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1.  TriTrypDB: a functional genomic resource for the Trypanosomatidae 
Nucleic Acids Research  2009;38(Database issue):D457-D462.
TriTrypDB ( is an integrated database providing access to genome-scale datasets for kinetoplastid parasites, and supporting a variety of complex queries driven by research and development needs. TriTrypDB is a collaborative project, utilizing the GUS/WDK computational infrastructure developed by the Eukaryotic Pathogen Bioinformatics Resource Center ( to integrate genome annotation and analyses from GeneDB and elsewhere with a wide variety of functional genomics datasets made available by members of the global research community, often pre-publication. Currently, TriTrypDB integrates datasets from Leishmania braziliensis, L. infantum, L. major, L. tarentolae, Trypanosoma brucei and T. cruzi. Users may examine individual genes or chromosomal spans in their genomic context, including syntenic alignments with other kinetoplastid organisms. Data within TriTrypDB can be interrogated utilizing a sophisticated search strategy system that enables a user to construct complex queries combining multiple data types. All search strategies are stored, allowing future access and integrated searches. ‘User Comments’ may be added to any gene page, enhancing available annotation; such comments become immediately searchable via the text search, and are forwarded to curators for incorporation into the reference annotation when appropriate.
PMCID: PMC2808979  PMID: 19843604
2.  Anatomy and evolution of telomeric and subtelomeric regions in the human protozoan parasite Trypanosoma cruzi 
BMC Genomics  2012;13:229.
The subtelomeres of many protozoa are highly enriched in genes with roles in niche adaptation. T. cruzi trypomastigotes express surface proteins from Trans-Sialidase (TS) and Dispersed Gene Family-1 (DGF-1) superfamilies which are implicated in host cell invasion. Single populations of T. cruzi may express different antigenic forms of TSs. Analysis of TS genes located at the telomeres suggests that chromosome ends could have been the sites where new TS variants were generated. The aim of this study is to characterize telomeric and subtelomeric regions of T. cruzi available in TriTrypDB and connect the sequences of telomeres to T. cruzi working draft sequence.
We first identified contigs carrying the telomeric repeat (TTAGGG). Of 49 contigs identified, 45 have telomeric repeats at one end, whereas in four contigs the repeats are located internally. All contigs display a conserved telomeric junction sequence adjacent to the hexamer repeats which represents a signature of T. cruzi chromosome ends. We found that 40 telomeric contigs are located on T. cruzi chromosome-sized scaffolds. In addition, we were able to map several telomeric ends to the chromosomal bands separated by pulsed-field gel electrophoresis.
The subtelomeric sequence structure varies widely, mainly as a result of large differences in the relative abundance and organization of genes encoding surface proteins (TS and DGF-1), retrotransposon hot spot genes (RHS), retrotransposon elements, RNA-helicase and N-acetyltransferase genes. While the subtelomeric regions are enriched in pseudogenes, they also contain complete gene sequences matching both known and unknown expressed genes, indicating that these regions do not consist of nonfunctional DNA but are instead functional parts of the expressed genome. The size of the subtelomeric regions varies from 5 to 182 kb; the smaller of these regions could have been generated by a recent chromosome breakage and telomere healing event.
The lack of synteny in the subtelomeric regions suggests that genes located in these regions are subject to recombination, which increases their variability, even among homologous chromosomes. The presence of typical subtelomeric genes can increase the chance of homologous recombination mechanisms or microhomology-mediated end joining, which may use these regions for the pairing and recombination of free ends.
PMCID: PMC3418195  PMID: 22681854
3.  Genome Size, Karyotype Polymorphism and Chromosomal Evolution in Trypanosoma cruzi 
PLoS ONE  2011;6(8):e23042.
The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites.
Methodology/Principal Findings
The genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (José-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively.
Despite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.
PMCID: PMC3155523  PMID: 21857989
4.  Chromosomal copy number variation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains 
BMC Genomics  2015;16(1):499.
Trypanosoma cruzi, the etiologic agent of Chagas disease, is currently divided into six discrete typing units (DTUs), named TcI–TcVI. CL Brener, the reference strain of the T. cruzi genome project, is a hybrid with a genome assembled into 41 putative chromosomes. Gene copy number variation (CNV) is well documented as an important mechanism to enhance gene expression and variability in T. cruzi. Chromosomal CNV (CCNV) is another level of gene CNV in which whole blocks of genes are expanded simultaneously. Although the T. cruzi karyotype is not well defined, several studies have demonstrated a significant variation in the size and content of chromosomes between different T. cruzi strains. Despite these studies, the extent of diversity in CCNV among T. cruzi strains based on a read depth coverage analysis has not been determined.
We identify the CCNV in T. cruzi strains from the TcI, TcII and TcIII DTUs, by analyzing the depth coverage of short reads from these strains using the 41 CL Brener chromosomes as reference. This study led to the identification of a broader extent of CCNV in T. cruzi than was previously speculated. The TcI DTU strains have very few aneuploidies, while the strains from TcII and TcIII DTUs present a high degree of chromosomal expansions. Chromosome 31, which is the only chromosome that is supernumerary in all six T. cruzi samples evaluated in this study, is enriched with genes related to glycosylation pathways, highlighting the importance of glycosylation to parasite survival.
Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression, which represents a strategy that may be crucial for parasites that mainly depend on post-transcriptional mechanisms to control gene expression.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1680-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4491234  PMID: 26141959
Chromosome copy number variation; Trypanosoma cruzi; Genomic plasticity
5.  A population study of the minicircles in Trypanosoma cruzi: predicting guide RNAs in the absence of empirical RNA editing 
BMC Genomics  2007;8:133.
The structurally complex network of minicircles and maxicircles comprising the mitochondrial DNA of kinetoplastids mirrors the complexity of the RNA editing process that is required for faithful expression of encrypted maxicircle genes. Although a few of the guide RNAs that direct this editing process have been discovered on maxicircles, guide RNAs are mostly found on the minicircles. The nuclear and maxicircle genomes have been sequenced and assembled for Trypanosoma cruzi, the causative agent of Chagas disease, however the complement of 1.4-kb minicircles, carrying four guide RNA genes per molecule in this parasite, has been less thoroughly characterised.
Fifty-four CL Brener and 53 Esmeraldo strain minicircle sequence reads were extracted from T. cruzi whole genome shotgun sequencing data. With these sequences and all published T. cruzi minicircle sequences, 108 unique guide RNAs from all known T. cruzi minicircle sequences and two guide RNAs from the CL Brener maxicircle were predicted using a local alignment algorithm and mapped onto predicted or experimentally determined sequences of edited maxicircle open reading frames. For half of the sequences no statistically significant guide RNA could be assigned. Likely positions of these unidentified gRNAs in T. cruzi minicircle sequences are estimated using a simple Hidden Markov Model. With the local alignment predictions as a standard, the HMM had an ~85% chance of correctly identifying at least 20 nucleotides of guide RNA from a given minicircle sequence. Inter-minicircle recombination was documented. Variable regions contain species-specific areas of distinct nucleotide preference. Two maxicircle guide RNA genes were found.
The identification of new minicircle sequences and the further characterization of all published minicircles are presented, including the first observation of recombination between minicircles. Extrapolation suggests a level of 4% recombinants in the population, supporting a relatively high recombination rate that may serve to minimize the persistence of gRNA pseudogenes. Characteristic nucleotide preferences observed within variable regions provide potential clues regarding the transcription and maturation of T. cruzi guide RNAs. Based on these preferences, a method of predicting T. cruzi guide RNAs using only primary minicircle sequence data was created.
PMCID: PMC1892023  PMID: 17524149
6.  Kinetoplastid PPEF phosphatases: Dual acylated proteins expressed in the endomembrane system of Leishmania 
Bioinformatic analyses have been used to identify potential downstream targets of the essential enzyme N-myristoyl transferase in the TriTryp species, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. These database searches predict ∼60 putative N-myristoylated proteins with high confidence, including both previously characterised and novel molecules. One of the latter is an N-myristoylated protein phosphatase which has high sequence similarity to the Protein Phosphatase with EF-Hand (PPEF) proteins identified in sensory cells of higher eukaryotes. In L. major and T. brucei, the PPEF-like phosphatases are encoded by single-copy genes and are constitutively expressed in all parasite life cycle stages. The N-terminus of LmPPEF is a substrate for N-myristoyl transferase and is also palmitoylated in vivo. The wild type protein has been localised to the endocytic system by immunofluorescence. The catalytic and fused C-terminal domains of the kinetoplastid and other eukaryotic PPEFs share high sequence similarity, but unlike their higher eukaryotic relatives, the C-terminal parasite EF-hand domains are degenerate and do not bind calcium.
PMCID: PMC1885993  PMID: 17169445
PPEF, Protein Phosphatase with EF-Hands; NMT, N-myristoyl transferase; BSF, bloodstream form; PCF, procyclic form; N-Myristoylation; Palmitoylation; Protein phosphatases; Bioinformatics
7.  Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection 
PLoS Pathogens  2016;12(4):e1005511.
Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our comprehensive, high resolution transcriptomic dataset provides a substantially more detailed interpretation of T. cruzi infection biology and offers a basis for future drug and vaccine discovery efforts.
Author Summary
In-depth knowledge of the functional processes governing host colonization and transmission of pathogenic microorganisms is essential for the advancement of effective intervention strategies. This study focuses on Trypanosoma cruzi, the vector-borne protozoan parasite responsible for human Chagas disease and the leading cause of infectious myocarditis worldwide. To gain global insights into the biology of this parasite and its interaction with mammalian host cells, we have exploited a deep-sequencing approach to generate comprehensive, high-resolution transcriptomic maps for mammalian-infective stages of T. cruzi with the simultaneous interrogation of the human host cell transcriptome across an infection time course. We demonstrate that the establishment of intracellular T. cruzi infection in mammalian host cells is accompanied by extensive remodeling of the parasite and host cell transcriptomes. Despite the lack of transcriptional control mechanisms in trypanosomatids, our analyses identified functionally-enriched processes within sets of developmentally-regulated transcripts in T. cruzi that align with known or predicted biological features of the parasite. The novel insights into the biology of intracellular T. cruzi infection and the regulation of amastigote development gained in this study establish a unique foundation for functional network analyses that will be instrumental in providing functional links between parasite dependencies and host functional pathways that have the potential to be exploited for intervention.
PMCID: PMC4821583  PMID: 27046031
8.  Ancestral Genomes, Sex, and the Population Structure of Trypanosoma cruzi 
PLoS Pathogens  2006;2(3):e24.
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, 24Sα 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 24Sα 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.
The parasite protozoan Trypanosoma cruzi causes Chagas disease, a malady that afflicts almost 20 million people in South America and Central America. Although the genome sequencing of T. cruzi has been recently completed, little is known about its population structure and evolution. Since 1999, two major evolutionary lineages presenting distinct epidemiological characteristics have been recognized in the parasite: T. cruzi I and T. cruzi II, the latter being much more associated with severe chronic cases of the disease. We describe new and important aspects of the population structure of the parasite, especially the characterization of a third ancestral lineage that we propose to call T. cruzi III. Through careful dissection of the genetic constitution of blocks of genes that are stably transmitted from generation to generation of the parasite we deduced at least two occurrences of the formation of hybrid strains from the parental lineages T. cruzi II and T. cruzi III, including the strain CLBrener, whose genome was sequenced. We did not find any hybrids originating from T. cruzi I. A fascinating finding was that both hybrids studied had the same mitochondrial DNA type as the T. cruzi III ancestral lineage, which was quite different from T.cruzi II.
PMCID: PMC1434789  PMID: 16609729
9.  Gene organization and sequence analyses of transfer RNA genes in Trypanosomatid parasites 
BMC Genomics  2009;10:232.
The protozoan pathogens Leishmania major, Trypanosoma brucei and Trypanosoma cruzi (the Tritryps) are parasites that produce devastating human diseases. These organisms show very unusual mechanisms of gene expression, such as polycistronic transcription. We are interested in the study of tRNA genes, which are transcribed by RNA polymerase III (Pol III). To analyze the sequences and genomic organization of tRNA genes and other Pol III-transcribed genes, we have performed an in silico analysis of the Tritryps genome sequences.
Our analysis indicated the presence of 83, 66 and 120 genes in L. major, T. brucei and T. cruzi, respectively. These numbers include several previously unannotated selenocysteine (Sec) tRNA genes. Most tRNA genes are organized into clusters of 2 to 10 genes that may contain other Pol III-transcribed genes. The distribution of genes in the L. major genome does not seem to be totally random, like in most organisms. While the majority of the tRNA clusters do not show synteny (conservation of gene order) between the Tritryps, a cluster of 13 Pol III genes that is highly syntenic was identified. We have determined consensus sequences for the putative promoter regions (Boxes A and B) of the Tritryps tRNA genes, and specific changes were found in tRNA-Sec genes. Analysis of transcription termination signals of the tRNAs (clusters of Ts) showed differences between T. cruzi and the other two species. We have also identified several tRNA isodecoder genes (having the same anticodon, but different sequences elsewhere in the tRNA body) in the Tritryps.
A low number of tRNA genes is present in Tritryps. The overall weak synteny that they show indicates a reduced importance of genome location of Pol III genes compared to protein-coding genes. The fact that some of the differences between isodecoder genes occur in the internal promoter elements suggests that differential control of the expression of some isoacceptor tRNA genes in Tritryps is possible. The special characteristics found in Boxes A and B from tRNA-Sec genes from Tritryps indicate that the mechanisms that regulate their transcription might be different from those of other tRNA genes.
PMCID: PMC2695483  PMID: 19450263
10.  Widespread, focal copy number variations (CNV) and whole chromosome aneuploidies in Trypanosoma cruzi strains revealed by array comparative genomic hybridization 
BMC Genomics  2011;12:139.
Trypanosoma cruzi is a protozoan parasite and the etiologic agent of Chagas disease, an important public health problem in Latin America. T. cruzi is diploid, almost exclusively asexual, and displays an extraordinarily diverse population structure both genetically and phenotypically. Yet, to date the genotypic diversity of T. cruzi and its relationship, if any, to biological diversity have not been studied at the whole genome level.
In this study, we used whole genome oligonucleotide tiling arrays to compare gene content in biologically disparate T. cruzi strains by comparative genomic hybridization (CGH). We observed that T. cruzi strains display widespread and focal copy number variations (CNV) and a substantially greater level of diversity than can be adequately defined by the current genetic typing methods. As expected, CNV were particularly frequent in gene family-rich regions containing mucins and trans-sialidases but were also evident in core genes. Gene groups that showed little variation in copy numbers among the strains tested included those encoding protein kinases and ribosomal proteins, suggesting these loci were less permissive to CNV. Moreover, frequent variation in chromosome copy numbers were observed, and chromosome-specific CNV signatures were shared by genetically divergent T. cruzi strains.
The large number of CNV, over 4,000, reported here uphold at a whole genome level the long held paradigm of extraordinary genome plasticity among T. cruzi strains. Moreover, the fact that these heritable markers do not parse T. cruzi strains along the same lines as traditional typing methods is strongly suggestive of genetic exchange playing a major role in T. cruzi population structure and biology.
PMCID: PMC3060142  PMID: 21385342
11.  A Genome-Wide Analysis of Genetic Diversity in Trypanosoma cruzi Intergenic Regions 
Trypanosoma cruzi is the causal agent of Chagas Disease. Recently, the genomes of representative strains from two major evolutionary lineages were sequenced, allowing the construction of a detailed genetic diversity map for this important parasite. However this map is focused on coding regions of the genome, leaving a vast space of regulatory regions uncharacterized in terms of their evolutionary conservation and/or divergence.
Using data from the hybrid CL Brener and Sylvio X10 genomes (from the TcVI and TcI Discrete Typing Units, respectively), we identified intergenic regions that share a common evolutionary ancestry, and are present in both CL Brener haplotypes (TcII-like and TcIII-like) and in the TcI genome; as well as intergenic regions that were conserved in only two of the three genomes/haplotypes analyzed. The genetic diversity in these regions was characterized in terms of the accumulation of indels and nucleotide changes.
Principal Findings
Based on this analysis we have identified i) a core of highly conserved intergenic regions, which remained essentially unchanged in independently evolving lineages; ii) intergenic regions that show high diversity in spite of still retaining their corresponding upstream and downstream coding sequences; iii) a number of defined sequence motifs that are shared by a number of unrelated intergenic regions. A fraction of indels explains the diversification of some intergenic regions by the expansion/contraction of microsatellite-like repeats.
Author Summary
Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and poses a serious public health problem in the America, with approximately 8 million people infected and 200,000 new cases reported annually. The disease has different clinical manifestations. The fact that infections by the same species cause different clinical outcomes is believed to be determined, at least in part, by the genetic background of the parasite (infection by different strains). Previous characterizations of the genetic diversity in Trypanosoma cruzi were carried out on the protein-coding portions of the genome. However, the genetic diversity of non-coding intergenic regions remained unexplored. These regions are particularly important in trypanosomes because they contain essential regulatory sequences that drive the process of mRNA maturation and that ultimately govern the expression of genes. In this study, we analyzed the genetic diversity present in non-coding regions of the genome, and provide a broad picture of the selective forces acting on this subset of the genome. Based on this analysis we identified a highly conserved core of intergenic regions, that were maintained essentially unchanged over large evolutionary periods of time, as well as a highly divergent set of intergenic regions.
PMCID: PMC4006747  PMID: 24784238
12.  TcTASV: A Novel Protein Family in Trypanosoma cruzi Identified from a Subtractive Trypomastigote cDNA Library 
The identification and characterization of antigens expressed in Trypanosoma cruzi stages that parasitize mammals are essential steps for the development of new vaccines and diagnostics. Genes that are preferentially expressed in trypomastigotes may be involved in key processes that define the biology of trypomastigotes, like cell invasion and immune system evasion.
Methodology/Principal Findings
With the initial aim of identifying trypomastigote-specific expressed tags, we constructed and sequenced an epimastigote-subtracted trypomastigote cDNA library (library TcT-E). More than 45% of the sequenced clones of the library could not be mapped to previously annotated mRNAs or proteins. We validated the presence of these transcripts by reverse northern blot and northern blot experiments, therefore providing novel information about the mRNA expression of these genes in trypomastigotes. A 280-bp consensus element (TcT-E element, TcT-Eelem) located at the 3′ untranslated region (3′ UTR) of many different open reading frames (ORFs) was identified after clustering the TcT-E dataset. Using an RT-PCR approach, we were able to amplify different mature mRNAs containing the same TcT-Eelem in the 3′ UTR. The proteins encoded by these ORFs are members of a novel surface protein family in T. cruzi, (which we named TcTASV for T. cruzi Trypomastigote, Alanine, Serine and Valine rich proteins). All members of the TcTASV family have conserved coding amino- and carboxy-termini, and a central variable core that allows partitioning of TcTASV proteins into three subfamilies. Analysis of the T. cruzi genome database resulted in the identification of 38 genes/ORFs for the whole TcTASV family in the reference CL-Brener strain (lineage II). Because this protein family was not found in other trypanosomatids, we also looked for the presence of TcTASV genes in other evolutionary lineages of T. cruzi, sequencing 48 and 28 TcTASVs members from the RA (lineage II) and Dm28 (lineage I) T. cruzi strains respectively. Detailed phylogenetic analyses of TcTASV gene products show that this gene family is different from previously characterized mucin (TcMUCII), mucin-like, and MASP protein families.
We identified TcTASV, a new gene family of surface proteins in T. cruzi.
Author Summary
Chagas' disease, caused by the kinetoplastid protozoan parasite Trypanosoma cruzi, is endemic in Latin America. At present there are neither vaccines for prevention nor totally effective drugs for the treatment of the disease. T. cruzi has a complex life cycle alternating between a reduviid insect (the vector) and a mammalian host, where different parasite stages are found. Differentially expressed genes are the hallmark of the specialized biology of each life cycle stage. The aim of this work was to identify genes expressed in the trypomastigote stage (a blood-circulating stage that invades new cells and spreads the infection in different organs of the mammalian host) that could be used to develop new vaccines or diagnostics. An initial screening of trypomastigote transcripts was performed by sequencing of an epimastigote-subtracted trypomastigote cDNA library. Besides identifying a large proportion of differentially expressed mRNAs, we discovered a novel protein family, which we denominated TcTASV.
PMCID: PMC2950142  PMID: 20957201
13.  Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion 
PLoS Neglected Tropical Diseases  2015;9(11):e0004216.
The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.
Methodology/ Principal Findings
Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca2+ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.
We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.
Author Summary
Trypanosoma cruzi is the etiologic agent of Chagas’ disease, which infects 6–7 million people worldwide, mostly in Latin America. Currently, there are no vaccines available, and the drugs used for treatment are toxic and are not fully effective. To infect mammalian hosts, T. cruzi relies on the ability to invade host cells, replicate intracellularly and spread the infection in different organs of the mammalian host. Knowledge of the structure and function of T. cruzi surface molecules is fundamental to understanding the mechanisms by which the parasite interacts with its host. T. cruzi infective forms engage a repertoire of surface and secreted molecules, some of which are involved in triggering signaling pathways both in the parasite and the host cell, leading to intracellular Ca2+ mobilization, a process essential for parasite internalization. Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), including their genomic distribution, expression and cellular localization. We studied the mechanism of action of TcSMP in host-cell invasion and proposed a triggering role for TcSMP in host-cell lysosome exocytosis during metacyclic internalization. TcSMP genes are conserved among different T. cruzi lineages and share orthologs in other Trypanosoma species. These results suggest that the diversification of TcSMP genes in mammalian trypanosomes occurred after continental drift. In T. cruzi this gene family expanded by gene duplication.
PMCID: PMC4643927  PMID: 26565791
14.  The Trypanosoma cruzi Sylvio X10 strain maxicircle sequence: the third musketeer 
BMC Genomics  2011;12:58.
Chagas disease has a diverse pathology caused by the parasite Trypanosoma cruzi, and is indigenous to Central and South America. A pronounced feature of the trypanosomes is the kinetoplast, which is comprised of catenated maxicircles and minicircles that provide the transcripts involved in uridine insertion/deletion RNA editing. T. cruzi exchange genetic material through a hybridization event. Extant strains are grouped into six discrete typing units by nuclear markers, and three clades, A, B, and C, based on maxicircle gene analysis. Clades A and B are the more closely related. Representative clade B and C maxicircles are known in their entirety, and portions of A, B, and C clades from multiple strains show intra-strain heterogeneity with the potential for maxicircle taxonomic markers that may correlate with clinical presentation.
To perform a genome-wide analysis of the three maxicircle clades, the coding region of clade A representative strain Sylvio X10 (a.k.a. Silvio X10) was sequenced by PCR amplification of specific fragments followed by assembly and comparison with the known CL Brener and Esmeraldo maxicircle sequences. The clade A rRNA and protein coding region maintained synteny with clades B and C. Amino acid analysis of non-edited and 5'-edited genes for Sylvio X10 showed the anticipated gene sequences, with notable frameshifts in the non-edited regions of Cyb and ND4. Comparisons of genes that undergo extensive uridine insertion and deletion display a high number of insertion/deletion mutations that are likely permissible due to the post-transcriptional activity of RNA editing.
Phylogenetic analysis of the entire maxicircle coding region supports the closer evolutionary relationship of clade B to A, consistent with uniparental mitochondrial inheritance from a discrete typing unit TcI parental strain and studies on smaller fragments of the mitochondrial genome. Gene variance that can be corrected by RNA editing hints at an unusual depth for maxicircle taxonomic markers, which will aid in the ability to distinguish strains, their corresponding symptoms, and further our understanding of the T. cruzi population structure. The prevalence of apparently compromised coding regions outside of normally edited regions hints at undescribed but active mechanisms of genetic exchange.
PMCID: PMC3040149  PMID: 21261994
15.  Trypanosoma cruzi: Molecular characterization of an RNA binding protein differentially expressed in the parasite life cycle 
Experimental parasitology  2007;117(1):99-105.
Molecular studies have shown several peculiarities in the regulatory mechanisms of gene expression in trypanosomatids. Protein coding genes are organized in long polycistronic units that seem to be constitutively transcribed. Therefore, post-transcriptional regulation of gene expression is considered to be the main point for control of transcript abundance and functionality. Here we describe the characterization of a 17 kDa RNA-binding protein from Trypanosoma cruzi (TcRBP19) containing an RNA recognition motive (RRM). This protein is coded by a single copy gene located in a high molecular weight chromosome of T. cruzi. Orthologous genes are present in the TriTryp genomes. TcRBP19 shows target selectivity since among the different homoribopolymers it preferentially binds polyC. TcRBP19 is a low expression protein only barely detected at the amastigote stage localizing in a diffuse pattern in the cytoplasm.
PMCID: PMC2020836  PMID: 17475252
Kinetoplastida; Trypanosoma cruzi; RNA binding proteins; RRM protein; TcRBP19
16.  A genomic scale map of genetic diversity in Trypanosoma cruzi 
BMC Genomics  2012;13:736.
Trypanosoma cruzi, the causal agent of Chagas Disease, affects more than 16 million people in Latin America. The clinical outcome of the disease results from a complex interplay between environmental factors and the genetic background of both the human host and the parasite. However, knowledge of the genetic diversity of the parasite, is currently limited to a number of highly studied loci. The availability of a number of genomes from different evolutionary lineages of T. cruzi provides an unprecedented opportunity to look at the genetic diversity of the parasite at a genomic scale.
Using a bioinformatic strategy, we have clustered T. cruzi sequence data available in the public domain and obtained multiple sequence alignments in which one or two alleles from the reference CL-Brener were included. These data covers 4 major evolutionary lineages (DTUs): TcI, TcII, TcIII, and the hybrid TcVI. Using these set of alignments we have identified 288,957 high quality single nucleotide polymorphisms and 1,480 indels. In a reduced re-sequencing study we were able to validate ~ 97% of high-quality SNPs identified in 47 loci. Analysis of how these changes affect encoded protein products showed a 0.77 ratio of synonymous to non-synonymous changes in the T. cruzi genome. We observed 113 changes that introduce or remove a stop codon, some causing significant functional changes, and a number of tri-allelic and tetra-allelic SNPs that could be exploited in strain typing assays. Based on an analysis of the observed nucleotide diversity we show that the T. cruzi genome contains a core set of genes that are under apparent purifying selection. Interestingly, orthologs of known druggable targets show statistically significant lower nucleotide diversity values.
This study provides the first look at the genetic diversity of T. cruzi at a genomic scale. The analysis covers an estimated ~ 60% of the genetic diversity present in the population, providing an essential resource for future studies on the development of new drugs and diagnostics, for Chagas Disease. These data is available through the TcSNP database (
PMCID: PMC3545726  PMID: 23270511
17.  Repetitive DNA is associated with centromeric domains in Trypanosoma brucei but not Trypanosoma cruzi 
Genome Biology  2007;8(3):R37.
Centromeres in Trypanosoma cruzi and Trypanosoma brucei can be localised to regions between directional gene clusters that contain degenerate retroelements, and in the case of T. brucei, repetitive DNA.
Trypanosomes are parasitic protozoa that diverged early from the main eukaryotic lineage. Their genomes display several unusual characteristics and, despite completion of the trypanosome genome projects, the location of centromeric DNA has not been identified.
We report evidence on the location and nature of centromeric DNA in Trypanosoma cruzi and Trypanosoma brucei. In T. cruzi, we used telomere-associated chromosome fragmentation and found that GC-rich transcriptional 'strand-switch' domains composed predominantly of degenerate retrotranposons are a shared feature of regions that confer mitotic stability. Consistent with this, etoposide-mediated topoisomerase-II cleavage, a biochemical marker for active centromeres, is concentrated at these domains. In the 'megabase-sized' chromosomes of T. brucei, topoisomerase-II activity is also focused at single loci that encompass regions between directional gene clusters that contain transposable elements. Unlike T. cruzi, however, these loci also contain arrays of AT-rich repeats stretching over several kilobases. The sites of topoisomerase-II activity on T. brucei chromosome 1 and T. cruzi chromosome 3 are syntenic, suggesting that centromere location has been conserved for more than 200 million years. The T. brucei intermediate and minichromosomes, which lack housekeeping genes, do not exhibit site-specific accumulation of topoisomerase-II, suggesting that segregation of these atypical chromosomes might involve a centromere-independent mechanism.
The localization of centromeric DNA in trypanosomes fills a major gap in our understanding of genome organization in these important human pathogens. These data are a significant step towards identifying and functionally characterizing other determinants of centromere function and provide a framework for dissecting the mechanisms of chromosome segregation.
PMCID: PMC1868937  PMID: 17352808
18.  Genomic variation of Trypanosoma cruzi: involvement of multicopy genes. 
Infection and Immunity  1990;58(10):3217-3224.
By using improved pulsed field gel conditions, the karyotypes of several strains of the protozoan parasite Trypanosoma cruzi were analyzed and compared with those of Leishmania major and two other members of the genus Trypanosoma. There was no difference in chromosome migration patterns between different life cycle stages of the T. cruzi strains analyzed. However, the sizes and numbers of chromosomal bands varied considerably among T. cruzi strains. This karyotype variation among T. cruzi strains was analyzed further at the chromosomal level by using multicopy genes as probes in Southern hybridizations. The chromosomal location of the genes encoding alpha- and beta-tubulin, ubiquitin, rRNA, spliced leader RNA, and an 85-kilodalton protein remained stable during developmental conversion of the parasite. The sizes and numbers of chromosomes containing these sequences varied among the different strains analyzed, implying multiple rearrangements of these genes during evolution of the parasites. During continuous in vitro cultivation of T. cruzi Y, the chromosomal location of the spliced leader gene shifted spontaneously. The spliced leader gene encodes a 35-nucleotide RNA that is spliced in trans from a 105-nucleotide donor RNA onto all mRNAs in T. cruzi. The spliced leader sequences changed in their physical location in both the cloned and uncloned Y strains. Associated with the complex changes was an increase in the infectivity of the rearranged variant for tissue culture cells. Our results indicate that the spliced leader gene clusters in T. cruzi undergo high-frequency genomic rearrangements.
PMCID: PMC313642  PMID: 2169461
19.  Identification of Strain-Specific B-cell Epitopes in Trypanosoma cruzi Using Genome-Scale Epitope Prediction and High-Throughput Immunoscreening with Peptide Arrays 
The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.
By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.
Findings and Conclusions
A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.
Author Summary
Serological tests are preferentially used for the diagnosis of Chagas disease during the chronic phase because of the low parasitemia and high anti-T. cruzi antibody titers. However, contradictory or inconclusive results, mainly related to the characteristics of the antigens used, are often observed. Additionally, the factors influencing variation in the clinical forms of Chagas disease have not been elucidated, although it is likely that host and parasite genetics are involved. Several studies attempting to correlate the parasite strain with the clinical forms have used hemoculture and/or PCR-based genotyping. However, both techniques have limitations. Hemoculture requires the isolation of parasites from patient blood and the growth of these parasites in animals or in vitro culture, thereby possibly selecting certain subpopulations. Moreover, the level of parasitemia in the chronic phase is very low, hindering the detection of parasites. Additionally, direct genotyping of parasites from infected tissues is an invasive procedure that requires medical care and hinders studies with a large number of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi on a genome-wide scale for use in the serodiagnosis and serotyping of Chagas disease using ELISA. Development of a serotyping method based on the detection of strain-specific antibodies may help to understand the relationship between the infecting strain and disease evolution.
PMCID: PMC3814679  PMID: 24205430
20.  Phylogenetic and syntenic data support a single horizontal transference to a Trypanosoma ancestor of a prokaryotic proline racemase implicated in parasite evasion from host defences 
Parasites & Vectors  2015;8:222.
Proline racemase (PRAC) enzymes of Trypanosoma cruzi (TcPRAC), the agent of Chagas disease, and Trypanosoma vivax (TvPRAC), the agent of livestock trypanosomosis, have been implicated in the B-cells polyclonal activation contributing to immunosuppression and the evasion of host defences. The similarity to prokaryotic PRAC and the absence in Trypanosoma brucei and Trypanosoma congolense have raised many questions about the origin, evolution, and functions of trypanosome PRAC (TryPRAC) enzymes.
We identified TryPRAC homologs as single copy genes per haploid genome in 12 of 15 Trypanosoma species, including T. cruzi and T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli, T. conorhini and T. lewisi, all parasites of mammals. Polymorphisms in TcPRAC genes matched T. cruzi genotypes: TcI-TcIV and Tcbat have unique genes, while the hybrids TcV and TcVI contain TcPRACA and TcPRACB from parental TcII and TcIII, respectively. PRAC homologs were identified in trypanosomes from anurans, snakes, crocodiles, lizards, and birds. Most trypanosomes have intact PRAC genes. T. rangeli possesses only pseudogenes, maybe in the process of being lost. T. brucei, T. congolense and their allied species, except the more distantly related T. vivax, have completely lost PRAC genes.
The genealogy of TryPRAC homologs supports an evolutionary history congruent with the Trypanosoma phylogeny. This finding, together with the synteny of PRAC loci, the relationships with prokaryotic PRAC inferred by taxon-rich phylogenetic analysis, and the absence in trypanosomatids of any other genera or in bodonids or euglenids suggest that a common ancestor of Trypanosoma gained PRAC gene by a single and ancient horizontal gene transfer (HGT) from a Firmicutes bacterium more closely related to Gemella and other species of Bacilli than to Clostridium as previously suggested. Our broad phylogenetic study allowed investigation of TryPRAC evolution over long and short timescales. TryPRAC genes diverged to become species-specific and genotype-specific for T. cruzi and T. rangeli, with resulting genealogies congruent with those obtained using vertically inherited genes. The inventory of TryPRAC genes described here is the first step toward the understanding of the roles of PRAC enzymes in trypanosomes differing in life cycles, virulence, and infection and immune evasion strategies.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0829-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4417235  PMID: 25890302
Proline racemase; Trypanosoma cruzi; Trypanosoma vivax; Trypanosoma rangeli; Horizontal gene transfer; Gene loss; Kinetoplastid evolution; Phylogeny; Synteny; Genotyping
21.  Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family 
PLoS ONE  2013;8(4):e60209.
Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.
PMCID: PMC3616161  PMID: 23560078
22.  Trypanosoma cruzi IIc: Phylogenetic and Phylogeographic Insights from Sequence and Microsatellite Analysis and Potential Impact on Emergent Chagas Disease 
Trypanosoma cruzi, the etiological agent of Chagas disease, is highly genetically diverse. Numerous lines of evidence point to the existence of six stable genetic lineages or DTUs: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe. Molecular dating suggests that T. cruzi is likely to have been an endemic infection of neotropical mammalian fauna for many millions of years. Here we have applied a panel of 49 polymorphic microsatellite markers developed from the online T. cruzi genome to document genetic diversity among 53 isolates belonging to TcIIc, a lineage so far recorded almost exclusively in silvatic transmission cycles but increasingly a potential source of human infection. These data are complemented by parallel analysis of sequence variation in a fragment of the glucose-6-phosphate isomerase gene. New isolates confirm that TcIIc is associated with terrestrial transmission cycles and armadillo reservoir hosts, and demonstrate that TcIIc is far more widespread than previously thought, with a distribution at least from Western Venezuela to the Argentine Chaco. We show that TcIIc is truly a discrete T. cruzi lineage, that it could have an ancient origin and that diversity occurs within the terrestrial niche independently of the host species. We also show that spatial structure among TcIIc isolates from its principal host, the armadillo Dasypus novemcinctus, is greater than that among TcI from Didelphis spp. opossums and link this observation to differences in ecology of their respective niches. Homozygosity in TcIIc populations and some linkage indices indicate the possibility of recombination but cannot yet be effectively discriminated from a high genome-wide frequency of gene conversion. Finally, we suggest that the derived TcIIc population genetic data have a vital role in determining the origin of the epidemiologically important hybrid lineages TcIId and TcIIe.
Author Summary
Trypanosoma cruzi, the etiological agent of Chagas disease, infects over 10 million people in Latin America. Six major genetic lineages of the parasite have been identified with differential geographic distributions, ecological associations and epidemiological importance. With the advent of the T. cruzi genome sequence, it is possible to examine the micro-epidemiology of T. cruzi using high resolution genetic markers that assess diversity within these major types. Here we examine the genetic diversity of TcIIc, a poorly understood T. cruzi genetic lineage found predominantly among wild cycles of parasite transmission infecting terrestrial mammals and triatomine vectors, but also a potentially important emergent human disease agent. Amongst a number of findings, we show that TcIIc genetic diversity is comparable to other ancient T. cruzi lineages, highly spatially structured, and that a stringent co-evolutionary relationship with its principal reservoir host can be ruled out. Additionally, TcIIc is one of the two parents of hybrid lineages TcIId and TcIIe, which cause most of the Chagas disease that occurs in the Southern Cone of South America. The system we have developed will help to clarify the ecological circumstances around the emergence of these epidemiologically important hybrids, and perhaps help predict similar events in the future.
PMCID: PMC2727949  PMID: 19721699
23.  Database of Trypanosoma cruzi repeated genes: 20 000 additional gene variants 
BMC Genomics  2007;8:391.
Repeats are present in all genomes, and often have important functions. However, in large genome sequencing projects, many repetitive regions remain uncharacterized. The genome of the protozoan parasite Trypanosoma cruzi consists of more than 50% repeats. These repeats include surface molecule genes, and several other gene families. In the T. cruzi genome sequencing project, it was clear that not all copies of repetitive genes were present in the assembly, due to collapse of nearly identical repeats. However, at the time of publication of the T. cruzi genome, it was not clear to what extent this had occurred.
We have developed a pipeline to estimate the genomic repeat content, where shotgun reads are aligned to the genomic sequence and the gene copy number is estimated using the average shotgun coverage. This method was applied to the genome of T. cruzi and copy numbers of all protein coding sequences and pseudogenes were estimated. The 22 640 results were stored in a database available online. 18% of all protein coding sequences and pseudogenes were estimated to exist in 14 or more copies in the T. cruzi CL Brener genome. The average coverage of the annotated protein coding sequences and pseudogenes indicate a total gene copy number, including allelic gene variants, of over 40 000.
Our results indicate that the number of protein coding sequences and pseudogenes in the T. cruzi genome may be twice the previous estimate. We have constructed a database of the T. cruzi gene repeat data that is available as a resource to the community. The main purpose of the database is to enable biologists interested in repeated, unfinished regions to closely examine and resolve these regions themselves using all available shotgun data, instead of having to rely on annotated consensus sequences that often are erroneous and possibly misleading. Five repetitive genes were studied in more detail, in order to illustrate how the database can be used to analyze and extract information about gene repeats with different characteristics in Trypanosoma cruzi.
PMCID: PMC2204015  PMID: 17963481
24.  A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product 
Trypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.
Methodology/Principal Findings
We present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI–TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.
Based on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.
Author Summary
Trypanosoma cruzi, the causative agent of Chagas Disease, infects approximately 8 million people in the Americas, with 200,000 new cases reported anually. The disease, in its chronic stage, has different manifestations: mega-colon, mega-esophagus, and cardiomyopathy, among others. The fact that infections by the same species cause these different clinical outcomes is believed to be determined, at least in part, by the genetic background of the parasite (infection by different strains). By analyzing a number of molecular markers, the population of the parasite has been divided into seven major evolutionary lineages, which evolve mostly independently, by clonal expansion with infrequent exchange of genetic material. Accurate classification of different strains and isolates into their corresponding evolutionary lineages is therefore essential to obtain a good map of biological, biochemical and ecoepidemiological features for the whole species. The current methods available to type T. cruzi stocks are either laborious and costly (requiring the amplification and sequencing of a variable number of genes or gene fragments), or limited in resolution. In this work we describe a number of key discriminant sites in a gene encoding a putative enzyme from the sterol pathway of the parasite, which were exploited to design a couple of alternative typing assays. Using these key discriminant sites, we can classify any T. cruzi stock into either six or seven evolutionary lineages using only one gene fragment, and in a matter of hours (depending on the assay used). To our knowledge, the proposed assays are the first typing assays that can discriminate T. cruzi stocks with such speed and low cost.
PMCID: PMC3409129  PMID: 22860154
25.  Trypanosoma cruzi in the Chicken Model: Chagas-Like Heart Disease in the Absence of Parasitism 
The administration of anti-trypanosome nitroderivatives curtails Trypanosoma cruzi infection in Chagas disease patients, but does not prevent destructive lesions in the heart. This observation suggests that an effective treatment for the disease requires understanding its pathogenesis.
Methodology/Principal Findings
To understand the origin of clinical manifestations of the heart disease we used a chicken model system in which infection can be initiated in the egg, but parasite persistence is precluded. T. cruzi inoculation into the air chamber of embryonated chicken eggs generated chicks that retained only the parasite mitochondrial kinetoplast DNA minicircle in their genome after eight days of gestation. Crossbreeding showed that minicircles were transferred vertically via the germ line to chicken progeny. Minicircle integration in coding regions was shown by targeted-primer thermal asymmetric interlaced PCR, and detected by direct genomic analysis. The kDNA-mutated chickens died with arrhythmias, shortness of breath, cyanosis and heart failure. These chickens with cardiomyopathy had rupture of the dystrophin and other genes that regulate cell growth and differentiation. Tissue pathology revealed inflammatory dilated cardiomegaly whereby immune system mononuclear cells lyse parasite-free target heart fibers. The heart cell destruction implicated a thymus-dependent, autoimmune; self-tissue rejection carried out by CD45+, CD8γδ+, and CD8α lymphocytes.
These results suggest that genetic alterations resulting from kDNA integration in the host genome lead to autoimmune-mediated destruction of heart tissue in the absence of T. cruzi parasites.
Author Summary
The Trypanosoma cruzi acute infections can be asymptomatic but approximately one third of the chronically infected cases may present Chagas disease. Parasite persistence and autoimmunity are theories trying to explain the clinical and pathological manifestations of Chagas disease in the heart and the digestive system. To clearly demonstrate roles played by parasite persistence and autoimmunity in Chagas disease we used a chicken model refractory to the T. cruzi. In this study we inoculated the invasive T. cruzi in the air chamber of embryonated eggs. The infection was eradicated by the innate immunity and the chicks were parasite-free at hatching, but they retained the parasitic mitochondrial kinetoplast DNA minicircle in their genome. We documented the kDNA minicircle integrated in the chicken genome by a targeted prime TAIL-PCR, Southern hybridizations, cloning and sequencing. The kDNA minicircles integrated in coding regions of various chromosomes, and mutated chickens developed an inflammatory cardiomyopathy hallmark of Chagas disease, whereby immune system mononuclear cells lyse parasite-free target heart fibers. Genotype alterations resulting from transfers of the parasitic DNA were associated with the tissue destruction carried out by effectors CD45+, CD8γδ+, CD8α lymphocytes. This research provides insights about a protozoan infection that can induce genetically driven autoimmune disease.
PMCID: PMC3066158  PMID: 21468314

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