Isolation and characterisation of genomic and cDNA clones coding for a serine-, alanine-, and proline-rich protein of Trypanosoma cruzi.

We report here the isolation and characterisation of genomic and cDNA clones encoding a Serine-, Alanine-, and Proline-rich protein (SAP) of Trypanosoma cruzi metacyclic trypomastigotes. The deduced peptides translated from these clones were characterised by a high content of residues of alanine, proline, serine, glycine, valine, and threonine distributed in several repeats: P(2-4), S(2-3), A(2-3), AS, SA, PA, AP, SP, PS, and TP. The repeats are partially homologous to the serine-, alanine-, and proline-containing motifs of Leishmania major and Leishmania mexicana proteophosphoglycans. Genes coding for SAP are part of a polymorphic family whose members are linked to members of gp85/sialidase and mucin-like gene families. This is consistent with the hypothesis that this genetic organisation could be a means by which T. cruzi co-ordinates the expression of major surface proteins.

A recombinant protein based on the Trypanosoma cruzi metacyclic trypomastigote 82-kilodalton antigen that induces and effective immune response to acute infection.

To further investigate the immunological properties of the stage-specific 82-kDa glycoprotein (gp82) of Trypanosoma cruzi metacyclic trypomastigotes, previously shown to induce antigen-specific humoral and T-cell responses in mice, we performed a series of experiments with recombinant proteins containing sequences of gp82 fused to glutathione S-transferase. Of five fusion proteins tested, only J18b and J18b1, the carboxyproximal peptides containing amino acids 224 to 516 and 303 to 516, respectively, were recognized by monoclonal antibody 3F6 as well as by various anti-T. cruzi antisera and, when administered to mice, were capable of eliciting antibodies directed to the native gp82. The amino-terminal peptide and other carboxyterminal recombinant proteins lacking the central domain of gp82 (amino acids 224 to 356), which is exposed on the surface of live metacyclic forms, did not display any of these properties. Spleen cells derived from mice immunized with any of the five recombinant proteins proliferated in vitro in the presence of native gp82.J18b was the most stimulatory, whereas J18b3, the peptide containing amino acids 408 to 516, elicited the weakest response. When BALB/c mice immunized with J18b antigen plus A1(OH)3 as adjuvant were challenged 10 5 metacyclic trypomastigotes, 85% of them resisted acute infection, in comparison with control mice that received glutathione S-transferase plus adjuvant. Antibodies induced by J18b protein lacked agglutinating or complement-dependent lytic activity and failed to neutralize parasite infectivity. On the other hand, CD4+T cells from the spleens of J18b-immunized mice displayed an intense proliferative activity upon stimulation with 1.25 microgram of native gp82 per ml, which resulted in increased production of gamma interferon, a cytokine associated with resistance to T. cruzi infection.

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.

Trypanosoma cruzi: characterization of in vitro and in vivo synthesized polypeptides from epimastigote forms of different strains.

Intact RNAs were isolated from epimastigote forms of different Trypanosoma cruzi strains. Translation of the mRNAs using rabbit reticulocyte lysate and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed protein profiles comparable to those observed by labeling cells in vivo. No major interstrain differences were observed in the patterns of the polypeptides synthesized in vitro and in vivo, indicating that metabolic proteins are similar among distinct strains. Several T. cruzi polypeptides produced in the rabbit reticulocyte lysate system were immunoprecipitated by specific antisera. The patterns of antigenic polypeptides recognized by antisera raised against epimastigotes from different strains were also very similar.

Protective immune response to Plasmodium chabaudi, developed by mice after drug controlled infection or vaccination with parasite extracts: analysis of stage specific antigens from the asexual blood cycle.

The protective immune response to asexual blood infection by Plasmodium chabaudi was studied in mice immunized either by drug controlled infection or by vaccination with preparations of merozoïtes or free parasites at different stages of development. Animals immunized by the first method developed a sterile immunity. The passive transfer of their serum protected naïve recipients from the lethal development of the infection, but affected only moderately the initial course of the parasitaemia. Animals immunized with either ring, schizont or merozoïte preparations exhibited a limited but significant resistance to infection: when challenged with 10(6) parasites of the homologous strain they exhibited a reduced parasitaemia as compared to control mice, and in addition, 50% of them recovered from the infection. Immunochemical analysis of parasite antigens showed that a family of high molecular weight proteins synthesized essentially at the schizont stage and conserved in the merozoites are important immunogens. Quantitative rather than qualitative differences were observed in the pattern of parasite proteins immunoprecipitated by serum of animals exhibiting sterile immunity or moderate protective immunity. A schizont specific polypeptide of mol. wt 82 Kd which is found in the surface of the merozoite is preferentially immunoprecipited by serum from animals exhibiting sterile immunity.

Cloning of cDNAS that code for Plasmodium chabaudi antigens of the erythrocytic forms and cross-hybridize with P. falciparum.

Several Plasmodium chabaudi antigens are synthesized and the corresponding mRNAs are detected by cell-free translation only during specific stages of the erythrocytic cycle. Ring stage mRNA was used to construct a cDNA library in the plasmid vector pBR 322. The library was screened by differential hybridization with cDNA specific for ring stage parasites or schizonts. Two clones which showed some stage specificity and seven which did not were retained. Three clones were characterized by hybrid-selected translation. Clones 451, 148 and 443 contain sequences homologous to the messages for a 27 kDa stage-specific antigen, a 37 kDa early antigen and a 24 kDa antigen, respectively. The nine clones cross-hybridize to various degrees with cDNA from P. falciparum but cross hybridization intensities do not reflect the strength of immunological cross-reactivity.

Identification of surface membrane proteins and surface membrane antigens of plasmodium chabaudi merozoites.

Surface membrane proteins of viable merozoites of Plasmodium chabaudi were iodinated by the Iodogen method and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirteen surface membrane proteins ranging from 22 to 270 kDa were thus identified. Most of these proteins could be immunoprecipitated by sera of mice immunized with extracts of P. chabaudi. A few, however, were precipitated only by sera of mice challenged with living parasites after immunization.

Proteins synthesized during specific stages of the schizogonic cycle and conserved in the merozoites of Plasmodium chabaudi.

Highly synchronized blood infections of Plasmodium chabaudi (strain IP-PCI) were induced in mice. Pulse-labelling in vitro experiments, using [35S]methionine, identified 28 proteins whose synthesis is exclusively or preferentially observed during specific stages of the erythrocytic cycle: 11 during the ring state, 16 from the mature trophozoite and schizont stages and 1 during the schizont stage. An analysis of merozoite proteins that are synthesized at various stages of parasite development indicated that most of the higher molecular weight proteins are synthesized during the schizont stage of the blood cycle.

Chemical composition of the plasma membrane from epimastigote forms of Trypanosoma cruzi.

The chemical composition of two plasma membrane fractions from epimastigote forms of Trypanosoma cruzi is reported. Fraction M, a preparation obtained by conventional methods of cell fractionation is composed of 31% proteins, 34% lipids, 16% carbohydrates and 3% of the lipopeptidophosphoglycan. Phospholipids and sterols account for 7.5 and 9%, respectively, of the total mass. Phosphatidylethanolamine is the major phospholipid in fraction M, representing 45% of the total membrane phospholipids. The other fraction, fraction V (vesicles), was obtained by treatment of the cell with a vesiculating agent. This fraction contains 42% lipids, 20% carbohydrates, 13% proteins and 21% of the lipopeptidophosphoglycan. Phospholipids and sterols make up 17 and 8%, respectively, of the total mass of this fraction. Phosphatidylcholine and phosphatidylethanolamine are the main phospholipids found in fraction V. Phosphonolipids and sialic acid have not been detected in either membrane fraction. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis show that the glycoproteins ABC and the lipopeptidophosphoglycan are 50- and 10-times more concentrated, respectively, in fractions V and M than in the whole cell homogenate. The high molar sterol/phospholipid ratio found in fraction M suggests that this fraction is less fluid than fraction V, perhaps reflecting a migration of certain membrane components in the presence of the vesiculating agent. Hence, fraction M is, probably, more representative of the epimastigote plasma membrane as a whole than fraction V.