Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 angstrom resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 angstrom. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

Background: The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. Methodology/Principal Findings: We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. Conclusions/Significance: Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature In Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Haplotype distribution of five nuclear genes based on network genealogies and Bayesian inference indicates that Trypanosoma cruzi hybrid strains are polyphyletic

Chagas disease is still a major public health problem in Latin America. Its causative agent, Trypanosoma cruzi, can be typed into three major groups, T. cruzi I, T. cruzi II and hybrids. These groups each have specific genetic characteristics and epidemiological distributions. Several highly virulent strains are found in the hybrid group; their origin is still a matter of debate. The null hypothesis is that the hybrids are of polyphyletic origin, evolving independently from various hybridization events. The alternative hypothesis is that all extant hybrid strains originated from a single hybridization event. We sequenced both alleles of genes encoding EF-1 alpha, actin and SSU rDNA of 26 T. cruzi strains and DHFR-TS and TR of 12 strains. This information was used for network genealogy analysis and Bayesian phylogenies. We found T. cruzi I and T. cruzi II to be monophyletic and that all hybrids had different combinations of T. cruzi I and T. cruzi II haplotypes plus hybrid-specific haplotypes. Bootstrap values (networks) and posterior probabilities (Bayesian phylogenies) of clades supporting the monophyly of hybrids were far below the 95% confidence interval, indicating that the hybrid group is polyphyletic. We hypothesize that T. cruzi I and T. cruzi II are two different species and that the hybrids are extant representatives of independent events of genome hybridization, which sporadically have sufficient fitness to impact on the epidemiology of 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

Relationship between occurrence of Panama disease in banana trees of cv. Nanicao and nutrients in soil and leaves

Relationship between occurrence of Panama disease in banana trees of cv. Nanicao and nutrients in soil and leaves The objective of the present work was to verify if the incited symptoms in banana trees cv. Nanicao, belonging to the subgroup Cavendish, in Vale do Ribeira, are related to levels of nutrients in soil and leaves. Sixteen areas in Vale do Ribeira were selected, one half with symptomatic plants and the other with healthy plants. In those areas the third leaf of five plants and the soil near those plants were collected, at depths from 0 to 20 cm and from 20 to 40 cm. At both depths of the sampled soil, levels of Ca, Mg, PO(4)(-3), S and cationic exchange capacity (CEC) were significantly different among the areas, and the low values of these elements were present in the areas containing symptomatic plants. At both depths, Mg, Al and H in relation to CEC were significantly different among the areas, and the low values of Mg and high of Al and H were present in the areas with symptomatic plants. The N, K and S in the leaves were significantly different among the areas. These elements showed low values in the areas containing symptomatic plants. Despite the fact that some amounts of macronutrients of the soil and of the leaves are present only in the areas containing plants of Nanicao with symptoms similar to fusariosis, proof of a possible occurrence of race of the pathogen should be looked for in Vale do Ribeira.

Enteroinvasive Escherichia coli vs. Shigella flexneri : how different patterns of gene expression affect virulence

Important features of the enteroinvasive Escherichia coli (EIEC) phenotype and gene expression likely to confer EIEC with a lower ability to cause disease than Shigella flexneri were described here for the first time. To confirm the lower pathogenicity of EIEC, we have analyzed the keratoconjunctivitis developed in guinea-pigs with EIEC or S. flexneri. Shigella flexneri induced a more pronounced proinflammatory response, whereas EIEC induced a mild form of the disease. EIEC showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Plaques formed by EIEC during intercellular spreading were four times smaller than those formed by S. flexneri. At the molecular level, the lower expression of virulence genes by EIEC during infection of Caco-2 cells highlighted the importance of effective gene transcription for bacterial pathogenicity.

Host-guest complexation of a nitroheterocyclic compound with cyclodextrins: a spectrofluorimetric and molecular modeling study

Nitroheterocyclic compounds (NC) were candidate drugs proposed for Chagas disease chemotherapy. In this study, we investigated the complexation of hydroxymethylnitrofurazone (NFOH), a potential antichagasic compound, with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), Hydroxypropyl-beta-cyclodextrin (HP-beta-CD), Dimethyl-beta-cyclodextrin (DM-beta-CD) and gamma-cyclodextrin (gamma-CD) by fluorescence spectroscopy and molecular modeling studies. Hildebrand-Benesi equation was used to calculate the formation constants of NFOH with cyclodextrins based on the fluorescence differences in the CDs solution. The complexing capacity of NFOH with different CDs was compared through the results of association constant according to the following order: DM-beta-CD > beta-CD > alpha-CD > HP-beta-CD > gamma-CD. Molecular modeling studies give support for the experimental assignments, in favor of the formation of an inclusion complex between cyclodextrins with NFOH. This is an important study to investigate the effects of different kinds of cyclodextrins on the inclusion complex formation with NFOH and to better characterize a potential formulations to be used as therapeutic options for the oral treatment of Chagas disease.

Mitochondrial function in the yeast form of the pathogenic fungus Paracoccidioides brasiliensis

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I-V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD(+)-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast`s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain

Trypanosoma cruzi is the etiological agent of Chagas` disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T cruzi strain Y solved at 2.2 angstrom resolution. DHODH is a flavin mononucleotide containing enzyme, which catalyses the oxidation Of L-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Genetic studies have shown that DHODH is essential for T cruzi survival, validating the idea that this enzyme can be considered an attractive target for the development of antichagasic drugs. In our work, a detailed analysis of T cruzi DHODH crystal structure has allowed us to suggest potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design. (c) 2008 Elsevier Inc. All rights reserved.

Trypanocidal Activity of Pimarane Diterpenes from Viguiera arenaria (Asteraceae)

Five structurally related pimarane diterpenes isolated from the roots of Viguiera arenaria and a further compound obtained by chemical derivatization were evaluated in vitro against the trypomastigote forms of Trypanosoma cruzi. The natural compound ent-15-pimarene-8 beta,19-diol and the derivative ent-8(14),15-pimaradiene-3 beta-acetoxy showed the highest trypanocidal activity, displaying IC50 values of 116.5 +/- 1.21 and 149.3 +/- 1.07 mu M, respectively, while the positive control, violet gentian, showed an IC50 of 76 mu M. Based on the results, it can be concluded that minor structural differences among the tested diterpenes influence significantly the trypanocidal activity, thus bringing new perspectives to the establishment of structure-activity relationships among this type of metabolites to the treatment of Chagas` disease. Copyright (C) 2008 John Wiley & Sons, Ltd.

Precursor system of liquid crystalline phase containing propolis microparticles for the treatment of periodontal disease: Development and characterization

Precursor systems of liquid crystalline phase were prepared using the surfactant PPG-5-Ceteth-20, isopropyl myristate, and water; gelatin microparticles containing propolis were then added into these systems. Homogeneity of dispersion, the in-system microparticle morphology, and sedimentation behavior of each formulation were evaluated. The rheological and mechanical properties (hardness, compressibility, and adhesiveness), the work of syringing, and the propolis release profile were also evaluated. All the formulations exhibited pseudoplastic flow and thixotropy, and they displayed storage modulus, loss modulus, dynamic viscosity, and loss tangent that depended on temperature, frequency, and composition. Mechanical properties varied significantly among the formulations being affected by changes in the composition and temperature. Raising the concentration of surfactant and adding propolis microparticles significantly decreased the work of syringing. The drug release was non-Fickian (anomalous) and there was no significant difference between the tested systems in the times required for 10%, 30%, and 50% release of the initial drug loading.

Reduction of parasitism tissue by treatment of mice chronically infected with Trypanosoma cruzi with lignano lactones

The reduction of parasitism tissue upon treatment with two lignano lactones, namely (-)- cubebin (CUB) and (-)-hinokinin (HNK), was evaluated in the chronic phase of Chagas` disease by quantifying the enzyme beta-galactosidase expressed by the CL B5 clone strain of Trypanosoma cruzi. Tissue karyometry was also performed. Treatment with the assessed lignans led to a larger reduction in parasitism tissue in all evaluated organs, compared with benznidazole (BZN). Oral treatment with CUB or HNK was more effective. Karyometry results demonstrated that the infected control animals had increased nuclear area compared with uninfected controls, indicating cellular hypertrophy. Results also revealed that use of CUB or HNK was able to significantly prevent this increase, and a slight decrease in the nuclear area was observed, compared with mice treated with BZN. Taken together, these data demonstrate that CUB and HNK could be considered as potential compounds for the development of new drugs for treatment of Chagas` disease.

Trypanosoma cruzi: evaluation of (-)-cubebin derivatives activity in the messenger RNAs processing

No fully effective treatment has been developed since the discovery of Chagas` disease. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effective in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing, which is unusual RNA processing reaction, and it implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. Cubebin derivatives seem to provide treatments with less collateral effects than benznidazole and showed similar or better trypanocidal activities than benznidazole. Therefore, the cubebin derivatives ((-)-6,6`-dinitrohinokinin (DNH) and (-)-hinokinin (HQ)) interference in the mRNA processing was evaluated using T. cruzi permeable cells (Y and BOL (Bolivia) strains) following by RNase protection reaction. These substances seem to intervene in any step of the RNA transcription, promoting alterations in the RNA synthesis, even though the RNA processing mechanism still occurs. Furthermore, HQ presented better activity against the parasites than DNH, meaning that BOL strain seems to be more resistant than Y.

Trypanocidal Activity of Limonoids and Triterpenes from Cedrela fissilis

Chagas` disease is an illness that affects millions of people in Central and South America, The search for both a prophylactic drug to be added to human blood as well as a safe and reliable therapeutic drug are greatly needed to control such disease. Herein, we report the trypanocidal activity of 15 crude extracts and 14 Compounds (limonoids and triterpenes) as well as the isolation of 25 known compounds (6 limonoids, 12 triterpenes, 1 sesquiterpene, 5 steroids, and 1 flavonoid) from Cedrela fissilis. The present study shows that this plant is a Promising Source of active compounds for the control of Chagas` disease. The inhibitory activity found for odoratol indicates that it is potentially useful as an alternative for the chemoprophylactic gentian violet.