Maria Júlia M. Alves

Trypanosoma cruzi: Shedding of surface antigens as membrane vesicles

Tissue culture-derived trypomastigotes from Trypanosoma cruzi spontaneously shed surface antigens into the culture medium. The shedding is a temperature- and time-dependent phenomenon and is independent of the presence of proteins or immune serum in the medium. The analysis of this process in four strains (Y, YuYu, CA1, and RA) showed differences in the amounts of polypeptides released. However, for all strains the liberation of the entire set of surface polypeptides ranging in molecular mass from 70 to 150 kDa was observed. Biochemical and electron microscopic data strongly suggest that most of the surface antigens are released as plasma membrane vesicles, ranging from 20 to 80 nm in diameter.

Partial inhibition of trypomastigote entry into cultured mammalian cells by monoclonal antibodies against a surface glycoprotein of Trypanosoma cruzi

Monoclonal antibodies were raised against the surface of trypomastigote forms of Trypanosoma cruzi. Although some of these antibodies reacted against antigens shared by trypomastigote and epimastigote or amastigote forms, the majority were trypomastigote-specific. Trypomastigote-specific monoclonal antibodies recognized all infective stages, including trypomastigotes from the bloodstream of infected mice, insect feces, tissue culture and those resulting from differentiation of epimastigotes in axenic culture media. The monoclonal antibodies H1A10 and 6A2, as well as Fab fragments from H1A10, partially prevented T. cruzi invasion of LLC-MK2 cell monolayers (inhibition of 50-70%) when present throughout the entire experiment. Both antibodies recognized an 85 kDa glycoprotein (Tc-85) of the trypomastigote surface which contains N-acetyl-D-glucosamine and/or sialic acid.

Cloning of a surface membrane glycoprotein specific for the infective form of Trypanosoma cruzi having adhesive properties to laminin

Trypomastigotes of Trypanosoma cruzi express a set of surface glycoproteins known, collectively, as Tc-85. A monoclonal antibody to these proteins, named H1A10, inhibits (50–90%) in vitro parasite interiorization into host cells, thus implicating these glycoproteins in the infection process. Two DNA inserts, a genomic DNA fragment and a full-length cDNA encoding the H1A10 epitope, have now been cloned and characterized. Results show that both have high sequence identity with all reported members of the gp85/trans-sialidase gene family, although the H1A10 epitope exists only in the Tc-85 subset of the family. The epitope has been mapped by competition of antibody binding to a Tc-85 recombinant protein with peptides having sequences predicted by the Tc-85 DNA sequence, which contains also putativeN-glycosylation sites and COOH-terminal glycosylphosphatidylinositol anchor insertion sites, as expected, since an N-glycan chain and a glycosylphosphatidylinositol anchor have been characterized previously in the Tc-85 subset. The protein encoded by the full-length cDNA insert binds to cells and in vitro to laminin, but not to gelatin or fibronectin, in a saturable manner. For the first time it was possible to assign a defined ligand to a sequenced glycoprotein belonging to the gp85 family. This fact, together with the reported binding of family members to cell surfaces, reinforces the hypothesis that this family encodes glycoproteins with similar sequences but differing enough as to bind to different ligands and thus forming a family of adhesion glycoproteins enabling the parasite to overcome the barriers interposed by cell membranes, extracellular matrices, and basal laminae.

L -Proline is essential for the intracellular differentiation of Trypanosoma cruzi

Using as the host cell, a proline‐requiring mutant of Chinese hamster ovary cell (CHO‐K1), it was possible to arrest the differentiation of amastigote forms of Trypanosoma cruzi at the intermediate intracellular epimastigote‐like stage. Complete differentiation to the trypomastigote stage was obtained by addition of l‐proline to the medium. This effect was more pronounced using the T. cruzi CL‐14 clone that differentiates fully at 33°C (permissive temperature) and poorly at 37°C (restrictive temperature). A synchronous differentiation of T. cruzi inside the host‐cell is then possible by temperature switching in the presence of proline. It was found that differentiation of intracellular epimastigotes and trypomastigote bursting were proline concentration dependent. The intracellular concentration of proline was measured as well as the transport capacity of proline by each stage of the parasite. Amastigotes have the highest concentration of free proline (8.09 ± 1.46 mM) when compared to trypomastigotes (3.81 ± 1.55) or intracellular epimastigote‐like forms (0.45 ± 0.06 mM). In spite of having the lowest content of intracellular free proline, intracellular epimastigotes maintained the highest levels of l‐proline transport compared to trypomastigotes and intracellular amastigotes, providing evidence for a high turnover for the l‐proline pool in that parasite stage. This is the first report to establish a relationship between proline concentration and intracellular differentiation of Trypanosoma cruzi in the mammalian host.

A lipopeptidophosphoglycan from Trypanosoma cruzi (epimastigota): Isolation, purification and carbohydate composition

A lipopeptidophosphoglycan was extracted from epimastigote forms of Trypanosoma cruzi by phenol (44%) treatment of sonicated cells. The substance was purified from other glycoproteins and nucleic acid as follows: ethanol fractionation. Bio-Gell P-150 column chromatography in the presence of 0.1% sodium dodecyl sulfate, extraction with chloroform/methanol/water (10 : 10 : 3) and precipitation of the pure compound by methanol. The substance migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis stained with periodic acid-Schiff and Coomassie blue. In the absence of sodium dodecyl sulfate very little or no migration was observef aggregates. In such gels a Sudan Black positive reaction coincident with the periodic acid-Schiff positive banc was obtained. Neutral sugars (60%, by phenol-sulfuric acid assay) were analysed by paper chromatography and gal-liquid chromatography. The following ratio was found: mannose : galactose : glucose = 35 : 22 : 1. Glucosamine, identified by paper chromatography, was colorimetrically estimated (0.8%). Sialic acid was not detected. Analysis by the biuret method gave 9.5% protein. All phosphorus present (2%) was released by hydrolysis, thus apparently excluding the possibility of an alkyl phosphonic acid as a structural component. Fatty acids were detected by thin layer chromatography in a hexane extract of the acid hydrolysate. Gas-liquid chromatography of the esterified mixture showed that the main component had the same retention time as palmitic acid methyl ester. The infrared spectrum was consistent with the general structure and indicated the presence of alpha-glycopyranosyl linkages. Low concentrations of the lipopeptidophosphoglycan were able to inhibit the concanavalin A-induced agglutination of epimastigotes.

Arginine kinase overexpression improves Trypanosoma cruzi survival capability

Arginine kinase catalyzes the reversible transphosphorylation between adenosine diphosphate (ADP) and phosphoarginine, which is involved in temporal and spatial adenosine triphosphate (ATP) buffering. Here we demonstrate that the homologous overexpression of the Trypanosoma cruzi arginine kinase improves the ability of the transfectant cells to grow and resist nutritional and pH stress conditions. The stable transfected parasites showed an increased cell density since day 10 of culture, when the carbon sources became scarce, which resulted 2.5‐fold higher than the control group on day 28. Additional stress conditions were also tested. We propose that arginine kinase is involved in the adaptation of the parasite to environmental changes.

Expression of trans-sialidase and 85-kDa glycoprotein genes in Trypanosoma cruzi is differentially regulated at the post-transcriptional level by labile protein factors.

To adapt to different environments,Trypanosoma cruzi, the protozoan parasite that causes Chagas’ disease, expresses a different set of proteins during development. To begin to understand the mechanism that controls this differential gene expression, we have analyzed the levels ofamastin and trans-sialidase mRNAs and the mRNAs encoding members of the 85-kDa glycoprotein gene family, which are differentially expressed in the T. cruzi stages found in the mammalian host. Amastin mRNA is expressed predominantly in intracellular and proliferative amastigotes.trans-Sialidase mRNAs are found mostly in forms undergoing transformation from amastigotes to trypomastigotes inside infected cells, whereas mRNAs encoding the 85-kDa glycoproteins appear only in the infective trypomastigotes released from the cells. The genes coding for these mRNA species are constitutively transcribed in all stages of T. cruzi cells, suggesting that expression is controlled post-transcriptionally during differentiation. Inhibition of transcription by actinomycin D revealed that each mRNA species has a relatively long half-life in stages where it accumulates. In the case of the trans-sialidase and 85-kDa glycoprotein genes, mRNA accumulation was induced by treatment with the protein synthesis inhibitor cycloheximide at the stages that preceded the normal accumulation. Therefore, mRNA stabilization may account for mRNA accumulation. mRNA degradation could be promoted by proteins with high turnover, or stabilization could be promoted by forming a complex with the translational machinery at defined times in development. Identification of the factors that induce mRNA degradation or stabilization is essential to the understanding of control of gene expression in these organisms.

Glycoproteins from Trypanosoma cruzi: Partial purification by gel chromatography

Glycoproteins from cell membranes have been related to several functions like antigenicity, cellular adhesion and hormone recognition, among others [ 11. Glycoproteins of unicellular eukaryotes have been much less studied when compared with higher organisms. In the cell surface of African trypanosomatids these macromolecules were related to antigenicity and host-parasite relationship [2-71. Recently, we reported a differential agglutination in the presence of concanavalin A between epimastigote and trypomastigote forms of Trypanosoma cruzi [8]. The agglutinability of the epimastigote forms suggested the existence of reacting glycoproteins toward con A in their cell surface. This report describes the partial purification and some properties of a glycoprotein complex of whole-cell epimastigote forms of T. cruzi.

Active Transport of l-Proline in Trypanosoma cruzi

Abstract l-proline is the main energy source in insect vector stages of most trypanosomatids, including Trypanosoma cruzi epimastigotes. This is the first biochemical description of two active proline transporter systems in T. cruzi. Uptake of this amino acid occurred by a low affinity system B and a high affinity system A. System B consistently appeared more specific than System A when excess competing amino acids were used in transport inhibition assays. Furthermore, the high affinity system is 70% inhibited by l-tryptophan, but the low affinity system is not. Both systems were found to be insensitive to the intracellular proline concentration and d-proline did not inhibit l-proline uptake showing that both systems are stereospecific. Both systems were Na+ and K+ independant but dependant on energy since ATP depletion impairs l-proline uptake. The combined action of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) and oligomycin, and the dependence of activity on pH, further differentiated between the two systems leading to the conclusion that the high affinity system is a H+ gradient-dependant transporter whereas the low affinity system depends directly on ATP.

A surface antigen of Trypanosoma cruzi involved in cell invasion (Tc-85) is heterogeneous in expression and molecular constitution.

A monoclonal antibody (McAb), H1A10, causes 46-96% inhibition of the invasion of tissue culture cells by trypomastigotes from different strains and clones of Trypanosoma cruzi. Higher inhibition was observed when axenic medium-derived metacyclic trypomastigotes (72-96%) were employed instead of tissue culture-derived trypomastigotes (46-60%). In contrast to the metacyclic population, in which all individuals reacted with the McAb, part of the tissue-cultured trypomastigote population did not bind the antibody. The molecular masses and isoelectric points of the glycoproteins recognized by the H1A10 McAb differed when tissue culture trypomastigotes of the Y strain and YuYu strain were analyzed. Variability between the strains was also observed when the antigens were metabolically labelled in the presence of tunicamycin. These results suggest that the differences described are probably not due solely to the carbohydrate portion of the molecule.