Synthesis of the O-linked pentasaccharide in glycoproteins of Trypanosoma cruzi and selective sialylation by recombinant trans-sialidase

The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infection and pathogenesis. The synthesis of the pentasaccharide, beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-beta-D-Galp-(1-->6)-[beta-D-Galf-(1-->4)]-D-GlcpNAc and the corresponding alditol, previously isolated by reductive beta-elimination of the mucins, is described. The key step was the 6-O-glycosylation of a easily accessible derivative of beta-D-Galf-(1-->4)-D-GlcpNAc with a beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-D-Galp donor using the trichloroacetimidate method. The beta-linkage was diastereoselectively obtained by the nitrile effect. The pentasaccharide is the major oligosaccharide in the mucins of T. cruzi, G strain and presents two terminal beta-D-Galp residues for possible sialylation by TcTS. A preparative sialylation reaction was performed with its benzyl glycoside and the sialylated product was isolated and characterized. NMR spectroscopic analysis showed that selective monosialylation occurred at the terminal (1-->3) linked galactopyranose.     

The amino terminal domain of a novel WD repeat protein from Trypanosoma cruzi contains a non-canonical mitochondrial targeting signal

WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to the beta-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome, Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions. TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of Trypanosoma brucei and Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that mitochondrial import is mediated by an alternative targeting signal.     

The Trypanosoma cruzi metacyclic-specific protein Met-III associates with the nucleolus and contains independent amino and carboxyl terminal targeting elements

Metacyclogenesis in Trypanosoma cruzi involves the differentiation of replicating non-infective epimastigotes into non-replicating metacyclic trypomastigotes. This pre-adapts parasites for infection of the mammalian host and is characterised by several morphological changes and structural alterations to the nucleus, including nucleolar disaggregation. Experimental investigation of these developmental processes has been hampered by a lack of robust molecular markers. Here, we describe the precise temporal expression of the T. cruzi-specific protein Met-III, in the genome reference strain CL Brener. Expression is restricted to metacyclics in the insect stages of the life-cycle and is rapidly down-regulated following invasion of mammalian cells. Met-III localises to dispersed foci typical of the disassembled nucleolus in metacyclics and to the discrete single nucleolus of cells soon after macrophage invasion. To identify elements that target Met-III, we generated a series of tagged green fluorescent protein fusion proteins and examined their sub-nuclear location in transformed parasites. These experiments demonstrated that amino and carboxyl terminal fragments, characterised by clusters of basic residues, could independently mediate nucleolar sequestration. To investigate the function of Met-III, we used gene deletion. This showed that Met-III is not required for the development of metacyclic trypomastigotes and that null mutants can complete the life-cycle in vitro.     

Highly specific inactivation of triosephosphate isomerase from Trypanosoma cruzi

We searched for molecules that selectively inactivate homodimeric triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the parasite that causes Chagas' disease. We found that some benzothiazoles inactivate the enzyme. The most potent were 3-(2-benzothiazolylthio)-propanesulfonic acid, 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid, and 2-(2-4(4-aminophenyl)benzothiazole-6-methylbenzothiazole-7-sulfonic acid. Half-maximal inactivation by these compounds was attained with 33, 56, and 8 microM, respectively; in human TIM, half-maximal inactivation required 422 microM, 3.3 mM, and 1.6 mM. In TcTIM, the effect of the benzothiazoles decreased as the concentration of the enzyme was increased. TcTIM has a cysteine (Cys 15) at the dimer interface, whereas human TIM has methionine in that position. In M15C human TIM, the benzothiazole concentrations that caused half-maximal inactivation were much lower than in the wild type. The overall findings suggest that the benzothiazoles perturb the interactions between the two subunits of TcTIM through a process in which the interface cysteine is central in their deleterious action.     

Molecular and functional characterization of a spermidine transporter (TcPAT12) from Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas' disease, is the only eukaryotic cell which lacks the ability to synthesize polyamines de novo. In this work, we describe for the first time the molecular and biochemical properties of a high-affinity spermidine transporter from T. cruzi. The transporter gene TcPAT12 was functionally expressed in Xenopus laevis oocytes, showing high levels of spermidine uptake. Similar apparent affinity constants for spermidine uptake were obtained when comparing T. cruzi epimastigotes and heterologous expressed TcPAT12 in X. laevis. In addition, TcPAT12 also transports putrescine and the amino acid l-arginine at lower rates than spermidine.     

Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis

Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.     

Molecular characterization of ribonucleoproteic antigens containing repeated amino acid sequences from Trypanosoma cruzi

Trypanosoma cruzi expresses several proteins containing antigenic amino acid repeats. Here we characterized TcRpL7a and TcRBP28, which carry similar repeat motifs and share homology to the eukaryotic L7a ribosomal protein and to a Trypanosoma brucei RNA binding protein, respectively. Analyses of the full length and truncated recombinant TcRpL7a showed that the humoral response of patients with Chagas disease is directed towards its repetitive domain. Sequence analyses of distinct copies of TcRpL7a genes present in the genome of six T. cruzi strains indicate that the number of repeats is higher in proteins from T. cruzi II than T. cruzi I strains. A serum panel of 59 T. cruzi infected patients showed that 73% reacted with TcRpL7a, 71% reacted with TcRBP28 and 80% reacted with 1:1 mixture of both antigens. Synthetic peptides harboring the TcRpL7a repeat motif reacted with 46% of the serum samples. Antibodies raised against both antigens identified equivalent amounts of the native proteins in all three stages of the parasite life cycle. Analyses of subcellular fractions indicated that TcRBP28 is present in the cytoplasm whereas TcRpL7a co-fractionates with polysomes. Confirming their predicted cellular localization, GFP fusions showed that, whereas GFP::TcRBP28 localizes in the cytoplasm, GFP::TcRpL7a accumulates in the nucleus, where ribosome biogenesis occurs.     

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cellinteractions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediatedinteractions. These physiological interactions are crucial for normal thymocytedifferentiation, but can be disrupted in pathological situations. Indeed, there is severethymic atrophy in animals acutely infected with Trypanosoma cruzi due toCD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together withchanges in ECM deposition and thymocyte migration. We studied an in vitro model of TECinfection by T. cruzi and found that infected TEC cultures show a reducednumber of cells, which was likely associated with decreased proliferative capacity, butnot with increased cell death, as demonstrated by bromodeoxyuridine and annexin-Vlabelling. The infected TEC cultures exhibited increased expression of fibronectin (FN),laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relativenumber of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted inlower infection rates. Consistent with these data, we observed increased thymocyteadhesion to infected TEC cultures. Overall, these results suggest that ECM molecules,particularly FN, facilitate infection of the thymic epithelium and that the consequentenhancement of ECM expression might be associated with changes in TEC-thymocyteinteractions.     

Chimeric synthetic peptides as antigens for detection of antibodies to Trypanosoma cruzi

Six chimeric synthetic peptides (QCha-1, QCha-2, QCha-3, QCha-4, QCha-5, and QCha-6) incorporating antigenic sequences of two immunodominant repeat B-cell epitopes of Trypanosoma cruzi were synthesized by conventional solid-phase peptide synthesis. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of positive Chagasic sera (n=82), while specificity was evaluated with samples from healthy blood donors (n=44) and patients with other infectious diseases (n=86). The antigenicity of the chimeric peptides in solid-phase immunoassays was compared with that of the monomeric peptides. Data demonstrated that the chimeric peptide QCha-5 was the most reactive because it detected antibodies to parasite efficiently. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of Chagas' disease.     

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.     

Preparative route to N-glycolylneuraminic acid phenyl 2-thioglycoside donor and synthesis of Neu5Gc-alpha-(2-->3')-lactosamine 3-aminopropyl glycoside

The spacer-armed trisaccharide, Neu5Gc-alpha-(2-->3')-lactosamine 3-aminopropyl glycoside, was synthesized by regio- and stereoselective sialylation of the suitably protected triol acceptor, 3-trifluoroacetamidopropyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(6-O-benzyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside, with the donor methyl [phenyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha,beta-D-galacto-2-nonulopyranosid]onate. The donor was obtained, in turn, from methyl [phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha,beta-D-galacto-2-nonulopyranosid]onate by N-tert-butoxycarbonylation of the acetamido group followed by total N- and O-deacetylation, per-O-acetylation, subsequent Boc group removal, and N-acetoxyacetylation.     

Trypanosoma cruzi: antiprotozoal activity of parthenolide obtained from Tanacetum parthenium (L.) Schultz Bip. (Asteraceae, Compositae) against epimastigote and amastigote forms

This study reports the activity of crude extracts, fractions and parthenolide (pure compound) obtained from Tanacetum parthenium against two forms of the parasite Trypanosoma cruzi. Feverfew is a traditional herbal medicine that has been used for the treatment of migraine, fever and arthritis. Activity against epimastigote forms was observed for crude extracts, fractions and parthenolide, and a progressive increase in the antitrypanosomal effect was observed in the course of the purification process. The pure compound showed IC50/96h and IC90/96h of 0.5 microg/ml and 1.25 microg/ml, respectively. The cytotoxic effect of parthenolide in LLMCK2 cells was 3.2 microg/ml (CC50/96h) and the selectivity index was 6.4. No hemolysis was detected for the pure compound. The internalization index of T. cruzi in LLMCK2 cells was reduced almost 51% at the concentration of 2 microg/ml of parthenolide, and 96.6% at 4 microg/ml. Scanning and transmission electron microscopy permitted observation of morphological modifications and ultrastructural alterations.     

Risk of Trypanosoma cruzi I (Kinetoplastida: Trypanosomatidae) transmission by Panstrongylus geniculatus (Hemiptera: Reduviidae) in Caracas (Metropolitan District) and neighboring States, Venezuela

The collection of Panstrongylus geniculatus bugs by inhabitants of dwellings in Caracas city (Metropolitan District) and in the neighboring Miranda and Vargas Sates, Venezuela, allowed for the gathering of data on the potential role of this sylvatic triatomine bug as a vector of Chagas disease in this area. The natural infection by Trypanosoma cruzi was recorded by examining fresh and stained faeces of the bugs. Additionally, a random amplification of polymorphic DNA technique for parasite identification and group typing was employed. A dot-ELISA test was used to identify the gut content of the triatomine bugs with the aim of assessing and quantifying the vector-human contact. Sixty-seven specimens (76.1%) were positive to T. cruzi (identified as T. cruzi I) and 60.2% (53/88) gave a positive reaction to the human antiserum. The human blood-positive samples included mixed blood meals with domestic animals (dog, pig and cow) (9.4%) and with mouse (3.8%). The overall Human Blood Index, measured as the percentage of bugs whose gut contents reacted with human antiserum on the total numbers of bugs that reacted with all the antisera tested, was 98.1%. Almost 41% of the bugs that had fed on humans were also positive for T. cruzi. These data show that the feeding of P. geniculatus on humans does not seem to be accidental and that its rate of infection by T. cruzi is high in this area which is not regarded as endemic for Chagas disease by the National Control Programme. This situation is particularly striking because it occurs in and around Caracas, the capital city, where 20% of the whole population of Venezuela live, human migrations from endemic areas are continuous, people in the crowded shantytown as well as people living in high-quality country houses are equally at risk and the epidemiological cycle Didelphis marsupialis/Rattus rattus-P. geniculatus-human does appear to occur successfully.     

Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium

The isolation of biological clones of Trypanosoma cruzi by microscopically dispensing individual organisms or by serial dilution is laborious and time consuming. The inability to resolve mixed T. cruzi infections, from vectors and hosts, and to isolate clones of slow growing genotypes by efficient plating on solid media, has hindered characterisation studies and downstream applications. We have devised and validated a sensitive, solid medium plating technique for rapid in vitro isolation of clones representative of all the recognised T. cruzi lineages (TCI, TCIIa-e), including the slow growing strain CANIII (TC IIa) and Trypanosoma rangeli, with high plating efficiencies. Furthermore, the method is effective for the isolation of clones directly from silvatic triatomine bugs and from experimentally infected mice harbouring mixed infections, allowing resolution of multiclonal infections from varied sources.     

In vitro and in vivo antiparasital activity against Trypanosoma cruzi of three novel 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one-based complexes

Conventional reactions of the versatile multidentate ligand 5-methyl-1,2,4-triazolo[1,5-a] pyrimidin-7(4H)-one (HmtpO) with metallic(II) perchlorate salts lead to three novel multidimensional complexes [Cu(HmtpO)₂(H₂O)₃](ClO₄)₂·H₂O (1), {[Cu(HmtpO)₂(H₂O)₂](ClO₄)₂ ·2HmtpO}_n (2) and {[Co(HmtpO)(H₂O)₃](ClO₄)₂·2H₂O}_n (3). We have tested the antiparasital activity in vitro and in vivo of the three new complexes against Trypanosoma cruzi showing very promising results and overcoming clearly the reference drug commonly used for the Chagas disease treatment, benznidazole.     

Risk factors associated with Trypanosoma cruzi exposure in domestic dogs from a rural community in Panama

Chagas disease, caused by Trypanosoma cruzi infection, is a zoonosisof humans, wild and domestic mammals, including dogs. In Panama, the main T.cruzi vector is hodnius pallescens, a triatomine bugwhose main natural habitat is the royal palm, Attalea butyracea. Inthis paper, we present results from three T. cruzi serological tests(immunochromatographic dipstick, indirect immunofluorescence and ELISA) performed in51 dogs from 24 houses in Trinidad de Las Minas, western Panama. We found that ninedogs were seropositive (17.6% prevalence). Dogs were 1.6 times more likely to becomeT. cruzi seropositive with each year of age and 11.6 times ifroyal palms where present in the peridomiciliary area of the dog''s household or itstwo nearest neighbours. Mouse-baited-adhesive traps were employed to evaluate 12peridomestic royal palms. All palms were found infested with R.pallescens with an average of 25.50 triatomines captured per palm. Of 35adult bugs analysed, 88.6% showed protozoa flagellates in their intestinal contents.In addition, dogs were five times more likely to be infected by the presence of anadditional domestic animal species in the dog''s peridomiciliary environment. Ourresults suggest that interventions focused on royal palms might reduce the exposureto T. cruzi infection.     

Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection

The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 10⁴ T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection.     

Na(+)-ATPase and protein kinase C are targets to 1-O-hexadecylphosphocoline (miltefosine) in Trypanosoma cruzi

Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na⁺-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2 mM), a specific inhibitor of Na⁺-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na⁺-ATPase activity with IC₅₀ = 18 ± 5 μg mL⁻¹. This effect was shown to be reversible, dependent on the pH and Ca²⁺. The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na⁺-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na⁺-ATPase and PKC activities.     

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 Å 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.