Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop.

A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

Natural programmed cell death in T. cruzi epimastigotes maintained in axenic cultures

Trypanosoma cruzi, a parasitic protozoan, is the agent of Chagas' disease or American trypanosomiasis, an endemic pathology in Latin America, affecting up to 18 million people, with high public health costs. Programmed cell death (PCD) has many functions in development and tissue remodeling in metazoans. In protozoa, it could represent concomitant or alternative mechanisms for clonal selection, immune response evasion, and population size regulation. In this work, we describe the natural occurrence of PCD in T. cruzi epimastigotes during the stationary phase of growth in axenic culture or under nutrient deprivation. Thus, we have observed phosphatidylserine externalization, cellular volume decrease, caspase-like protein activity, and DNA fragmentation. Additionally, serum deprivation also induces autophagic characteristics such as monodansylcadaverine-labeled vesicles accumulation and redistribution of proteins homologous to Atg8. In agreement with our results, apoptosis may play an important role in parasite survival. Then, identification and modulation of molecular targets inducing programmed cell death in T. cruzi may lead to new potential therapeutic approaches for Chagas' disease.     

Trypanosoma cruzi infection disrupts vinculin costameres in cardiomyocytes

Chagas' disease cardiomyopathy is an important manifestation of Trypanosoma cruzi infection, leading to cardiac dysfunction and serious arrhythmias. We have here investigated by indirect immunofluorescence assay the distribution of vinculin, a focal adhesion protein with a major role in the transmission of contraction force, during the T. cruzi-cardiomyocyte infection in vitro and in vivo. No change in vinculin distribution was observed after 24 h of infection, where control and T. cruzi-infected cardiomyocytes displayed vinculin localized at costameres and intercalated discs. On the other hand, a clear disruption of vinculin costameric distribution was noted after 72 h of infection. A significant reduction in the levels of vinculin expression was observed at all times of infection. In murine experimental Chagas' disease, alteration in the vinculin distribution was also detected in the infected myocardium, with no costameric staining in infected myocytes and irregular alignment of intercalated discs in cardiac fibers. These data suggest that the disruption of costameric vinculin distribution and the enlargement of interstitial space due to inflammatory infiltration may contribute to the reduction of transmission of cardiac contraction force, leading to alterations in the heart function in Chagas' disease.     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

Trypanosoma cruzi: Correlation of Growth Kinetics to Zymodeme Type in Clones Derived from Various Sources*†

SYNOPSIS. Trypanosoma ( Schizotrypanum ) cruzi clones were derived from isolates of an acute human case of Chagas' disease (strain Esmereldo), a human case of T. cruzi infection (strain CAN-III) and from a naturally infected opossum (strain WA-250). The isoenzyme patterns and growth rates of the clones were stable during long-term cultivation, by serial passages, of the parasites in liquid medium. Both clones of strain Esmereldo were zymodeme II; the 2 clones of strain CAN-III, zymodeme III; and the 5 clones of strain WA-250, zymodeme I. The range in doubling times of the parasite populations in liquid medium were 36–49. 7 h (strain WA-250), 117.2–133.7 h (Esmereldo clones) and 169–208 h (CAN-III clones).     

Humoral autoimmune response in Chagas' disease: Trypanosoma cruzi ribosomal antigens as immunizing agents

Abstract Molecular expression cloning techniques have revealed that patients with chronic Chagas' heart disease (cChHD) present a strong humoral response against the cloned C-terminal portions of the four Trypanosoma cruzi ribosomal P proteins TcP1, TcP2α (TcP2b), TcP2β (TcPJL5), and TcP0. This protein family presents several features that may be important in the immunopathology of Chagas disease. Their exposed location on the ribosome, and the amplification of their parasite-specific, Ser free C-terminal domain, generate a strong anti-parasite P response that may induce anti-P autoimmunity. Evidences indicate that the serological pattern of the anti-P response from chagasic patients may be the consequence of a chronic immunization with T. cruzi ribosomal antigens.     

High correlation between Chagas' disease serology and PCR-based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area

Abstract The detection of Trypanosoma cruzi kinetoplast DNA by polymerase chain reaction (PCR) amplification is a potentially powerful tool for the parasitological diagnosis of Chagas' disease. We have applied this technique in a field situation in Bolivia, where 45 children from a primary school were subjected to serological testing, buffy coat analysis and PCR diagnosis. 26 of the 28 serology-positive individuals were also positive by PCR. In addition, two serology-negative children gave a positive result by PCR, including one who was positive in the buffy coat test. These results suggest that PCR detection of T. cruzi DNA in blood can be a very useful complement to serology in Chagas' disease diagnosis in Bolivia.     

Production of hydrogen peroxide by peripheral blood monocytes and specific macrophages during experimental infection with Trypanosoma cruzi in vivo

Acute Chagas' disease triggers potent inflammatory reaction characterized by great increase of peripheral blood monocyte (PBM) and macrophage numbers. We studied the respiratory burst responses of PBM and peritoneal and splenic macrophages to in vivo infection (rats). The ultrastructure of heart inflammatory macrophages was also investigated. The infection increased the hydrogen peroxide (H2O2) production by PBM and splenic macrophages but not by peritoneal macrophages. Accordingly, the PBM and spleen cell numbers increased but the total number of peritoneal cells was similar to controls. Heart macrophages of infected rats exhibited increase (number and size) and activated morphology in parallel to high cardiomyocyte parasitism. Our data highlight the importance of innate immunity and H2O2production to host resistance during acute phase of T. cruzi infection. A novel finding is that H2O2production seems related to specific types of monocytes/macrophages that are able to release this agent when in presence of high parasite load.     

Effects of cyclooxygenase inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection

Prostaglandins are known to be produced by macrophages when challenged with Trypanosoma cruzi, the etiological agent of Chagas' disease. It is not known whether these lipid mediators play a role in oxidative stress in host defenses against this important protozoan parasite. In this study, we demonstrated that inducible cyclooxygenase-mediated prostaglandin production is a key chemical mediator in the control of parasite burden and erythrocyte oxidative stress during T. cruzi infection in C57BL/6 and BALB/c mice, prototype hosts for the study of resistance and susceptibility in murine Chagas' disease. The results suggested the existence of at least two mechanisms of oxidative stress, dependent or independent with regard to the nitric oxide and cyclooxygenase pathway, where one or the other is more evident depending on the mouse strain.     

Cloning of Trypanosoma cruzi trans-Sialidase and Expression in Pichia pastoris

Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme that transfers sialic acids among glycoproteins and glycolipids both from the host cell surface and its own surface. This enzyme, called trans-sialidase, is different from higher eukaryotic sialyltransferases in that it does not accept cytidine 5′-monophospho-N-acetylneuraminic acid as a donor substrate. Also, the common glycosyltransferase structure is not present. To study this enzyme, an active member was cloned and expressed in higher eukaryotic cells. Expression of recombinant enzyme was achieved in the methylotrophic yeast Pichia pastoris. The N-terminal fusion of a secretion signal and the C-terminal addition of an epitope tag resulted not only in high expression levels, but also enabled easy detection and purification. Using P. pastoris, we obtained about 5 mg of enzymatically active trans-sialidase per liter of induced culture medium.     

Mapping of B cell epitopes in an immunodominant antigen of Trypanosoma cruzi using fusions to the Escherichia coli LamB protein

The JL8 protein antigen from Trypanosoma cruzi, a dominant immunogen in man, has been characterized as containing tandem amino acid repeats. Here, we describe the use of the LamB protein of Escherichia coli as a carrier of JL8 derived sequences in order to map the immunodominant B cell epitopes in this antigen. Five different sequences of JL8 were inserted in the LamB protein and the JL8-LamB fusion proteins were tested by ELISA with human chronic chagasic sera. The fusion carrying the sequence AEKQKAAEATKVAE was recognized by most sera. This protein was also capable of inhibiting the binding of human chagasic antibodies to GST-JL8 in competitive ELISA suggesting that it contains an immunodominant B cell epitope of JL8.     

Analysis and expression of the Borrelia burgdorferi P/Gau fla gene: Identification of heterogeneity with the B31 strain

The flagellin gene from the P/Gau strain of Borrelia burgdorferi was cloned and sequenced. The translated P/Gau flagellin protein differed from the flagellin of the B31 strain at 13 of 336 amino acids. This includes seven differences between amino acids 190-234, an immunodominant and specific region for B. burgdorferi. The entire flagellin molecule, as well as peptides of the internal portion of the protein which is more specific for B. burgdorferi, has been expressed in Escherichia coli using a pET7HIS.2 expression system. These peptides may be of great value for the development of sensitive and specific recombinant-based serological assays.     

Natural Chagas disease in four baboons

Background  Chagas disease is common in Central and South America and the southern United States. The causative agent is Trypanosoma cruzi (order Kinetoplastida, family Trypanosomatidae), a kinetoplastid protozoan parasite of humans and other vertebrates. It is a serious public health issue and the leading cause of heart disease and cardiovascular death in Central and South America. In 1984, a colony baboon was discovered to be infected with T. cruzi .
Methods  As the initial diagnosis was made by microscopic observation of the amastigote forms of T. cruzi in myocardial fibers, T. cruzi amastigotes have been identified in three additional baboons.
Results  The primary findings were similar in all four baboons and were congestive heart failure with edema of dependent areas, hydrothorax, hydropericardium, and multifocal to diffuse lymphoplasmacytic myocarditis.
Conclusions  A baboon animal model of Chagas disease could contribute significantly to the development of therapies for the disease in humans.     

Trypanosoma cruzi infection by oral route: how the interplay between parasite and host components modulates infectivity

Trypanosoma cruzi infection by oral route constitutes the most important mode of transmission in some geographical regions, as illustrated by reports on microepidemics and outbreaks of acute Chagas' disease acquired by ingestion of food contaminated with parasites from triatomine insects. In the mouse model, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium, a unique portal of entry for systemic infection. High efficiency of metacyclic forms in establishing infection by oral route is associated with expression of gp82, a stage-specific surface molecule that binds to gastric mucin and to epithelial cells. Gp82 promotes parasite entry by triggering the signaling cascades leading to intracellular Ca²⁺ mobilization. T. cruzi strains deficient in gp82 can effectively invade cells in vitro, by engaging the Ca²⁺ signal-inducing surface glycoprotein gp30. However, they are poorly infective in mice by oral route because gp30 has low affinity for gastric mucin. Metacyclic forms also express gp90, a stage-specific surface glycoprotein that binds to host cells and acts as a negative regulator of invasion. T. cruzi strains expressing gp90 at high levels, in addition to gp82 and gp30, are all poor cell invaders in vitro. Notwithstanding, their infectivity by oral route may vary because, unlike gp82 and gp30, which resist degradation by pepsin in the gastric milieu, the gp90 isoforms of different strains have varying susceptibility to peptic digestion. For instance, in a T. cruzi isolate, derived from an acute case of Chagas' disease acquired by oral route, gp90 is extensively degraded by gastric juice in the mouse stomach and this renders the parasite highly invasive towards target cells. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of the disease reported in outbreaks of oral infection.     

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas' disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas' disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas' disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens.     

Chemical Constituents from Aerial Parts of Baccharis sphenophylla and Effects against Intracellular Forms of Trypanosoma cruzi

The hexane extract from aerial parts Baccharis sphenophylla Dusén ex Malme (Asteraceae) displayed activity against amastigote forms of Trypanossoma cruzi and was subjected to chromatographic steps to afford one unreported – 7α-hydroxy-ent-abieta-8(14),13(15)-dien-16,12β-olide ( 1 ) and three known diterpenes – ent-kaur-16-en-19-oic acid, ( 2 ), grandifloric acid ( 3 ), and 15β-tiglinoyloxy-ent-kaur-16-en-19-oic acid ( 4 ), two sesquiterpenes – spathulenol ( 5 ) and oplopanone ( 6 ) – as well as hexacosyl p-coumarate ( 7 ). Isolated compounds were characterized by NMR and ESI-HR-MS spectra and were evaluated in vitro for activity against amastigote forms of the parasite T. cruzi – the relevant clinical form in the chronic phase of Chagas disease. In addition, the activity of compounds 1 – 7 against NCTC cells was evaluated. Compounds 1 and 7 showed effectiveness with EC₅₀ values of 21.3 and 16.9 μM, respectively. Both compounds also exhibited reduced toxicity against NCTC cells (CC₅₀>200 μM) with SI values higher than 9.4 and 11.9. Obtained results suggest that the new ent-abietane diterpene 1 and alkyl coumarate 7 could be used as prototypes for the development of novel and selective semisynthetic derivatives against intracellular forms of T. cruzi.     

A postgenomic view of the heat shock proteins in kinetoplastids

The kinetoplastids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi are causative agents of a diverse spectrum of human diseases: leishmaniasis, sleeping sickness and Chagas' disease, respectively. These protozoa possess digenetic life cycles that involve development in mammalian and insect hosts. It is generally accepted that temperature is a triggering factor of the developmental programme allowing the adaptation of the parasite to the mammalian conditions. The heat shock response is a general homeostatic mechanism that protects cells from the deleterious effects of environmental stresses, such as heat. This response is universal and includes the synthesis of the heat-shock proteins (HSPs). In this review, we summarize the salient features of the different HSP families and describe their main cellular functions. In parallel, we analyse the composition of these families in kinetoplastids according to literature data and our understanding of genome sequence data. The genome sequences of these parasites have been recently completed. The HSP families described here are: HSP110, HSP104, group I chaperonins, HSP90, HSP70, HSP40 and small HSPs. All these families are widely represented in these parasites. In particular, kinetoplastids possess an unprecedented number of members of the HSP70, HSP60 and HSP40 families, suggesting key roles for these HSPs in their biology.     

Presence of an unusually high concentration of an ubiquitinated histone-like protein in Trypanosoma cruzi

The conjugation of ubiquitin to histones H2A and H2B has been established in higher eukaryotes and has been related to changes in chromatin organization. In Trypanosoma cruzi, no condensation of chromatin occurs during mitosis. In order to determine the presence of histone ubiquitination in T. cruzi epimastigotes, histones were extracted from chromatin and analyzed by three electrophoretic systems: acid-urea, triton-acid-urea and sodium-dodecyl-sulphate polyacrylamide gel. The immunochemical detection of ubiquitin-histone conjugates by Western blotting showed a strong reaction with a slow migrating band of M_r 19 kDa. The high percentage of ubiquitin-histone conjugates present in T. cruzi chromatin may be related to the inability of this parasite to condense chromatin into a 30 nm fiber. J. Cell. Biochem. 66:433–440, 1997. © 1997 Wiley-Liss, Inc.     

Trypanosoma cruzi ubiquitin as an antigen in the differential diagnosis of Chagas disease and leishmaniasis

In the present report we describe Trypanosoma cruzi ubiquitin as an antigen to be utilized in the differential diagnosis of Chagas disease and leishmaniasis. Initially, recombinant T. cruzi ubiquitin was evaluated against a panel of sera by phage dot immunoassay, showing a good performance against chagasic sera. However, the presence of a carboxy-terminal tail region encoding a ribosomal protein homologous to a related protein present in the genome of Leishmania sp. gave significant cross-reactivity with leishmanial sera. Therefore, ubiquitin was purified by a simple biochemical protocol and its immunoreactivity was studied by enzyme-linked immunosorbent assay. Analysis of 104 sera indicates that the response to ubiquitin is very sensitive towards chronic chagasic sera (98%) and, more important, highly species-specific, presenting better performance compared to the use of the recombinant protein or the total epimastigote extracts when tested against a panel of leishmanial sera, where out of a total of 70 sera tested, only five sera from the mucocutaneous form of the disease reacted with T. cruzi ubiquitin. On the other hand, Leishmania ubiquitin was not recognized by chagasic sera, but was recognized by sera from different forms of leishmaniasis. These results make ubiquitin an excellent candidate to be used in the differential diagnosis of these two parasitic diseases. The molecular basis for this highly species-specific response is discussed.     

Lactose derivatives are inhibitors of Trypanosoma cruzi trans-sialidase activity toward conventional substrates in vitro and in vivo

Chagas' disease, caused by Trypanosoma cruzi, affects about 18 million people in Latin America, and no effective treatment is available to date. To acquire sialic acid from the host glycoconjugates, T. cruzi expresses an unusual surface sialidase with trans-sialidase activity (TcTS) that transfers the sugar to parasite mucins. Surface sialic acid was shown to have relevant functions in protection of the parasite against the lysis by complement and in mammalian host cell invasion. The recently determined 3D structure of TcTS allowed a detailed analysis of its catalytic site and showed the presence of a lactose-binding site where the beta-linked galactose accepting the sialic acid is placed. In this article, the acceptor substrate specificity of lactose derivatives was studied by high pH anion-exchange chromatography with pulse amperometric detection. The lactose open chain derivatives lactitol and lactobionic acid, as well as other derivatives, were found to be good acceptors of sialic acid. Lactitol, which was the best of the ones tested, effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Furthermore, lactitol inhibited parasite mucins re-sialylation when incubated with live trypanosomes and TcTS. Lactitol also diminished the T. cruzi infection in cultured Vero cells by 20-27%. These results indicate that compounds directed to the lactose binding site might be good inhibitors of TcTS.