Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite.

Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite.

A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases.

Several genes encode members of the Trypanosoma cruzi (Tc) trans-sialidase (TS) family. These proteins contain an enzymatic domain on the N terminus, the only one required for TS activity, and an antigenic domain (SAPA (shed acute phase antigen) amino acid (aa) repeats) on the C terminus. Only some members of this glycoprotein family are enzymatically active. The complete sequence of two clones encoding the enzymatic domain of active and inactive protein from each of two Tc strains has now been obtained. Comparison of these sequences showed a limited divergence among them: 20 out of the 642 deduced aa in the enzymatic domain were found to differ. From these 20 aa, only one was found to be essential for enzymatic activity. A Tyr342 residue is deduced in both active proteins while a His342 is present in both inactive ones. This naturally occurring Tyr342-->His substitution completely abolished the TS activity. In addition to Tyr342, a second deduced aa, Pro231, was found to be necessary for full enzymatic TS activity; a Pro231-->Ala change rendered the TS protein partially active. Fourteen aa residues, including Tyr342, out of the 16 aa in the active site of a sialidase from Salmonella typhimurium are present at the same or very similar positions in the Tc TS.

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes.

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2-4 different chromosomes in several parasite isolates.

Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms.

Both, culture-derived and metacyclic trypomastigotes of Trypanosoma cruzi shed a glycoprotein, the shed acute phase antigen, that is responsible for the trans-sialidase activity. In the present work the structure of the glycosylphosphatidylinositol membrane anchor of the trans-sialidase isolated from metacyclic forms was determined. Parasites were metabolically labelled with [9, 10(n)3H]-palmitic acid and the glycoprotein was purified by immunoprecipitation with a monoclonal antibody directed against the repetitive aminoacid sequence. Treatment of the glycoprotein with phosphatidylinositol phospholipase C (PI-PLC) from Bacillus thuringiensis rendered a lipid that comigrated in TLC with a standard of ceramide. No alkylglycerol was detected in contrast with the results previously obtained for the trans-sialidase isolated from culture-derived trypomastigotes where both lipids were found. Chemical and chromatographic analysis showed that the lipid moiety is palmitoyldihydrosphingosine with a minor amount of stearoyldihydrosphingosine. The glycan constituent of the glycosylphosphatidylinositol-anchor was analysed by nitrous acid deamination of the aqueous phase of the PI-PLC treatment, followed by reduction with NaBH4 and hydrolysis of the phosphodiester with aqueous hydrofluoric acid. A major oligosaccharide was obtained and enzymatic treatment with exoglycosidases and further chromatography in a high pH anion exchange system showed that the trimannosyl core backbone is substituted by an alpha-galactose. A comparison between the lipid constituent of the glycosylphosphatidylinositol anchor of several proteins and their spontaneous shedding by the action of an endogenous PI-PLC was made.

The reactivity of sera from chagasic patients against different fragments of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, suggests the presence of defined antigenic and catalytic domains.

Three fragments of cruzipain, expressed separately in bacterial vectors, were used to detect antibodies in sera from patients with chronic Chagas' disease. Most antibodies directed against the enzyme were found to react with the C-terminal extension, thus suggesting the presence of immunodominant B-cell epitopes within this protein domain. Immunoprecipitation with these antibodies did not impair enzyme activity. It is suggested that cruzipain consists of an enzymatic domain and a non-enzymatic, immunodominant domain, which corresponds to the C-terminal extension.

A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi.

Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.

In vivo Junin virus-mouse macrophages interaction.

The role of mononuclear phagocytic cells in extraneural infection of the mouse with Junin virus (JV) was studied. Endpoint susceptibility (4 days of life) was evaluated by intraperitoneal (i.p.) inoculation of suckling mice. By means of immunofluorescence (IF) and C3 receptor assays, it was found that macrophages were permissive to viral replication in vivo and fostered the recruitment of inflammatory cells as evidenced by the absence of C3 marker. In support, in vitro infection failed to induce alterations of this receptor. Throughout, both in vivo and in vitro, there were no signs of C3-mediated phagocytosis. Silica treatment had no effect on either resistance or susceptibility, suggesting that the "macrophage-barrier" failed to hinder or favour the course of disease. Differences with other JV models are discussed.

Immune system pathogenesis is prevented by the neutralization of the systemic trans-sialidase from Trypanosoma cruzi during severe infections.

During the acute phase of Trypanosoma cruzi infection, strong haematological and immune system alterations are observed. The parasite expresses trans-sialidase, a virulence factor responsible for the sialylation of its surface glycoconjugates. This enzyme is also shed to the bloodstream where it is associated with immune system alterations triggered during the infection. During experimental and human infections, the host elicits antibodies able to neutralize the enzyme activity that would be responsible for restricting systemic trans-sialidase to the early steps of the infection, when major immune alterations are induced. The actual relevance of these antibodies was tested by passive transference of monoclonal neutralizing antibodies in acute infection models displaying extreme sensitivity to the infection. Mice were inoculated with virulent parasite strains that induce high parasitaemia, early mortality and strong immune tissue abnormalities. The trans-sialidase-neutralizing antibodies were able to preserve B cell areas both in ganglia and spleen as well as the thymus architecture even in these extreme models. Although no differences between control and treated mice regarding animal survival were found, a major role for the humoral response in controlling the damage of the immune system induced by a systemically distributed virulence factor was defined in an infection with a eukaryotic pathogen.

Junin virus-induced non-specific suppressor cells interact with unrelated antigen-specific suppressor cells.

Junin virus (JV) infection of adult (resistant) BALB/c mice induces antigen non-specific delayed-type hypersensitivity (DTH) suppressor T cells, termed Tsv, bearing the Thy-1+, Ly-1+2- phenotype. These cells may be related to survival to infection since DTH reaction is associated with lethal meningoencephalitis. Employing several xenogeneic red blood cell (RBC) sensitization schedules to induce different cell subpopulations, we have attempted to establish the target of JV-induced suppressor cells (Tsv). The target of Tsv cells was actually included in the antigen-specific suppressor cell compartment, as demonstrated for the RBC system. Tsv cells were able to trigger suppressor cells to act without loss of their specificity. The presence of two sets of sheep RBC-induced DTH suppressor cells bearing the Ly-1+2- and Ly-1-2+ phenotypes was disclosed in low (10(6))-dose sensitized mice. Both sets were simultaneously required by Tsv to achieve DTH suppression. In contrast, in high (10(8] SRBC-dose sensitized animals treated with cyclophosphamide (doses of 50 mg/kg), a single Ly-1-2+ suppressor cell was required.

Junin virus-induced delayed-type hypersensitivity suppression in adult mice.

Junin virus (JV) infection of suckling mice leads to lethal meningoencephalitis consistent with a delayed-type hypersensitivity (DTH)-like immune response. In contrast, there are no central nervous system (CNS) alterations, and high antibody titers are induced in resistant adult mice. As a possible explanation, JV infection in adult mice may provoke DTH depression. Thus in this work we study the alterations induced by JV in the immune response of adult mice by using sheep red blood cells (SRBC) as an unrelated indicator antigen. JV infection was found to abrogate DTH significantly, regardless of SRBC priming time, virus strain attenuation, and viral route of inoculation. The effect proved viral dose-dependent and required live and infectious virus. However, the humoral response to SRBC, as determined by splenic "plaque-forming cell" count was found higher than controls. These results are consistent with adult mouse response to JV infection. In contrast to the guinea pig model, there is no destruction of immunocompetent cells. T-cell percentage in the spleen was high, suggesting involvement with DTH-suppressive action. We suggest that the immune response to SRBC in adult infected mice may be used for understanding the mechanisms involved in resistance.

Correlation between cyclophosphamide-induced viral susceptibility and depletion of Junin virus-induced suppressor populations.

In contrast to lymphocytic choriomeningitis virus, another arenavirus, Junin virus (JV), the etiologic agent of Argentine hemorrhagic fever, when inoculated into suckling mice, induces lethal meningoencephalitis characterized by a delayed-type hypersensitivity (DTH)-like immune response. However, the adult BALB/c mouse is resistant to infection and no DTH reaction can be seen. This different viral sensitivity may be related to the development of an antigen non-specific DTH-suppressor cell pathway at work in the adult mouse. When the resistant mice are treated with cyclophosphamide (Cy) (50 mg/kg each dose) given at days -1,+1,+4 (zero: infection day), animals become susceptible and develop DTH reaction in brain that leads to death. We analyze the influence of the timing of Cy administration on the suppressor system developing after infection. It was found that Cy depletes the previously described JV-induced suppressor populations (Tsv) but a new suppressor cell (Tsv*) is disclosed bearing the Thy 1+ Ly1+2- phenotype which is unable to depress DTH in Cy-treated animals. With only two doses of Cy corresponding to days -1 and +1, the target of Tsv* cells is depleted but the third dose is still required to achieve full depletion of Tsv cells which are able to employ the Cy-resistant antigen-specific suppressor cells as targets. Since the Cy treatment is able to deplete the Tsv population together with the target of Tsv* cells, animals became unable to regulate lethal DTH reaction. Thus, a cellular explanation for an empirically established Cy schedule able to abrogate the adult mouse resistance to JV is proposed.

Suppressor T-cell population induced by Junin virus in adult mice.

Intracerebral (i.c.) Junin virus (JV) infection of adult BALB/c mice is characterized by the absence of morbidity and a low mortality (barely 8-10%). In contrast, the suckling mouse model exhibits almost 100% mortality following central nervous system (CNS) alterations consistent with a delayed-type hypersensitivity (DTH)-like immune response. Besides, JV infection of adult (resistant) mice leads to immunosuppression of DTH to unrelated antigens. Here we present evidence demonstrating that such suppression is mediated by JV-induced cells present in spleen from 24 hr to 24 days post-infection, bearing the Thy-1+, Ly-1+2- phenotype and reactive to an unrelated antigen such as sheep red blood cells (SRBC). No evidence of suppressor factors was found. A relatively low number of total splenic cells (5 x 10(6) cells/mouse) was enough to transfer suppression. Therefore, this cell population may be involved in adult mouse survival to JV infection.

Medium scale production and purification to homogeneity of a recombinant trans-sialidase from Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas' disease, presents an enzyme that catalyzes the transfer of sialic acid among glycoproteins and glycolipids known as trans-sialidase (TS), displaying some interesting features: 1) It differs from all other eucaryotic sialyltransferases, both kinetically and in substrate specificity and 2) it is involved in the parasite's mechanism of mammalian host cell invasion. We report here the production and purification to homogeneity of an enzymatically active recombinant TS (rTS) lacking the C-terminal amino acid repeats, using iminodiacetic metal affinity chromatography. Initial ratios of non-fusion recombinant versus total protein were very low in several expression systems tested, mainly due to high degradation rate. However, after purifying 1,330 times, we were able to obtain an essentially homogeneous preparation of rTS with a final yield of 29%. After minor changes, a modified protocol for a medium scale production was designed obtaining 0.5 mg of homogeneous rTS per liter of bacterial culture. The purified rTS behaved as a homogeneous protein in silver-stained denaturing gels, isoelectrofocusing and N-terminal sequencing having identical pH and temperature optima as the natural enzyme. Conditions to keep the rTS for long periods without a significant loss of activity were identified.