Patricia Cuervo

Analysis of an acute Chagas disease outbreak in the Brazilian Amazon: human cases, triatomines, reservoir mammals and parasites

An outbreak of Chagas disease occurred in Mazagão, Amapá, Brazilian Amazon in 1996. Seventeen of 26 inhabitants presented symptoms compatible with acute Chagas disease and were submitted to parasitological and serological tests. All 17 were positive in at least one parasitological test and 11 were also IgM or IgG anti-Trypanosoma cruzi positive. The nine asymptomatic patients were negative for parasites and one was positive for IgG anti-T. cruzi. Sixty-eight triatomines were captured (66 Rhodnius pictipes; two Panstrongylus geniculatus); 45 were infected with T. cruzi (43 R. pictipes; two P. geniculatus). Thirteen trypanosomatid strains were isolated: eight from humans and five from R. pictipes. Four were genotyped as T. cruzi I (two from humans; two from R. pictipes), seven as T. cruzi Z3 (six from humans; one from R. pictipes) and two as T. cruzi Z3 and T. rangeli (from R. pictipes). Treatment started for all patients leading to a decrease in parasitaemia in 16 during the follow-up period (6 months, 1, 5 and 7 years). All were serologically negative 7 years post-treatment. There was an overlap of genotypes in the same ecotope, raising the possibility of transmission through the oral route and the need for early therapeutic intervention for better patient management in the Brazilian Amazon.

Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes.

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host-parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1beta, and tryparedoxin peroxidase. Data mining showed that approximately 5% of identified proteins present a classical secretion signal whereas approximately 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.

Cross sectional study reveals a high percentage of asymptomatic Plasmodium vivax infection in the Amazon Rio Negro area, Brazil

A parasitological, clinical, serological and molecular cross-sectional study carried out in a highly endemic malaria area of Rio Negro in the Amazon State, Brazil, revealed a high prevalence of asymptomatic Plasmodium vivax infection. A total of 109 persons from 25 families were studied in five villages. Ninety-nine inhabitants (90.8%) had at least one previous episode of malaria. Serology showed 85.7% and 46.9% of positivity when P. falciparum antigens and P. vivax MSP-1, respectively, were used. Twenty blood samples were PCR positive for P. vivax (20.4%) and no P. falciparum infection was evidenced by this technique. No individual presenting positive PCR reaction had clinical malaria during the survey neither in the six months before nor after, confirming that they were cases of asymptomatic infection. Only one 12 year old girl presented a positive thick blood smear for P. vivax. This is the first description of asymptomatic Plasmodium infection in this area studied.

Proteomics of trypanosomatids of human medical importance.

Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei are protozoan parasites that cause a spectrum of fatal human diseases around the world. Recent completion of the genomic sequencing of these parasites has enormous relevance to the study of their biology and the pathogenesis of the diseases they cause because it opens the door to high-throughput proteomic technologies. This review encompasses studies using diverse proteomic approaches with these organisms to describe and catalogue global protein profiles, reveal changes in protein expression during development, elucidate the subcellular localisation of gene products, and evaluate host-parasite interactions.

Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

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.

Differential soluble protein expression between Trichomonas vaginalis isolates exhibiting low and high virulence phenotypes.

A comparative analysis of proteomic maps of long-term grown and fresh clinical Trichomonas vaginalis isolates exhibiting low and high virulence phenotypes, respectively, was performed using two-dimensional gel electrophoresis and mass spectrometry. Of 29 protein spots differentially expressed between the isolates, 19 were over-expressed in the isolate exhibiting high virulence phenotype: proteins associated with cytoskeletal dynamics, such as coronin and several isoforms of actin, as well as proteins involved in signal transduction, protein turnover, proteolysis, and energetic and polyamine metabolisms were identified. Some malate dehydrogenase, fructose-1,6-bisphosphate aldolase and ornithine cyclodeamidase isoforms were exclusively expressed by the highly virulent isolate. During interaction assays with VEC, parasites exhibiting high virulence phenotype rapidly adhered and switched to amoeboid forms. In contrast, low adhesion and no morphological transformation were observed in parasites displaying low virulence phenotype. Our findings demonstrate that expression of specific proteins by high and low virulence parasites could be associated with the ability of each isolate to undergo morphological transformation and interact with host cells. Such data represent an important step towards understanding of the complex interaction network of proteins that participate in the mechanism of pathogenesis of this protozoan.

Cysteine peptidase expression in Trichomonas vaginalis isolates displaying high- and low-virulence phenotypes.

In the present study, we identified and characterized the cysteine peptidase (CP) profiles of Trichomonas vaginalis isolates exhibiting high- and low-virulence phenotypes using a combination of two-dimensional SDS-PAGE (2DE), tandem mass spectrometry (MS/MS), and data mining. Seven of the eight CPs identified belong to Clan CA, family C1, cathepsin L-like CP, and one belongs to Clan CD, family C13, asparaginyl endopeptidase-like CP. Quantitative and qualitative differences in CP expression were detected between the isolates. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of differentially expressed CPs showed identity or high homology to previously described CP cDNA clones CP1, CP3, CP4, and to a secreted CP fraction of 30 kDa involved in apoptosis of vaginal epithelial cells. One- and two-dimensional-substrate gel analyses revealed the differential CP profiles between the isolates, indicating that the combination of zymography with 2DE and MS/MS might be a powerful experimental approach to map and identify active peptidases in T. vaginalis. Toxicity exerted upon HeLa cells by high- and low-virulence isolates was 98.3% and 31%, respectively. Pretreatment of parasites with specific Clan CA papain-like CP inhibitor l-3-carboxy-2,3-trans-epoxypropionyl-leucylamido(4-guanidino)butane (E-64) drastically reduced the cytotoxic effect to 21.7% and 0.8%, respectively, suggesting that T. vaginalis papain-like CPs are the main factors involved in the cellular damage.

A zymographic study of metalloprotease activities in extracts and extracellular secretions of Leishmania (Viannia) braziliensis strains

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.

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate-SDS-PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6-8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl-methyl sulfonyl-fluoride and N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin-like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin-like serine proteases occur during the developmental cycle of A. aegypti.

T-Cell Populations and Cytokine Expression Are Impaired in Thymus and Spleen of Protein Malnourished BALB/c Mice Infected with Leishmania infantum

Visceral leishmaniasis (VL) is a parasitic infectious disease that causes significant morbidity and mortality in the tropical and subtropical regions of the world. Although infections with visceralizing Leishmania may be asymptomatic, factors such as undernutrition increase the likelihood of progressing to clinical disease. Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered as a primary risk factor for the development of clinical VL. However, data regarding the immunological basis of this association are scarce. With the aim to analyze the effects of protein malnutrition on Leishmania infantum infection, we used BALB/c mice subjected to control or low protein isocaloric diets. Each animal group was divided into two subgroups and one was infected with L. infantum resulting in four study groups: animals fed 14% protein diet (CP), animals fed 4% protein diet (LP), animals fed 14% protein diet and infected (CPi), and animals fed 4% protein diet and infected (LPi).The susceptibility to L. infantum infection and immune responses were assessed in terms of body and lymphoid organ weight, parasite load, lymphocyte subpopulations, and cytokine expression. LPi mice had a significant reduction of body and lymphoid organ weight and exhibited a severe decrease of lymphoid follicles in the spleen. Moreover, LPi animals showed a significant decrease in CD4+CD8+ T cells in the thymus, whereas there was an increase of CD4+ and CD8+ T cells percentages in the spleen. Notably, the cytokine mRNA levels in the thymus and spleen of protein malnourished-infected animals were altered compared to the CP mice. Protein malnutrition results in a drastic dysregulation of T cells and cytokine expression in the thymus and spleen of L. infantum-infected BALB/c mice, which may lead to defective regulation of the thymocyte population and an impaired splenic immune response, accelerating the events of a normal course of infection.