Caracterizacao de cepas do Trypanosoma cruzi isoladas de doencas nos quais foi realizado transplante de coracao / Caracterization of Trypanosoma cruzi strains isolated from patients with heart transplantation

Tres cepas do Trypanosoma cruzi foram isoladas de pacientes com doenca de Chagas cronica, receptores de coracao por meio de transplante. Houve a caracterizacao dela atraves de modelo experimental baseado no emprego de camundongos, com avaliacao de parasitismo, mortalidade e intensidade da inflamacao no coracao, alem de analise do grau de parasitismo nesse orgao. Como controle, teve lugar comparacao com o comportamento da ja bem conhecida cepa Y. Ficaram caracterizadas atuacoes diferentes das cepas, ao serem valorizados os parametros citados, sem correlacao rigorosa com as evolucoes pos-transplantes, que sofrem a influencia de varios fatores, entre os quais podem estar particularidades vinculadas ao parasitismo.

Characterization of a glycosyl-phosphatidylinositol-anchored membrane protein from Trypanosoma cruzi.

Four monoclonal antibodies (MAbs) specific for Trypanosoma cruzi were obtained. Flow cytometry analysis showed that these four MAbs stained the membranes of the three main morphological forms of T. cruzi: amastigotes, trypomastigotes, and epimastigotes. The four MAbs seemed to recognize the same 50- to 55-kDa antigen that was revealed by immunoblotting. Competition experiments revealed that they defined at least two different epitopes on the molecule. The antigen was detected on the external surface of the membrane by immunoelectron microscopy. Several experiments indicated that the 50- to 55-kDa antigen recognized by these four MAbs was a glycosyl-phosphatidylinositol-anchored membrane protein. (i) The antigen could be removed from the cell surface by treatment with proteases, NaOH, HNO2, and phosphatidylinositol-specific phospholipase C (PI-PLC). (ii) The phase distribution of the antigen in Triton X-114 solutions changed drastically upon treatment with PI-PLC. The antigen was found mainly in the detergent phase in nontreated samples and in the aqueous phase in PI-PLC-digested samples. (iii) A cross-reacting determinant that was found in other glycosyl-phosphatidylinositol-anchored membrane proteins appeared after PI-PLC treatment.

Contribuiçao ao estudo da infecçao de morcegos por hemoflagelados do gênero trypanosoma gruby, 1843 / Contribution to the study of bats infected with bloodflagelates of the genus trypanosoma gruby, 1843

O presente estudo foi realizado no período de julho de 1983 a dezembro de 1984, nos municípios cearenses de Russas, Palhano, Limoeiro do Norte, Morada Nova, Canindé, Quixadá e Pereiro, os quais correspondem a parte da área endêmica da tripanossomíase americana. Do total de 15 espécies de morcegos coletadas, apenas artibeus planirostris, phyllostomus hastatus e phyllostomus discolor foram encontradas infectadas por tripanossomos. Os tripanossomos isolados de A. planirostris e de alguns exemplares de P. hastatus assemelham-se, por sua forma, dimensoes e comportamento, a trypanosoma cruzi. No caso de amostras isoladas de P. discolor e de alguns exemplares de P. hastatus, a forma e dimensoes dos tripanossomos e o fato de nao infectarem camundongos permitem identificá-los como Trypanosoma cruzi var.marinkellei

[SDS-PAGE analysis of surface proteins and antigens evidences high heterogenity in natural clones of Trypanosoma cruzi, correlated with isoenzyme variability]. / L'analyse SDS-PAGE des protéines et antigènes de surface révèle une forte hétérogénéité chez les clones naturels de Trypanosoma cruzi, corrélée à la variabilité isoenzymatique.

Surface protein and surface antigen patterns of 19 Trypanosoma cruzi laboratory clones, representing 17 different isozymic profiles (zymodemes), were compared by SDS-PAGE analysis. Surface protein patterns were found to be complex and heterogeneous. According to the number of common bands, we calculated similarity coefficients of surface protein patterns on the one hand, and of surface antigen patterns on the other hand, for 33 stock pairwise comparisons. In both cases, these coefficients were statistically correlated to the isozyme index of genetic identity. Such a correlation between independent genetic markers favours the clonal structure of T. cruzi natural populations previously evidenced. Moreover, we did not observe any notable differences in the surface antigen pattern among 4 T. cruzi cloned stocks precipitated by homologous as well as heterologous hyperimmune sera. The immunological significance of the molecular weight variability in surface antigen patterns among different zymodemes is discussed.

Isolation and immunological analysis of Trypanosoma cruzi glycolipids.

A water-soluble glycolipidic fraction from Trypanosoma cruzi was isolated using a mixture of hexane and isopropanol. Analysis by SDS-polyacrylamide gel electrophoresis, after staining by silver and Sudan Black B, showed that the fraction contained one band with a relatively high mobility. Its reactivity and specificity with human chagasic sera and T. cruzi infected mouse sera or with sera from patients with several other pathologies was determined by an immunoradiometric assay. The glycolipid-based radioimmunoassay for the detection of T. cruzi antigens provided a sensitive measure of its activity. However, cross-reactivity with several sera from patients with visceral and cutaneous leishmaniasis was detected.

Characterization of glycosyl phosphatidylinositol lipids of parasitic protozoans: Leishmania mexicana mexicana promastigotes, Trypanosoma cruzi Peru epimastigotes and Tritrichomonas foetus.

Glycosyl phosphatidylinositol lipids of cultured L.mex, mexicana LV732 promastigotes, T. cruzi Peru epimastigotes and Tritrichomonas foetus have been isolated and characterized using metabolic labelling and chromatographic and mass spectrometric (MS) techniques. TLC of the unsaponifiable lipid fractions of L. mex. mexicana and T. cruzi obtained from DEAE Sephadex A-25 followed by Iatrobead column chromatography showed three inositol phosphate-containing lipid components. [3H]myo-inositol, [3H]palmitic acid or H3 32PO4 lipid precursors were incorporated into these three lipid components. Fraction 2 (LM2 and TCP-2) comprises inositol phosphate ceramides. The other two fractions appear to contain mono-O-alkyl and di-O-alkyl glycerol inositol phosphates. Lyso-1-O-alkyl phosphatidylinositols could be cleaved by treatment of PI-specific phosphalipase C. The di-O-alkyl-phospho inositols of these parasites being the first dialkylglycerol lipids reported from eukaryotic membranes raises the possibility of chemotherapy for leishmaniasis and trypanosomiasis based upon functional impairment of alkyl ether lipids. Tritrichomonas foetus contains two major glycophosphosphingolipids, designated TF1 and TF2, which are metabolically labelled with [3H]myo-inositol and H3 32PO4. Both lipids contained ceramides. The major ceramide contains the 18:0 and 18:1 bases and 16:0 N-acyl group. The major glycolipid fraction (TF1) contains fucose linked to inositol diphosphate; one of the phosphates being linked to the ceramide moiety, and the other to ethanolamine. TF1 appears to be a novel class of glycophosphosphingolipid, which may be a part of a membrane anchor.

Growth characteristics in axenic and cell cultures, protein profiles, and zymodeme typing of three Trypanosoma cruzi isolates from Louisiana mammals.

Rates of trypomastigote adherence, interiorization, amastigote division in, and trypomastigote release from Vero cells were measured for Trypanosoma cruzi isolates from a dog (Tc-D), opossum (Tc-O), and an armadillo (Tc-A) from Louisiana. Because the Tc-O and Tc-A (wild isolates) trypomastigotes became interiorized rapidly, the media were quickly depleted of trypomastigotes thus reducing the numbers available to adhere to cells. In contrast, the Tc-D trypomastigote interiorization rate was slower. Intracellular amastigote division rate was slower for the Tc-D than the wild isolates. The Tc-D trypomastigotes were released from cells approximately 2 days later than wild isolate trypomastigotes, but twice the number were released. Growth rate for Tc-D epimastigotes in liver infusion tryptose media was faster than that of wild isolates. The doubling times for Tc-D, Tc-O, and Tc-A were 48.0, 69.0, and 67.4 hr, respectively. Soluble parasite extract was produced from epimastigotes of each isolate by freeze/thawing, sonication, and high-speed centrifugation. Proteins were separated on an SDS-PAGE slab gel and stained with Coomassie blue. Although similar bands were present in each preparation, the general pattern of staining was similar only between the Tc-O and Tc-A preparations, which showed some differences from the Tc-D preparation. Each isolate was zymodeme typed using 5 enzymes in lysates produced from epimastigotes of each isolate. Enzymes were separated electrophoretically and stained. Wild isolates showed similar patterns as zymodeme 1 reference stock, whereas the Tc-D isolate produced a pattern that did not resemble any of the reference stocks examined.

Structural features of the lipopeptidophosphoglycan from Trypanosoma cruzi common with the glycophosphatidylinositol anchors.

The lipopeptidophosphoglycan (LPPG) from Trypanosoma cruzi, a major constituent of the plasma membrane of epimastigote forms, has been now extracted with butanol/water from delipidated cells and purified by hydrophobic chromatography. We have found that the LPPG undergoes two reactions, characteristic of the glycosylphosphatidylinositol anchors: (a) cleavage of the ceramide by phosphatidylinositol-specific phospholipase C (PtdIns-specific phospholipase C) from Bacillus thuringiensis, (b) nitrous acid deamination of the non-N-acylated glucosamine. Palmitoylsphinganine, palmitoylsphingosine, lignoceroylsphinganine and, as minor components, the stearoylceramides were identified by gas liquid chromatography/mass spectrometry. The presence of cross reacting determinant (CRD) epitopes in the glycophosphoinositol released by PtdIns-specific phospholipase C was investigated by direct and inhibition ELISA. A sample of glycophosphoinositol containing 5 micrograms carbohydrate caused 60% inhibition of the binding of anti-CRD antibodies raised against the soluble form of variant surface glycoprotein.

DNA methylation in Trypanosoma cruzi.

DNA isolated from the protozoan Trypanosoma cruzi has been found to contain 5-methylcytosine. Analysis of T. cruzi DNA by both HpaII and MspI restriction endonucleases suggests that the sequence -CCGG- is not methylated. Probably T. cruzi DNA also contains N6-methyladenine. This report constitutes the first clear demonstration of the presence of methylated bases in the nuclear DNA from trypanosomes.

Trypanosoma cruzi histones. Further characterization and comparison with higher eukaryotes.

Histones extracted from T. cruzi chromatin were analyzed in three electrophoretic systems. Our results show that a basic protein with some properties similar to those of histone H1 from higher eukaryotes is present in T. cruzi. However this protein presents different electrophoretic mobilities than H1 histone from higher eukaryotes in all three electrophoretic systems tested. Considering the marked differences observed in the electrophoretic mobilities of T. cruzi histones as compared with those from higher eukaryotes, it is proposed that histones are conservative proteins primarily with regard to their function.

Purification of a glycoprotein excreted by Trypanosoma cruzi to increase the permeability of the host-cell membrane.

During invasion of the prospective host cell, metacyclic forms of Trypanosoma cruzi render the membrane of HeLa cells permeable to the alpha-sarcin toxin, by excreting a glycoprotein with N-acetyl-D-glucosamine residues. The molecular weight of the glycoprotein is 64,000 dalton and its isoelectric point is 4.8.

Localization of sugar-binding proteins in Trypanosoma cruzi using gold-labeled neoglycoproteins.

Neoglycoproteins labeled with colloidal gold particles were used for the ultrastructural localization of sugar-binding sites in epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi. Binding sites for N-acetyl-D-glucosamine and D-galactose were seen on the surface of about 80% and 5 to 10% of the trypomastigote forms, respectively. They were inhibited by addition of the respective monosaccharides to the incubation medium. Binding sites for D-mannose were not observed in trypomastigotes. No labeling of the surface of amastigote and epimastigote forms was observed with the neoglycoproteins which bind to N-acetyl-D-glucosamine, D-galactose and D-mannose-binding sites. Labeling of the nucleus and the kinetoplast with the neoglycoprotein which recognizes N-acetyl-D-glucosamine binding sites was observed. The results obtained are discussed in relation to the possible role which surface sugar binding sites play in the process of T. cruzi-host cell interaction.

Localization of sugar-binding proteins in Trypanosoma cruzi using gold-labeled neoglycoproteins

Neoglicoproteínas marcadas com ouro coloidal foram utilizadas para localizaçäo de sítios de ligaçäo de carboidratos em Trypanosoma cruzi. Sítios de ligaçäo para N-acetil-D-glicosamina e D-galactose foram observadas na superfície de cerca de 80 e 5 a 10% das formas tripomastigotas, respectivamente. Sítios de ligaçäo para D-manose näo foram observados na superfície de tripomastigotas. Neoglicoproteínas que reconhecem N-acetil-D-glicosamina, D-galactose e D-manose näo se ligam á superficie das formas epimastigota e amastigota. Marcaçäo de núcleo e cinetoplasto foi observada com a neoglicoproteína que reconhece N-acetil-D-glicosamina. Os resultados obtidos säo discutidos levando em consideraçäo o papel que proteínas que se ligam a carboidratos podem desempenhar no processo de interaçäo entre T. cruzi e a célula hospedeira

Localization of sugar-binding proteins in Trypanosoma cruzi using gold-labeled neoglycoproteins

Neoglicoproteínas marcadas com ouro coloidal foram utilizadas para localizaþõo de sítios de ligaþõo de carboidratos em Trypanosoma cruzi. Sítios de ligaþõo para N-acetil-D-glicosamina e D-galactose foram observadas na superfície de cerca de 80 e 5 a 10% das formas tripomastigotas, respectivamente. Sítios de ligaþõo para D-manose nõo foram observados na superfície de tripomastigotas. Neoglicoproteínas que reconhecem N-acetil-D-glicosamina, D-galactose e D-manose nõo se ligam á superficie das formas epimastigota e amastigota. Marcaþõo de núcleo e cinetoplasto foi observada com a neoglicoproteína que reconhece N-acetil-D-glicosamina. Os resultados obtidos sõo discutidos levando em consideraþõo o papel que proteínas que se ligam a carboidratos podem desempenhar no processo de interaþõo entre T. cruzi e a célula hospedeira (AU)

Molecular cloning of mtp70, a mitochondrial member of the hsp70 family.

We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family. Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication. This relationship to DnaK is especially relevant in view of the intracellular location of the protein. Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa. Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs. In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E. coli.

A novel flagellar Ca2+-binding protein in trypanosomes.

A 24-kDa protein of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is recognized by antisera from both humans and experimental animals infected with this organism. Near its C terminus are two regions that have sequence similarity with several Ca2+-binding proteins and that conform to the "E-F hand" Ca2+-binding structure. We expressed a cDNA encoding this protein in Escherichia coli and showed that both the recombinant protein and the 24-kDa native trypanosome protein do indeed bind Ca2+. The protein's low Ca2+-binding capacity (less than 2 mol of Ca2+/mol of protein) and high Ca2+-binding affinity (apparent Kd less than 50 microM Ca2+) are consistent with binding of Ca2+ via the E-F hand structures. Immunofluorescence assays using a mouse antiserum directed against the fusion protein localized the native protein to the trypanosome's flagellum. The protein's abundance, Ca2+-binding property, and flagellar localization suggest that it participates in molecular processes associated with the high motility of the parasite.

Terminal beta-D-galactofuranosyl epitopes recognized by antibodies that inhibit Trypanosoma cruzi internalization into mammalian cells.

Polyclonal antisera to 80 - 90-kDa and to 50 - 60-kDa polypeptides of tissue-culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL-14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being beta-D-galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the beta-D-galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide beta-D-galactofuranose (1----3)-alpha-D-mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.

Changes in fatty acid composition associated with differentiation of Trypanosoma cruzi.

The fatty acid composition during the transformation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigotes (metacyclogenesis) was analysed by gas-liquid chromatography and mass spectrometry. Significant qualitative and quantitative changes in the fatty acid composition occurred during incubation of epimastigotes derived from LIT medium in the triatomine artificial urine (TAU). Metacyclogenesis was also followed by alterations in the fatty acid pattern but these were considerably less pronounced when compared to the pattern obtained for TAU-incubated epimastigotes. These results suggest that changes in the lipid composition precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.

Fatty acid composition of amastigote and trypomastigote forms of Trypanosoma cruzi.

The fatty acid composition of total lipids from trypomastigote and amastigote forms of Trypanosoma cruzi and of Vero cells before and after parasite infection were analyzed by gas-liquid chromatography and mass spectrometry. Even-numbered, saturated, monoenoic and polyenoic acids ranging from C-12 to C-18 were characterized in both T. cruzi development stages. Significant changes in the fatty acid composition occurred during the T. cruzi life cycle. Oleic and linoleic acids were prominent in trypomastigote forms, whereas palmitic acid was the major fatty acid of amastigotes. Other differences include higher stearic acid and lower palmitoleic and linolenic acid levels as well as the absence of lauric acid in amastigotes as compared with trypomastigote forms. The fatty acid pattern of Vero cells before T. cruzi infection as compared with that after infection showed mostly qualitative differences. Linoleic and linolenic acids were observed only in T. cruzi infected cells.

Characterization of a protein from Trypanosoma cruzi trypomastigotes that cleaves non-immune IgG bound through its Fab fragment.

A protein from Trypanosoma cruzi bloodstream trypomastigotes that binds IgG from man and several other animal species was isolated and characterized. The 52-kDa protein obtained from different T. cruzi cell extracts showed saturable binding with a K of 3.72 nM. Immunoblot analysis revealed that Fab, but not Fc, fragments of the Ig were recognized. When the protein was added to an unrelated C-fixation reaction, lysis was abolished in a dose-dependent fashion. When freshly prepared T. cruzi extracts were run in a 10% acrylamide SDS gel into which normal rabbit IgG was incorporated before polymerization, proteolytic activity, as seen by a transparent band after Coomassie blue staining, migrated in the same m.w. range of the 52-kDa protein. These data provide further clues to the mechanisms through which this pathogen escapes the host's immune response, thus maintaining a long-standing infection.