transcriptional and post-translational levels, so that a poor correlation is observed between mRNA content and translated proteins. The completion of the genomic sequencing of several Leishmania species has enormous relevance to the study of the leishmaniasis pathogenesis. The combination of the available genomic resources of these parasites with powerful high-throughput proteomic analysis has shed light on various aspects of Leishmania biology as well as on the mechanisms underlying the disease. Diverse proteomic approaches have been used to describe and catalogue global protein profiles of Leishmania spp., reveal changes in protein expression during development, determine the subcellular localization of gene products, evaluate host-parasite interactions and elucidate drug resistance mechanisms. The characterization of these proteins has advanced, although many fundamental questions remain unanswered. Here, we present a historic review summarizing the different proteomic technologies applied to the study of Leishmania parasites during the last decades and we discuss the proteomic discoveries that have contributed to the understanding of Leishmania parasites biology and leishmaniasis.
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Molecules involved in pheromone biosynthesis may represent alternative targets for insect population control. This may be particularly useful in managing the reproduction of Lutzomyia longipalpis, the main vector of the protozoan parasite... more
Molecules involved in pheromone biosynthesis may represent alternative targets for insect population control. This may be particularly useful in managing the reproduction of Lutzomyia longipalpis, the main vector of the protozoan parasite Leishmania infantum in Latin America. Besides the chemical identity of the major components of the L. longipalpis sex pheromone, there is no information regarding the molecular biology behind its production. To understand this process, obtaining information on which genes are expressed in the pheromone gland is essential. In this study we used a transcriptomic approach to explore the pheromone gland and adjacent abdominal tergites in order to obtain substantial general sequence information. We used a laboratory-reared L. longipalpis (one spot, 9-Methyl GermacreneB) population, captured in Lapinha Cave, state of Minas Gerais, Brazil for this analysis. From a total of 3,547 cDNA clones, 2,502 high quality sequences from the pheromone gland and adjacent tissues were obtained and assembled into 1,387 contigs. Through blast searches of public databases, a group of transcripts encoding proteins potentially involved in the production of terpenoid precursors were identified in the 4th abdominal tergite, the segment containing the pheromone gland. Among them, protein-coding transcripts for four enzymes of the mevalonate pathway such as 3-hydroxyl-3-methyl glutaryl CoA reductase, phosphomevalonate kinase, diphosphomevalonate descarboxylase, and isopentenyl pyrophosphate isomerase were identified. Moreover, transcripts coding for farnesyl diphosphate synthase and NADP+ dependent farnesol dehydrogenase were also found in the same tergite. Additionally, genes potentially involved in pheromone transportation were identified from the three abdominal tergites analyzed. This study constitutes the first transcriptomic analysis exploring the repertoire of genes expressed in the tissue containing the L. longipalpis pheromone gland as well as the flanking tissues. Using a comparative approach, a set of molecules potentially present in the mevalonate pathway emerge as interesting subjects for further study regarding their association to pheromone biosynthesis. The sequences presented here may be used as a reference set for future research on pheromone production or other characteristics of pheromone communication in this insect. Moreover, some matches for transcripts of unknown function may provide fertile ground of an in-depth study of pheromone-gland specific molecules.
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Protozoan parasites are responsible for an impressive disease burden in developing and less-developed countries. The development of vaccines and effective new therapies for dealing with these organisms are among the main gaps to be filled... more
Protozoan parasites are responsible for an impressive disease burden in developing and less-developed countries. The development of vaccines and effective new therapies for dealing with these organisms are among the main gaps to be filled in the control of protozoan parasite diseases. Programmed cell death (PCD) pathways have gained attention in recent years because they comprise complex signalling pathways that
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Ten pairs of Leishmania (Viannia) strains isolated from mucosal and cutaneous lesions of the same patient were analyzed genotypically in order to determine whether populations that had metastasized to mucosal sites differed from those in... more
Ten pairs of Leishmania (Viannia) strains isolated from mucosal and cutaneous lesions of the same patient were analyzed genotypically in order to determine whether populations that had metastasized to mucosal sites differed from those in the cutaneous lesion. The strains were previously characterized by multi locus enzyme electrophoresis and/or monoclonal antibodies reactivity, and, for this study, only isolates from the same patient which were identified as the same species were employed. PCR-RFLP of internal transcribed spacer (ITS) rDNA, random amplified polymorphic DNA (RAPD), and schizodeme analyses were conducted. All genotyping methods revealed microheterogeneity between cutaneous and mucosal isolates from the same patient. The PCR-RFLP of the ITS rDNA and RAPD analysis were numerically analyzed through similarity coefficients and dendrograms were generated. All phenograms clustered cutaneous and mucosal strains of the same patient in one branch with a high degree of similari...
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American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of... more
American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.
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We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex... more
We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 microM), a specific inhibitor of chymotrypsin-like serine proteases.
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Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that encompasses a broad spectrum of clinical manifestations. In a previous study, we showed that Brazilian and Colombian L.... more
Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that encompasses a broad spectrum of clinical manifestations. In a previous study, we showed that Brazilian and Colombian L. braziliensis strains, isolated from patients with distinct clinical manifestations, display different pattern of metalloprotease activities. Following these results, we investigated the cellular localization of these molecules and their relation to the major surface protease (gp63) of Leishmania. Comparative analyses of metalloprotease expression among different clinical isolates as well as an evaluation of the effect of long-term in vitro passage on the expression pattern of these metalloproteases were also performed. Western blot analysis, using an anti-gp63 antibody, revealed polypeptide patterns with a similar profile to that observed in zymographic analysis. Flow cytometry and fluorescence microscopy analyses corroborated the presence of metallo...
Research Interests: Colombia, Flow Cytometry, Fluorescence Microscopy, Brazil, Confocal Microscopy, and 13 moreWestern blotting, Biological Sciences, Leishmaniasis, Humans, Animals, Flagella, Spectrum, In vitro culture, Western blot, Protozoan Proteins, Proteome, American Tegumentary Leishmaniasis, and Proteolytic activity
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The presence of iron in the extracellular medium is essential for both in vivo and in vitro survival of pathogenic microorganisms, including Trichomonas vaginalis and Tritrichomonas foetus. In these parasites, iron is directly involved in... more
The presence of iron in the extracellular medium is essential for both in vivo and in vitro survival of pathogenic microorganisms, including Trichomonas vaginalis and Tritrichomonas foetus. In these parasites, iron is directly involved in the proliferation, protein expression and activation of critical enzymes. The purpose of this study was to investigate the role of iron in ecto-ATPase, ecto-phophatase and secreted phosphatase activities of these trichomonads. We observed that trichomonads grown in iron-depleted medium exhibited a remarkable decrease in both ecto-ATPase and ecto-phosphatase activities, when compared to those cultivated under control conditions (iron-rich medium). Furthermore, parasites grown in iron-depleted medium restored their enzyme activities when they were re-inoculated into fresh iron-rich medium. We demonstrated that modulation of ecto-phosphohydrolase activities is due neither to enzyme-iron nor to substrate-iron complex formation, since iron addition directly to the medium where the enzymatic reactions occurred did not alter their activities. Previously, we had reported that a fresh clinical isolate of T. vaginalis was much more cytotoxic to epithelial cell monolayers than a long-term cultured one. In this study we witnessed that the fresh isolate of T. vaginalis presented higher activities to all herein investigated enzymes than the long-term cultured one. Altogether, our data clearly point out that iron has a pivotal role in the expression of phosphohydrolases in both trichomonads.
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Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and... more
Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and extracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5.5 to 9.0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L. (V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation.
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Research Interests: Mass Spectrometry, Biological Sciences, Molecular, Animals, New World, and 12 moreBiological Process, Protein Expression, Virulence factor, Proteome analysis, Protozoan Proteins, Gel electrophoresis, Two-Dimensional Gel Electrophoresis, Drug Targeting, Two dimensional Gel Electrophoresis, Proteome, Clinical Diagnosis, and Isoelectric point
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Research Interests: Mass Spectrometry, Proteomics, Signal Transduction, Virulence, Protein Turnover, and 14 moreAnimals, Trichomonas vaginalis, Comparative Analysis, Solubility, Phenotype, Protein Expression, Proteome analysis, Protozoan Proteins, Two-Dimensional Gel Electrophoresis, Two dimensional Gel Electrophoresis, Biochemistry and cell biology, Gene Expression Regulation, Interaction Network, and Differential expression
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Research Interests: Chemical Engineering, Analytical Chemistry, Mass Spectrometry, Animal Production, Time-of-flight mass spectrometry, and 18 moreOrganelles, Celiac Disease, Mass, Raw materials, Animals, Trichomonas vaginalis, Process Analysis, Clinical Sciences, CHEMICAL SCIENCES, Electrophoresis, Production Process, Food Sciences, Proteome analysis, Protozoan Proteins, Two-Dimensional Gel Electrophoresis, Proteome, Biochemistry and cell biology, and Isoelectric point
The sequence, genomic organization, and transcription of the gene encoding the H2A histone protein of the protozoan parasite Trypanosoma rangeli is described in this paper. The locus encoding the T. rangeli H2A protein is formed by at... more
The sequence, genomic organization, and transcription of the gene encoding the H2A histone protein of the protozoan parasite Trypanosoma rangeli is described in this paper. The locus encoding the T. rangeli H2A protein is formed by at least 11 gene units measuring 790 nucleotides in length, organized in tandem, and located in a single chromosome of approximately 1.9 Mb. The gene