Lytic antibodies elicited by Trypanosoma cruzi infection recognize epitopes present on both bloodstream trypomastigote and epimastigote forms of parasite

Sera of Chaga's disease patients containing anti-T. cruzi lytic antibodies were submitted to affinity chromatography using Sepharose 4B conjugated with antigen extracted from epimasiigote or trypomasiigote forms of the parasite. Epimastigotes were obtained from culture at the exponential growth phase and the trypomastigotes from blood of infected and immunosuppressed mice. Antigen of both parasite forms was obtained by sonication of the parasites followed by centrifugation. Both antigens were then conjugated to activated Sepharose 4B. Affinity chromatography was performed by passing sera from chagasic patients through an immunoadsorbent column containing either epimasiigote or trypomasiigote antigens. Antibodies bound to the column were eluted with cold 0,2 M glycine buffer pH 2,8. The eluted antibodies were analysed regarding their isotype and lytic activity. The results showed that anti-T. cruzi lytic antibodies present in sera from chagasic patients are mainly located in the IgG isotype and recognize epitopes present in both trypomasiigote and epimastigote forms. A brief report of this work has already been published12.

Chagas' disease: IgA, IgM and IgG antibodies to T. cruzi amastigote, trypomastigote and epimastigote antigens in acute and in different chronic forms of the disease

In an attempt to find a better T. cruzi antigen and possible immunological markers for the diagnosis of different clinical forms of Chagas' disease, amastigote and trypomastigote antigens obtained from immunosuppressed mice infected with T. cruzi (Y strain) were assessed in comparison with conventional epimastigote antigens. A total of 506 serum samples from patients with acute and with chronic (indeterminate, cardiac and digestive) forms, from nonchagasic infections, and from healthy individuals were assayed in immunofluorescence (IF) tests, to search for IgG, IgM and IgA antibodies. Amastigote proved to be the most convenient antigen for our purposes, providing higher relative efficiency indexes of 0.946, 0.871 and 0.914 for IgG, IgM and IgA IF tests, respectively. Anti-amastigote antibodies presented higher geometric mean titers (GMT) than anti-trypomastigote and anti-epimastigote. Anti-amastigote IgG antibodies were found in all forms of Chagas' disease, and predominantly IgA antibodies, in chronic digestive and in acute forms, as well as IgM antibodies, in latter forms. Thus, tests with amastigote antigen could be helpful for screening chagasic infections in blood banks. Practical and economical aspects in obtaining amastigotes as here described speak in favour of its use in developing countries, since those from other sources require more complex system of substruction, specialized personnel or equipment.

Alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) applied to dot-ELISA

The alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) was assessed in dot-ELISA for the diagnosis of Chagas' disease. Serum samples (355) from chagasic and non-chagasic patients were studied, and IgG antibodies to ASEA were found in all patients with chronic Chagas' disease. In non-chagasic patients 95.6% were negative, except for those with leishmaniasis (visceral and mucocutaneous), and some patients from control group reacted in low titers. The data indicate that dot-ELISA using ASEA is suitable for seroepidemiologic surveys to be employed in endemic areas for Chagas' disease.

Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera

Soluble antigens from epimastigotes of Trypanosoma cruzi were analyzed by western blot in terms of their reactivity with sera from patients with Chagas' disease. In addition, sera from patients with visceral (AVL) and tegumentar leishmaniasis (ATL) were also tested in order to identify cross-reactivities with Trypanosoma cruzy antigens. Twenty eight polypeptides with molecular weights ranging from 14 kDa to 113 kDa were identified with sera from Chagas' disease patients. An extensive cross-reactivity was observed when sera from human visceral leishmaniasis were used, while only a slight cross-reaction was observed with sera from tegumentar leishmaniasis. On the other hand, 10 polypeptidesspecifically reacting with sera from Chagas' disease patients were identified. Among them, the antigens with molecular weights of 46 kDa and 25 kDa reacted with all sera teste and may be good candidates for specific immunodiagnosis of Chagas' disease.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.

Trypanocidal activity of Brazilian plants against epimastigote forms from Y and Bolivia strains of Trypanosoma cruzi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Trypanocidal activity of Brazilian plants against epimastigote forms from Y and Bolivia strains of Trypanosoma cruzi

Chagas disease is one of the main public health problems in Latin America. Since the available treatments for this disease are not effective in providing cure, the screening of potential antiprotozoal agents is essential, mainly of those obtained from natural sources. This study aimed to provide an evaluation of the trypanocidal activity of 92 ethanol extracts from species belonging to the families Annonaceae, Apiaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Moraceae, Nyctaginaceae, and Verbenaceae against the Y and Bolivia strains of Trypanosoma cruzi. Additionally, cytotoxic activity on LLCMK2 fibroblasts was evaluated. Both the trypanocidal activity and cytotoxicity were evaluated using the MTT method, in the following concentrations: 500, 350, 250, and 100 µg/mL. Benznidazole was used for positive control. The best results among the 92 samples evaluated were obtained with ethanol extracts of Ocotea paranapiacabensis (Am93) and Aegiphila lhotzkiana (Am160). Am93 showed trypanocidal activity against epimastigote forms of the Bolivia strain and was moderately toxic to LLCMK2 cells, its Selectivity Index (SI) being 14.56, while Am160 showed moderate trypanocidal activity against the Bolivia strain and moderate toxicicity, its SI being equal to 1.15. The screening of Brazilian plants has indicated the potential effect of ethanol extracts obtained from Ocotea paranapiacabensis and Aegiphila lhotzkiana against Chagas disease.

A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei

A 'two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3' flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins.

The anticancer drug tamoxifen is active against Trypanosoma cruzi in vitro but ineffective in the treatment of the acute phase of Chagas disease in mice

The activity of the antineoplastic drug tamoxifen was evaluated against Trypanosoma cruzi. In vitro activity was determined against epimastigote, trypomastigote and amastigote forms of CL14, Y and Y benznidazole resistant T. cruzi strains. Regardless of the strain used, the drug was active against all life-cycle stages of the parasite with a half maximal effective concentration ranging from 0.7-17.9 µM. Two experimental models of acute Chagas disease were used to evaluate the in vivo efficacy of treatment with tamoxifen. No differences in parasitemia and mortality were observed between control mock-treated and tamoxifen-treated mice.

Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease

Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC50 to BZ between 7.6-32 µM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC50 of the strains varied from 15.6 ± 3-51.4 ± 1 µM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.

Trypanocidal activity of Piper arboreum and Piper tuberculatum (Piperaceae)

In the scope of our ongoing research on bioactive agents from Brazilian flora, twenty-four extracts and fractions obtained from Piper arboreum Aub. and Piper tuberculatum Jacq. (Piperaceae) were screened for trypanocidal activity by using MTT colorimetric assay. The strongest activity was found in hexane fractions from the leaves of P. arboreum (IC50= 13.3 µg/ mL) and P. tuberculatum (IC50 = 17.2 µg/mL). Hexane fractions of the fruits of P. tuberculatum and P. arboreum showed potent toxic effects on epimastigote forms of Trypanosoma cruzi, with values of IC50 (µg/mL) of 32.2 and 31.3, respectively. Additionally, the phytochemical study of the hexane fraction of P. arboreum leaves furnished two pyrrolidine amides, piperyline (1) and 4,5-dihydropiperyline (2), which could be responsible, at least in part for the observed antiprotozoal activity.

Genome Survey Sequence Analysis and Identification of Homologs of Major Surface Protease (gp63) Genes in Trypanosoma rangeli

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

Actions of a Proline Analogue, L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC(50) = 0.89+/-0.02 mM at 28 degrees C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54+/-0.01 mM at 37 degrees C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress.

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

Nitric oxide donor trans-[RuCl([15]aneN(4))NO]2+as a possible therapeutic approach for Chagas` disease

Background and purpose: Benznidazole (Bz) is the therapy currently available for clinical treatment of Chagas` disease. However, many strains of Trypanosoma cruzi parasites are naturally resistant. Nitric oxide (NO) produced by activated macrophages is crucial to the intracellular killing of parasites. Here, we investigate the in vitro and in vivo activities against T. cruzi, of the NO donor, trans-[RuCl([15]aneN(4))NO]2+. Experimental approach: Trans-[RuCl([15]aneN(4))NO]2+ was incubated with a partially drug-resistant T. cruzi Y strain and the anti-proliferative (epimastigote form) and trypanocidal activities (trypomastigote and amastigote) evaluated. Mice were treated during the acute phase of Chagas` disease. The anti-T. cruzi activity was evaluated by parasitaemia, survival rate, cardiac parasitism, myocarditis and the curative rate. Key results: Trans-[RuCl([15]aneN(4))NO]2+ was 10- and 100-fold more active than Bz against amastigotes and trypomastigotes respectively. Further, trans-[RuCl([15]aneN(4))NO]2+ (0.1 mM) induced 100% of trypanocidal activity (trypomastigotes forms) in vitro. Trans-[RuCl([15]aneN(4))NO]2+ induced permanent suppression of parasitaemia and 100% survival in a murine model of acute Chagas` disease. When the drugs were given alone, parasitological cures were confirmed in only 30 and 40% of the animals treated with the NO donor (3.33 mu mol center dot kg-1 center dot day-1) and Bz (385 mu mol center dot kg-1 center dot day-1), respectively, but when given together, 80% of the animals were parasitologically cured. The cured animals showed an absence of myocarditis and a normalisation of cytokine production in the sera. In addition, no in vitro toxicity was observed at the tested doses. Conclusions and implications: These findings indicate that trans-[RuCl([15]aneN(4))NO]2+ is a promising lead compound for the treatment of human Chagas` disease. This article is commented on by Machado et al., pp. 258-259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00662.x and to view a related paper in this issue by Silva et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00524.x.