Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a highly debilitating human pathology that affects millions of people in the Americas. The sequencing of this parasite's genome reveals that trans-sialidase/trans-sialidase-like (TcS), a polymorphic protein family known to be involved in several aspects of T. cruzi biology, is the largest T. cruzi gene family, encoding more than 1,400 genes. Despite the fact that four TcS groups are well characterized and only one of the groups contains active trans-sialidases, all members of the family are annotated in the T. cruzi genome database as trans-sialidase. After performing sequence clustering analysis with all TcS complete genes, we identified four additional groups, demonstrating that the TcS family is even more heterogeneous than previously thought. Interestingly, members of distinct TcS groups show distinctive patterns of chromosome localization. Members of the TcSgroupII, which harbor proteins involved in host cell attachment/invasion, are preferentially located in subtelomeric regions, whereas members of the largest and new TcSgroupV have internal chromosomal locations. Real-time RT-PCR confirms the expression of genes derived from new groups and shows that the pattern of expression is not similar within and between groups. We also performed B-cell epitope prediction on the family and constructed a TcS specific peptide array, which was screened with sera from T. cruzi-infected mice. We demonstrated that all seven groups represented in the array are antigenic. A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection. Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.     

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: Mutations in the 3' untranslated region of calmodulin gene are specific for lineages T. cruzi I, T. cruzi II, and the Zymodeme III isolates

Populations of Trypanosoma cruzi can be clustered in two main phylogenetic lineages, T. cruzi I and T. cruzi II and a third group denominated Zymodeme III (ZIII) has been described. Using 23 isolates representing the two major T. cruzi groups and the Zymodeme III, the 3' untranslated region (3'UTR) of the calmodulin gene was analyzed. Several mutations located on a 330 bp segment of this 3'UTR were observed, among which three important insertion/deletion events, namely (1) a dinucleotide AG present only in ZIII isolates; (2) a 13 bases purine block missing only in ZIII; and (3) a five base GT block in T. cruzi II. Minimum free energy dot plots show that T. cruzi I and T. cruzi ZIII exhibit similar patterns of optimal and sub-optimal folding of this segment. These mutations in 3'UTR of calmodulin raise the possibility that T. cruzi I and ZIII group are sharing common functional routes.     

The importance of the opossum (Didelphis albiventris) as a reservoir for Trypanosoma cruzi in Bambuí, Minas Gerais State.

In a survey realized on the sylvatic and peridomestic environments at Bambuí county, Minas Gerais State, 44 (37.9%) out of 116 opossums (Didelphis albiventris) captured were found to be naturally infected with Trypanosoma cruzi. One hundred and forty three parasite samples were obtained from 43 infected opossums using simultaneously hemoculture, xenodiagnosis (Triatoma infestans, Panstrongylus megistus and Rhodnius neglectus) and examination of anal glands contents. The parasite samples were characterized according to six isoenzyme patterns. All samples, independently of the method of isolation, presented an isoenzyme pattern similar to the standard T. cruzi Z1, showing that either xenodiagnosis or hemoculture can used without selecting parasite subpopulation from naturally infected opossums. Previous isoenzyme patterns reported for human T. cruzi isolates from the same region were completely different. This isoenzyme dissimilarity between sylvatic and domiciliar environments suggests the existence of two independent T. cruzi transmission cycles in Bambuí. The epidemiological implications of these results are discussed.     

Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.     

Trypanosoma cruzi (kinetoplastida Trypanosomatidae): biological heterogeneity in the isolates derived from wild hosts

The course of experimental infection of Swiss mice with 95 sylvatic Trypanosoma cruzi isolates included in TCI or TCII genotype was characterized. The purpose was to verify biological properties and its eventual correspondence with original host species, genotype or zymodeme. The isolates of T. cruzi were 100% infective, 55% resulted in patent parasitemia with 69% (36/52) of mortality. A meaningful biological heterogeneity was observed in both, TCI and TCII isolates. TCII isolates resulted in higher patent parasitemia 64% (38/59), in contrast to the 41% TCI infected Swiss mice (14/34). Parasitemia was not always associated to mortality. Higher biological heterogeneity was observed in T. cruzi II isolates derived from L. rosalia from the Atlantic Coastal Rain forest. TCII isolates derived from marsupials resulted in very similar infection profile in Swiss mice.     

Characterization of a candidate Trypanosoma rangeli small nucleolar RNA gene and its application in a PCR-based parasite detection

In this study, we report the isolation and characterization of a candidate Trypanosoma rangeli small nucleolar RNA (snoRNA) gene, and the development of a PCR assay for detection of the parasite based on its nucleotide sequence. This gene, isolated from a T. rangeli genomic sub-library, was named snoRNA-cl1 and is encoded by a multi-copy gene of 801bp in length. Computer sequence analysis of snoRNA-cl1 showed the presence of two sequence motifs, box C and box D, as well as of two long stretches that perfectly complement the universal core region of the mature rRNA 28S, suggesting that cl1 encodes for a Box C/D snoRNA from the parasite. Hybridization analysis using cl1 as probe, showed a weak hybridization signal with Trypanosoma cruzi DNA, demonstrating the existence of differences in this locus between these two species. Two oligonucleotide primers from this gene, which specifically amplified a 620-bp fragment in KP1 (+) and KP1 (-) strains of T. rangeli, were used in a PCR assay. The amplification allowed the detection of 1pg of DNA in the presence of heterologous DNA and no amplification was observed with different T. cruzi strains (groups I and II). In addition, the PCR assay reported here is able to detect T. rangeli in the presence of T. cruzi DNA, and is useful for detection of the parasite in samples from infected vectors.     

Evolutionary relationships in Trypanosoma cruzi: molecular phylogenetics supports the existence of a new major lineage of strains

For the purpose of investigating the evolutionary relationships among strains of the human parasite Trypanosoma cruzi, we have determined the nucleotide sequence, in 16 T. cruzi stocks, of a DNA fragment having approximately 1030 nucleotides in length. Phylogenetic analyses show the presence of at least three major groups of T. cruzi strains, a result that contradicts previous phylogenetic inferences based on polymorphism data. We also performed an analysis of the relative extent of nucleotide divergence among T. cruzi strains compared to the divergence between Leishmania species, using the gene encoding pteridine reductase. The results presented in this work show that the divergence among the most distant T. cruzi strains is at least as high as the divergence between two different species complexes of Leishmania, those containing L. major and L. mexicana.     

Minicircle organization and diversity in Trypanosoma cruzi populations

Trypanosoma cruzi strains and isolates can be divided into at least two groups using biochemical and molecular markers such as isoenzymes, ribosomal DNA, mini-exon gene spacers and some maxicircle genes. Despite the accumulating evidence that these major groups are phylogenetically distinct, their kinetoplast minicircle overall organization (i.e. number of conserved regions per length of minicircle molecule) remains conserved in all T. cruzi isolates studied so far, including the two T. cruzi major lineages -T. cruzi I and T. cruzi II - and a third group of uncertain taxonomic status, T. cruzi ZIII. Thus far, despite the extensive intra- and inter-minicircle sequence polymorphism, no group clustering has been observed.     

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.     

Trypanosoma cruzi in a caviomorph rodent: parasitological and pathological features of the experimental infection of Trichomys apereoides (Rodentia, Echimyidae)

To understand the interaction of Trypanosoma cruzi with caviomorph rodents, which supposedly have an ancient co-evolutionary history with this parasite, experimental infection of laboratory reared Trichomys apereoides with several isolates of both genotypes of the parasite was studied. Parasitemia, pattern of hematic cells, specific humoral immune response, histopathological features and parasite clearance were appraised. T. apereoides maintained stable infections independent of the T. cruzi genotype as demonstrated by positive PCR results in analyses of several tissues after a 5 months follow-up. The acute phase was characterized by abundant and disseminated presence of amastigotes, vacuolization and/or myocytolysis. Lymphocytosis was a common feature. The chronic phase was characterized mainly by lymphomacroeosinophilic infiltrates independent of the inoculated T. cruzi isolate. T. cruzi of different genotypes did not show any tissular preference in T. apereoides.     

The characterization of Chilean and Bolivian Trypanosoma cruzi stocks from Triatoma infestans by isoelectrofocusing

Culture forms of 12 Chilean and 9 Bolivian Trypanosoma cruzi stocks were compared isoenzymatically by the following enzymes: non-specific esterase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and alcohol dehydrogenase. On the basis of the electrophoretic mobility of these enzymes the stocks were classified into two main groups. Ten Chilean stocks were characterized as group II; two stocks showed enzyme patterns of group I. In contrast, five Bolivian stocks were classified as belonging to group I, the other four to group II. The results show that the two groups of T. cruzi overlap in Triatoma infestans suggesting that both groups of T. cruzi are infective for man. The classification of stocks into two groups is discussed in the light of published results of Brazilian T. cruzi stocks. A strong association of groups with the transmission cycles as it seems to be in Brazil does not exist in Chile and Bolivia.     

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.     

Cryptic epitope explains the failure of a monoclonal antibody to bind to certain isolates of Trypanosoma cruzi

A mouse monoclonal antibody, WIC 29.26 Ab, has previously been characterized as recognizing a carbohydrate epitope on a 72,000 m.w. glycoprotein (GP72) expressed on the surface of Trypanosoma cruzi epimastigotes and metacyclic trypomastigotes. This molecule has been implicated as a receptor in the control of parasite transformation, and when used as an immunogen in mice, partially protects against T. cruzi infection. In previous experiments in which a radioimmunoassay was used, WIC 29.26 Ab was found to react with approximately 50% of T. cruzi strains and clones derived from a variety of sources. In this study, we attempted to determine whether the WIC 29.26 Ab-nonreactive isolates lack the entire GP72 or merely lack the epitope recognized by this monoclonal antibody. WIC 226.4 Ab, a monoclonal antibody raised against periodate-treated GP72, reacted in an immunofluorescence assay with all strains and clones studied, including those which had not reacted with WIC 29.26 Ab. Likewise, two polyvalent rabbit sera, directed specifically against GP72, bound to all T. cruzi isolates tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent lysates of surface-labeled epimastigotes immunoprecipitated with WIC 29.26 Ab showed that the epitope bound by this antibody was present in all but one of the parasites that were surface-nonreactive, as well as in all those that were surface-reactive. WIC 29.26 Ab precipitated a single 72K Mr band from most strains and clones, but in several cases 79K Mr and 66K Mr bands were seen. Isolates from both the surface-reactive and the surface-nonreactive groups showed the latter pattern. These results demonstrate that GP72, or similar electrophoretic variants--and with one exception, the carbohydrate epitope bound by WIC 29.26 Ab--are present in the surface membrane of all strains and clones tested. This observation suggests that in intact epimastigotes of the surface-nonreactive isolates, the epitope is not accessible because of structural changes in the molecule itself or because of differences in the membrane environment of GP72.     

Trypanosoma cruzi: exploring the nuclear genome of zymodeme 3 stocks by chromosome size polymorphism

Chagas disease is emerging in the Brazilian Amazon. We evaluated the position of eight zymodeme 3 isolates from Amazonian sylvatic vectors and one human case in relation to Trypanosoma cruzi I and II major groups and hybrid strains by chromosome size polymorphism. Nineteen isolates were analyzed by mapping nine coding sequences on chromosomal bands (0.6-3.3Mbp). Numerical analysis was based on the absolute chromosomal size difference index (aCSDI). A dendrogram was obtained applying the minimum evolution criterion and considering the aCSDI values to estimate the branch lengths. The isolates were distributed in four groups. Group A clustered hybrid isolates; Groups B and C, T. cruzi II and T. cruzi I isolates, respectively. Seven Z3 stocks were clustered in Group D, which showed low intra-group diversity and was the most divergent. The proportion of two different-sized homologous chromosomes was determined. Wild vectors harboring Z3 stocks constitute a potential reservoir of human infection in the Amazon.     

Ancestral genomes, sex, and the population structure of Trypanosoma cruzi

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.     

Molecular characterization of the short interspersed repetitive element SIRE in the six discrete typing units (DTUs) of Trypanosoma cruzi

Repetitive sequences constitute an important proportion of the Trypanosoma cruzi genome; hence, they have been used as molecular markers and as amplification targets to identify the parasite presence via PCR. In this study, a molecular characterization of the SIRE repetitive element was performed in the six discrete typing units (DTUs) of T. cruzi. The results evidenced that this element, located in multiple chromosomes, was interspersed in the genome of all DTUs of the parasite. The presence of several motifs implicated in element insertion, duplication, and functionality suggests that SIRE could be an active element in the parasite genome. Of interest, there were SIRE specific Alu I fragments that allowed to discriminate DTU I from the others DTUs. Moreover, an UPGMA phenetic tree constructed from fragment sharing Southern blot data showed that T. cruzi I isolates conform a cluster separated from the T. cruzi II-VI isolates. When the relative number of SIRE copies was determined, a variation from 105 to 2,000 copies per haploid genome was observed among the different isolates without kept a DTU-relationship. In all, these findings suggest that SIRE sequence is a good target for parasite DNA amplification.     

Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages

It has been proposed that isolates of Trypanosoma cruzi, the agent of American trypanosomiasis, can be ordered into two primary phylogenetic lineages, first based on multilocus enzyme electrophoresis and random amplified polymorphic DNA, and subsequently based on the 24Salpha rRNA and mini-exon genes. Recent multilocus enzyme electrophoresis and random amplified polymorphic DNA data have additionally shown that the major multilocus enzyme electrophoresis/random amplified polymorphic DNA lineage II is further subdivided into five smaller lineages, designated IIa-IIe. In this study, the precise correspondence between the multilocus enzyme electrophoresis/random amplified polymorphic DNA and rRNA/mini-exon lineages was investigated. Using the 24Salpha rRNA and mini-exon markers in combination, five sets of strains were distinguished, corresponding to the multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I, IIa, IIc, IId and to lineages IIb/IIe together, respectively. The previous categorisation into only two primary lineages based on 24Salpha rRNA and mini-exon characterisation is explained, in part, by the lack of representativeness of the breadth of T. cruzi diversity in earlier study samples. Additionally, a PCR assay based on a length-variable region of the 18S rRNA gene distinguished lineage IIe from lineage IIb. Thus, the six multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages could be readily identified by combining data from the 24Salpha rRNA, mini-exon and 18S rRNA characterisation assays, further supporting the relevance of these genetic units for T. cruzi strain classification and subspecific nomenclature. The recently proposed groups T. cruzi I and T. cruzi II correspond to multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I and IIb, respectively. Our findings show that T. cruzi lineage characterisation based on a single marker (either mini-exon or 24Salpha rRNA) has insufficient resolution, and leads to important reinterpretations of recent epidemiological and evolutionary studies based on the oversimplified rRNA/mini-exon dichotomic classification of T. cruzi isolates.     

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization.