Identification of the telomere in Trypanosoma cruzi reveals highly heterogeneous telomere lengths in different parasite strains.

Here we describe the cloning and characterisation of the Trypanosoma cruzi telomere. In the Y strain, it is formed by typical GGGTTA repeats with a mean size of approximately 500 bp. Adjacent to the telomere repeats we found a DNA sequence with significant homology to the T.cruzi 85 kDa surface antigen (gp85). Examination of the telomere in nine T.cruzi strains reveals differences in the organisation of chromosome ends. In one group of strains the size of the telomere repeat is relatively homogeneous and short (0.5-1.5 kb) as in the Y strain, while in the other, the length of the repeat is very heterogeneous and significantly longer, ranging in size from 1 to >10 kb. These different strains can be grouped similarly to previously existing classifications based on isoenzyme loci, rRNA genes, mini-exon gene sequences, randomly amplified polymorphic DNA and rRNA promoter sequences, suggesting that differential control of telomere length and organisation appeared as an early event in T. cruzi evolution. Two-dimensional pulsed field gel electrophoresis analysis shows that some chromosomes carry telomeres which are significantly larger than the mean telomere length. Importantly, the T.cruzi telomeres are organised in nucleosomal and non-nucleosomal chromatin.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.     

Vaccination with epimastigotes of different strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection

In our laboratory, we have developed a model of vaccination in mice with Trypanosoma rangeli, a non-pathogenic parasite that shares many antigens with Trypanosoma cruzi. The vaccinated mice were protected against infection with virulent T. cruzi. The goal of the present work was to study the protective activity of strains of T. rangeli of different origin, with the aim of analysing whether this protective capacity is a common feature of T. rangeli. BALB/c mice were vaccinated with live or fixed epimastigotes of two T. rangeli strains, Choachi and SC-58. Vaccinated (VM) and control mice (CM) were infected with virulent T. cruzi, Tulahuen strain. The results showed that the levels of parasitemia of VM, vaccinated with the two strains of T. rangeli were significantly lower than those developed in CM. The survival rate of VM was higher than that CM. Histological studies revealed many amastigote nests and severe inflammatory infiltrates in the heart and skeletal muscles of CM, whereas in the VM only moderate lymphomonocytic infiltrates were detected. Altogether, the results of the present work as well as previous studies show that the antigens involved in the protection induced by T. rangeli are expressed in different strains of this parasite. These findings could prove useful in vaccine preparation.     

Characterization of glycosomal RING finger proteins of trypanosomatids

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins-PEX2, PEX10 and PEX12-comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.     

Immunoblot analysis of trypomastigote excreted-secreted antigens as a tool for the characterization of Trypanosoma cruzi strains and isolates

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted-secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.     

Antigenic make-up of Trypanosoma cruzi culture forms: identification of a specific component.

A comparison of Trypanosoma cruzi water soluble antigens with those of stercorarian and salivarian trypanosomes, and Leishmania using immunoprecipitation in gels and immunoelectrophoresis, with the aid of hyperimmune rabbit serum and heterologous adsorptions showed the following. 1) There is a high complexity of soluble antigens of T. cruzi and T. rangeli. 2) At the intraspecific level our results demonstrated the antigenic stability of T. cruzi when maintained in vitro, and that there was quantitative antigenic consistency of the culture forms of different strains of T. cruzi from diverse geographic and parasite sources. At the interspecific level, the antigenic relationships between T. cruzi and the other Trypanosomatidae were established, as follows: 6/10ths of the antigens are shared by stercorarian species (T. dionisii, T. rangeli); 4/10ths by a salivarian trypanosome (T. brucei); and 3/10ths by Leishmania (L. donovani, L. mexicana). 3) Among the 4/10ths of antigenic components specific to T. cruzi, one component was characterized by its antigenicity and immunogenicity in natural and experimental infections, and in immunization experiments; this component was specific to T. cruzi when compared to the other Trypanosomatidae antigens.     

Trypanosoma cruzi from the Paraguayan Chaco: isoenzyme profiles of strains isolated at Makthlawaiya

At Makthlawaiya, in the Paraguayan Chaco, the prevalence of Trypanosoma (Schizotrypanum) cruzi infection among both domestic Triatoma infestans and domestic dogs was 38%, and IgG anti-T. cruzi antibody was detected by the quantitative enzyme-linked immunosorbent assay (ELISA) in 80% (105/133) of human sera. Ninety percent (25/28) of T. cruzi strains isolated from both T. infestans and dogs showed heterozygous isoenzyme profiles for glucose phosphate isomerase, phosphoglucomutase and 6-phosphogluconate dehydrogenase. These strains appeared to be closely related to Bolivian zymodeme 2. Three Paraguayan T. cruzi strains showed homozygous isoenzyme profiles, similar to those of major Brazilian zymodemes. It was concluded that T. cruzi strains with heterozygous isoenzyme profiles predominate in domestic transmission cycles in this highly endemic area of the Paraguayan Chaco.     

The karyotype of Trypanosoma cruzi Dm 28c: comparison with other T. cruzi strains and trypanosomatids

Chromosome-sized DNA molecules from Trypanosoma cruzi clone Dm 28c were analyzed and compared with other T. cruzi strains and monogenetic trypanosomatids by orthogonal field alteration gel electrophoresis. The results showed that T. cruzi Dm 28c displays at least 18 chromosomes ranging from 550 to more than 1500 kb and that in general the trypanosomatids have smaller chromosomes distributed in the size range from 300 to 1500 kb. With the exception of T. cruzi strain G49, there is no evidence of minichromosomes, suggesting they are not widely distributed among different isolates of the parasite. The hybridization of T. cruzi chromosomal Southern blots with probes for T. cruzi-specific genes showed that their location can change from one strain to another, supporting the idea of the plasticity of the parasite genome. Furthermore, the chromosome pattern is strictly conserved during the transformation of T. cruzi Dm 28c epimastigotes to metacyclic trypomastigotes, suggesting that extensive chromosomal rearrangements do not occur during at least part of the life cycle of the parasite.     

Critical contribution of immunoproteasomes in the induction of protective immunity against Trypanosoma cruzi in mice vaccinated with a plasmid encoding a CTL epitope fused to green fluorescence protein

Acquired immunity against infection with Trypanosoma cruzi is dependent on CD8(+)T cells. Here, to develop a vaccine strategy taking advantage of activated CD8(+)T cells, we constructed a DNA vaccine, designated pGFP-TSA1, encoding a fusion protein linking GFP to a single CTL epitope of TSA1, a leading candidate for vaccine against T. cruzi. C57BL/6 mice vaccinated with this plasmid showed suppressed parasitemia and prolonged survival. Vaccination with pGFP-TSA1 enhanced epitope-specific cytotoxicity and IFN-gamma secretion by CD8(+)T cells. Furthermore, the depletion of CD8(+)T cells prior to challenge infection with T. cruzi completely abolished this protection, indicating that CD8(+)T cells are the principal effector T cells involved. When mice deficient in the proteasome activator PA28alpha/beta or the immunoproteasome subunits LMP2 and LMP7 were used, the protective immunity against infection was profoundly attenuated. Our findings clearly demonstrate that vaccination with pGFP-TSA1 successfully induces protection dependent on CD8(+)T cell activation, in which immunoproteasomes play a crucial role. It is noteworthy to document that physical binding of the epitope and GFP is required for induction of this protection, since mice vaccinated with pTSA1-IRES-GFP failed to acquire resistance, probably because the epitope and GFP are separately expressed in the antigen-presenting cells.     

The amino terminal domain of a novel WD repeat protein from Trypanosoma cruzi contains a non-canonical mitochondrial targeting signal

WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to the beta-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome, Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions. TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of Trypanosoma brucei and Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that mitochondrial import is mediated by an alternative targeting signal.     

Do commercial serologic tests for Trypanosoma cruzi infection detect Mexican strains in women and newborns?

We sought to determine the serological test that could be used for Trypanosoma cruzi seroprevalence studies in Mexico, where lineage I predominates. In a previous study among pregnant women and their newborns in the states of Yucatan and Guanajuato, we reported a 0.8-0.9% of prevalence for T. cruzi -specific antibodies by Stat-Pak and Wiener ELISA. We have expanded this study here by performing an additional non-commercial ELISA and confirming the seropositives with Western blot, using whole antigens of a local parasite strain. We found a seroprevalence of 0.6% (3/500) in Merida and 0.4% in Guanajuato (2/488). The 5 seropositive umbilical cord samples reacted to both non-commercial ELISA and Western blot tests, and only 1 of the maternal samples was not reactive to non-commercial ELISA. A follow-up of the newborns at 10 mo was performed in Yucatan to determine the presence of T. cruzi antibodies in children as evidence of congenital infection. None of the children was seropositive. One newborn from an infected mother died at 2 wk of age of cardiac arrest, but T. cruzi infection was not confirmed. The T. cruzi seroprevalence data obtained with both commercial tests (Stat-Pak and ELISA Wiener) are similar to those from non-commercial tests using a local Mexican strain of T. cruzi.     

Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis

Diagnosis of individuals infected by Trypanosoma cruzi is performed mainly by serological tests using crude antigens, which might crossreact with other infections. In the past ten years, many recombinant T. cruzi proteins and synthetic peptides have been described, and some are already on the market. Managers of laboratories and blood banks need to make decisions on a cost-benefit basis whether to include these new-generation tests. Here, we indicate antigens that are likely to prove most useful.     

Anti-striated muscle antibody activity produced by Trypanosoma cruzi

We have previously shown that Trypanosoma cruzi shares antigenic determinants with preparations of the calcium-sequestering adenosine triphosphatase of sarcoplasmic reticulum. The cross-reacting antigen (SRA) is also apparently present on the sarcolemma of cardiac myofibers. Using highly specific reference antisera to either the small membranes of T. cruzi or to a tryptic fragment of striated muscle SRA, it was shown that SRA is present in the striated muscle of animals representative of the evolutionary scale ranging from nonhuman primate to fish. The small membranes of nine different T. cruzi strains isolated from widely divergent areas of the American continents also reacted with the reference antisera. This indicates that SRA is present in these T. cruzi strains and may be prevalent among all T. cruzi strains. The shared T. cruzi-striated muscle antigen, SRA, may be a heteroantigen present in all T. cruzi strains and in the striated muscle of all classes of animals. Immunization of rabbits (three of five) or chickens (five pairs of five pairs) with striated muscle membrane preparations of different classes of animals, particularly those of nonhuman primate, chicken, and turtle, gave rise to IgG anti-allogeneic striated muscle antibody activity. Immunization of rabbits (four of nine) and chickens (five pairs of six pairs) with the small membranes of different T. cruzi strains also produced IgG anti-allogeneic striated muscle. These data indicate that T. cruzi shares cross-immunogenicity with striated muscle SRA. Since SRA is apparently present on the sarcolemma of cardiac myofibers, it may be implicated in the immunopathogenesis of Chagas' disease.     

Post genomic analysis of permeases from the amino acid/auxin family in protozoan parasites

The "amino acid/auxin permeases" is probably the most represented family of transporters in the Trypanosoma cruzi genome. Using a high-throughput searching routine and preliminary data from the T. cruzi genome project, more than 15,000 sequences were iteratively assembled into contigs, and 60 open reading frames corresponding to different putative amino acid transporters, clustered in 12 groups, were detected and characterized in silico. T. cruzi genomic organization of such sequences showed that these putative amino acid transporter genes are in an unusually large number and arranged in repeat clusters comprising about 0.2% of the genome. These data suggest that the family has evolved following tandem duplication events and constitutes a novel family of variable proteins in protozoan organisms. The mRNA expression of the predicted genes was demonstrated in infective and non-infective parasite forms. Orthologous sequences were also identified in other unicellular parasites such as Leishmania spp., Plasmodium spp., and Trypanosoma brucei.