Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture

Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.

Methylene blue vital staining for Trypanosoma cruzi trypomastigotes and epimastigotes

The morphological identification of Trypanosoma cruzi is currently considered to have a high specificity, but its sensitivity, which depends on the volume of the sample examined, is rather low. Trypanosome developmental stages suspended in blood, reduviid feces, and culture media are routinely searched for by means of fresh film examination (about 2 µL). High speed centrifugation of blood samples separates the buffy coat, where most trypomastigotes concentrate. As the parasites are transparent and colorless, their detection is mostly dependent on their motility. The fluorescent vital stain acridine orange has been used to enhance image contrast, as exemplified by the QBC (Quantitative Buffy Coat) technique. Staining blood, buffy coat, reduviid feces, and culture media samples with methylene blue (also a vital dye) is a means of producing sharp, well contrasted images of motile or non-motile T. cruzi developmental stages, only standard laboratory microscopes being required. Slides previously coated with a thin layer of methylene blue are used to stain fresh blood films. Photomicrographs exemplify the results of methylene blue staining applied to living and fixed parasites.

Morphological evidence by scanning electron microscopy of excretion of metacyclic forms of Trypanosoma cruzi in vector's urine

Comparision by scanning electron microscopy (SEM) of Trypanosoma cruzi flagellates attached to the cuticle of the rectal gland of infected Dipetalogaster maxima nymphs, showed marked differences before amd after feeding. Before feeding numerous metacyclic trypomastigotes were observed among the abundant epimastigotes that formed the carpet of flagellates. On the other hand, in insects that were allowed to urinate for 24 hours after a meal, the metacyclics were scarce,indicating that they had been detached by the urine flow. An asymetric type of cell division, probably originating both an epi-and a trypomastigote, was occasionally observed. The occurrence of swellings at different levels of the flagella of epimastigotes suggests that secondary sites of attachment may be common.

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37°C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.

Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite’s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Morphological, biological and molecular characterization of three strains of Trypanosoma cruzi Chagas, 1909 (Kinetoplastida, Trypanosomatidae) isolated from Triatoma sordida (Stal) 1859 (Hemiptera, Reduviidae) and a domestic cat

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A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (Ki) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 ± 2.47 µmol L-1. The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease.

GPIomics: global analysis of glycosylphosphatidylinositol-anchored molecules of Trypanosoma cruzi

Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole-low femtomole range) method that uses liquid chromatography-tandem mass spectrometry (LC-MS(n)) for the first large-scale analysis of GPI-anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome-wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI-anchored proteins. By analyzing the GPIome of T. cruzi insect-dwelling epimastigote stage using LC-MS(n), we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes. Molecular Systems Biology 7 April 2009; doi:10.1038/msb.2009.13

Kinetics of Trypanosoma cruzi destruction in the mouse spleen

Massive destruction of parasitized splenic macrophages was histologically observed at the height of a virulent infection caused by Trypanosoma cruzi (Y strain) in the mouse. This was coincident with a sudden drop in parasitemic curve. Most of the animals died at this point, probably due to the liberation of toxic products, such as TNF, following the massive destruction of parasitized cells. However, parasitized-cell destruction indicated the transition from susceptibility to resistance. Although it has been extensively studied in vitro, this study contributes with the morphological counterpart observed in vivo by optical and electron microscopy. When infected animals were specifically treated during early infection transition to chronic phase was immediately observed without splenic parasitism. Animals that apparently recovered from massive cell-destruction in the spleen showed evidences of a rapid restoration of splenic architecture.

American tripanosomiasis: a study on the prevalence of Trypanosoma cruzi and Trypanosoma cruzi-like organisms in wild rodents in San Luis province, Argentina

INTRODUCTION: Chagas disease is caused by Trypanosoma cruzi. Wild and perianthropic mammals maintain the infection/transmission cycle, both in their natural habitat and in the peridomestic area. The aim of this paper was to present the results from a study on wild rodents in the central and northern regions of San Luis province, Argentina, in order to evaluate the prevalence of this infection. METHODS: Sherman traps were set up in capture areas located between latitudes 32º and 33º S, and longitudes 65º and 66º W. The captured rodents were taxonomically identified and hemoflagellates were isolated. Morphological, biometric and molecular studies and in vitro cultures were performed. Infection of laboratory animals and histological examination of the cardiac muscle and inoculation area were also carried out. Parasites were detected in circulating blood in Calomys musculinus, Graomys griseoflavus, Phyllotis darwini and Akodon molinae. The parasites were identified using biological criteria. Molecular PCR studies were performed on some isolates, which confirmed the characterization of these hemoflagellates as Trypanosoma cruzi. RESULTS AND CONCLUSIONS: Forty-four percent of the 25 isolates were identified as Trypanosoma cruzi, and the remaining 56% as Trypanosoma cruzi-like. These findings provide evidence that wild rats infected with Trypanosoma cruzi and Trypanosoma cruzi-like organisms are important in areas of low endemicity.

Epidemiological factors related to the transmission risk of Trypanosoma cruzi in a Quilombola community, State of Mato Grosso do Sul, Brazil

INTRODUCTION: This work was an epidemiological investigation of the risk of Trypanosoma cruzi transmission in the rural Quilombola community of Furnas do Dionízio, State of Mato Grosso do Sul, Brazil. METHODS: Of the 71 animals examined, seven were captured (two opossums, Didelphis albiventris; four rats, Rattus rattus; and one nine-banded armadillo, Dasypus novemcinctus) and 64 were domestic (one canine, Canis familiaris; five pigs, Sus scrofa; two bovines, Bos taurus; five caprines, Capra sp.; and 51 ovines, Ovis aries). Parasitological tests were performed to detect parasites in the blood and to identify the morphology of flagellates. These methods included fresh examinations, buffy coat tests and blood cultures. Molecular analysis of DNA for identification of trypanosomatids was performed by polymerase chain reaction (PCR) with primers S35 and S36. RESULTS: The parasitological tests showed flagellates in an opossum and two cattle. The molecular tests showed DNA from T. cruzi in an opossum and a pig. Triatoma sordida was the only triatomine species found in the community, and it colonized households (four specimens) and the surrounding areas (124 specimens). Twenty-three specimens tested positive for flagellates, which were subsequently identified as T. cruzi by PCR. CONCLUSIONS: Data analysis demonstrated that T. cruzi has a peridomestic life cycle that involves both domestic and wild mammals.