Molecular characterization of ribonucleoproteic antigens containing repeated amino acid sequences from Trypanosoma cruzi

Trypanosoma cruzi expresses several proteins containing antigenic amino acid repeats. Here we characterized TcRpL7a and TcRBP28, which carry similar repeat motifs and share homology to the eukaryotic L7a ribosomal protein and to a Trypanosoma brucei RNA binding protein, respectively. Analyses of the full length and truncated recombinant TcRpL7a showed that the humoral response of patients with Chagas disease is directed towards its repetitive domain. Sequence analyses of distinct copies of TcRpL7a genes present in the genome of six T. cruzi strains indicate that the number of repeats is higher in proteins from T. cruzi II than T. cruzi I strains. A serum panel of 59 T. cruzi infected patients showed that 73% reacted with TcRpL7a, 71% reacted with TcRBP28 and 80% reacted with 1:1 mixture of both antigens. Synthetic peptides harboring the TcRpL7a repeat motif reacted with 46% of the serum samples. Antibodies raised against both antigens identified equivalent amounts of the native proteins in all three stages of the parasite life cycle. Analyses of subcellular fractions indicated that TcRBP28 is present in the cytoplasm whereas TcRpL7a co-fractionates with polysomes. Confirming their predicted cellular localization, GFP fusions showed that, whereas GFP::TcRBP28 localizes in the cytoplasm, GFP::TcRpL7a accumulates in the nucleus, where ribosome biogenesis occurs.     

Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi

The dodecamer universal minicircle sequence is a conserved sequence present in minicircles of trypanosomatid kinetoplast DNA studied so far. This sequence is recognised by a protein named universal minicircle sequence binding protein, described for Crithidia fasciculata, involved in minicircle DNA replication. We have identified a Trypanosoma cruzi gene homologue of the Crithidia fasciculata universal minicircle sequence binding protein. Similar to the Crithidia fasciculata universal minicircle sequence binding protein, the Trypanosoma cruzi protein, named PDZ5, contains five zinc finger motifs. Pulsed field gel electrophoresis indicated that the pdz5 gene is located in the chromosomal band XX of the Trypanosoma cruzi genome. The predicted amino acid sequence of PDZ5 shows a high degree of similarity with several trypanosomatid zinc finger proteins. Specific antibody raised against Crithidia fasciculata universal minicircle sequence binding protein recognises both the recombinant and endogenous PDZ5. The complete pdz5 coding sequence cloned in bacteria expresses a recombinant PDZ5 protein that binds specifically to the universal minicircle sequence dodecamer. These data strongly suggest that PDZ5 represents a Trypanosoma cruzi universal minicircle sequence binding protein.     

Polymorphic and differential expression of the Trypanosoma cruzi alleles containing universal minicircle binding protein

DNA replication mechanisms are poorly understood in most of trypanosomatids, in particular the replication of the peculiar mitochondrial DNA, the kinetoplast DNA (kDNA). To contribute to the knowledge on the mechanism of kDNA replication in Trypanosoma cruzi, we have previously characterized the Universal Minicircle Sequence Binding Protein of this parasite (TcUMSBP), which was first called PDZ5 [E.R. Coelho, T.P. Urmenyi, J. Franco da Silveira, E. Rondinelli, R. Silva, Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi, Int. J. Parasitol. 33 (2003) 853-858]. In this work, we describe two highly polymorphic alleles of the TcUMSBP locus in the T. cruzi reference clone CL Brener and the differential expression pattern of these alleles. A 62 bp sequence in the TcUMSBP upstream intergenic region in one of its alleles affects the efficiency of polycistronic RNA processing and the polyadenylation sites, and therefore regulates the differential expression of TcUMSBP alleles of this locus.     

Evidence for multiple hybrid groups in Trypanosoma cruzi

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.     

Trypanosoma cruzi: sequence polymorphism of the gene encoding the Tc52 immunoregulatory-released factor in relation to the phylogenetic diversity of the species

We have previously identified a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin family involved in thiol-disulfide redox reactions. Gene targeting strategy and immunological studies allowed showing that Tc52 is among T. cruzi virulence factors. Taking into account that T. cruzi has a genetic variability that might be important determinant that governs the different behaviour of T. cruzi clones in vitro and in vivo, we thought it was of interest to analyse the sequence polymorphism of Tc52 gene in several reference clones. The DNA sequences of 12 clones which represent the whole genetic diversity of T. cruzi allowed showing that 40 amino-acid positions over 400 analysed are targets for mutations. A number of residues corresponding to putative amino-acids playing a role in GSH binding and/or enzymatic function and others located nearby are subject to mutations. Although the immunological analysis showed that Tc52 is present in parasite extracts from different clones, it is possible that the amino-acid differences could affect the enzymatic and/or the immunomodulatory function of Tc52 variants and therefore the parasite phenotype.     

Stage-specific translational efficiency and protein stability regulate the developmental expression of p37, an RNA binding protein from Trypanosoma brucei

We have previously characterized two novel RNA binding proteins, p34 and p37, from Trypanosoma brucei. Their sequences do not show significant homology to other proteins but are highly homologous to one another. The p34 and p37 proteins are developmentally regulated, with p34 the predominant protein in the procyclic stage and p37 nearly exclusively expressed in the bloodstream cells. In vivo metabolic labeling of procyclic cells showed that p34 and p37 were differentially translated, with levels of p34 approximately fourfold higher than p37. The newly synthesized p34 and p37 exhibited differential stability in the procyclic stage. In vitro analysis confirmed this observation and further suggested that this differential stability may be due to a trypsin-like cysteine protease activity in procyclic extracts that selectively degraded the p37 protein. Taken together, these results indicate that the developmental regulation of the T. brucei RNA binding protein, p37, occurs at both translational and post-translational levels.     

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.     

Trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate dehydrogenase and small subunit ribosomal RNA

The genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major have been sequenced, but the phylogenetic relationships of these three protozoa remain uncertain. We have constructed trypanosomatid phylogenies based on genes for glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and small subunit ribosomal RNA (SSU rRNA). Trees based on gGAPDH nucleotide and amino acid sequences (51 taxa) robustly support monophyly of genus Trypanosoma, which is revealed to be a relatively late-evolving lineage of the family Trypanosomatidae. Other trypanosomatids, including genus Leishmania, branch paraphyletically at the base of the trypanosome clade. On the other hand, analysis of the SSU rRNA gene data produced equivocal results, as trees either robustly support or reject monophyly depending on the range of taxa included in the alignment. We conclude that the SSU rRNA gene is not a reliable marker for inferring deep level trypanosome phylogeny. The gGAPDH results support the hypothesis that trypanosomes evolved from an ancestral insect parasite, which adapted to a vertebrate/insect transmission cycle. This implies that the switch from terrestrial insect to aquatic leech vectors for fish and some amphibian trypanosomes was secondary. We conclude that the three sequenced pathogens, T. brucei, T. cruzi and L. major, are only distantly related and have distinct evolutionary histories.     

Antiserum against perimicrovillar membranes and midgut tissue reduces the development of Trypanosoma cruzi in the insect vector, Rhodnius prolixus

Antiserum raised against Rhodnius prolixus perimicrovillar membranes (PMM) and midgut tissue interfered with the midgut structural organization and reduced the development of Trypanosoma cruzi in the R. prolixus insect vector. SDS-PAGE and Western blot analyses confirmed the specific recognition of midgut proteins by the antibody. Feeding, mortality, molt, and oviposition of the insects were unaffected by feeding with the antiserum. However, the eclosion of the eggs were reduced from R. prolixus females treated with antiserum. Additionally, in vivo evaluation showed that after oral treatment with the antiserum, the intensity of infection with the Dm-28c clone of T. cruzi decreased in the digestive tract of fifth-instar nymphs and in the excretions of R. prolixus adults. These results suggest that the changes observed in the PMM organization in the posterior midgut of R. prolixus may not be important for triatomine survival but the antiserum acts as a transmission-reduction vaccine able to induce significant decreases in T. cruzi infection in the vector.