Identification of novel interspersed DNA repetitive elements in the Trypanosoma cruzi genome associated with the 3′UTRs of surface multigenic families

Trypanosoma cruzi is the etiological agent of Chagas disease, which affects millions of people in Latin America. No transcriptional control of gene expression has been demonstrated in this organism, and 50% of its genome consists of repetitive elements and members of multigenic families. In this study, we applied a novel bioinformatics approach to predict new repetitive elements in the genome sequence of T. cruzi. A new repetitive sequence measuring 241 nt was identified and found to be interspersed along the genome sequence from strains of different DTUs. This new repeat was mostly on intergenic regions, and upstream and downstream regions of the 241 nt repeat were enriched in surface protein genes. RNAseq analysis revealed that the repeat was part of processed mRNAs and was predominantly found in the 3′ untranslated regions (UTRs) of genes of multigenic families encoding surface proteins. Moreover, we detected a correlation between the presence of the repeat in the 3′UTR of multigenic family genes and the level of differential expression of these genes when comparing epimastigote and trypomastigote transcriptomes. These data suggest that this sequence plays a role in the posttranscriptional regulation of the expression of multigenic families.

Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion.

BACKGROUND: 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. CONCLUSION/SIGNIFICANCE: 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.

Molecular Characterization of Trypanosoma cruzi Tc8.2 Gene Indicates Two Differential Locations for the Encoded Protein in Epimastigote and Trypomastigote Forms.

This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.

The TryPIKinome of five human pathogenic trypanosomatids: Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Leishmania braziliensis and Leishmania infantum--new tools for designing specific inhibitors.

Phosphatidylinositol (PI) kinases are at the heart of one of the major pathways of intracellular signal transduction. Herein, we present the first report on a survey made by similarity searches against the five human pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Leishmania braziliensis and Leishmania infantum genomes available to date for phosphatidylinositol- and related-kinases (TryPIKs). In addition to generating a panel called "The TryPIKinome", we propose a model of signaling pathways for these TryPIKs. The involvement of TryPIKs in fundamental pathways, such as intracellular signal transduction and host invasion processes, makes the study of TryPIKs an important area for further inquiry. New subtype-specific inhibitors are expected to work on individual members of the PIK family and, therefore, can presumably neutralize trypanosomatid invasion processes.

Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags.

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.

Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32 percent are not related to any other sequences in database and 68 percent presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.

A novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi

We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions

The Trypanosoma cruzi genome project: nuclear karyotype and gene mapping of clone CL Brener

By using improved pulsed field gel electrophoresis conditions, the molecular karyotype of the reference chromosomal bands ranging in size from 0.45 to 3.5 Magabase pairs (Mbp) were resolved in a single run. The weighted sum of the chromosomal bands was approximately 87 Mbp. Chromoblots were hybridized with 39 different homologous probes, 13 of which identified single chromosomes. Several markers linkage and four different linkage groups were identified, each comprising two markers. Densitometric analysis suggests that most of the chromosomal bands contain two or more chromosomes representing either homologous chromosomes and/or heterologous chromosomes with similar sizes.

Parasite genome projects and the Trypanosoma cruzi genome initiative

Since the start of the human genome project, a great number of genome projects on other "model" organism have been initiated, some of them already completed. Several initiatives have been started on parasite genomes, mainly through support from WHO/TDR, involving North-South and South-South collaborations, and great hopes are vested in that these initiatives will lead to new tools for disease control and prevention, as well as to the establishment of genomic research technology in developing countries. The Trypanosoma cruzi genome project, using the clone CL-Brener as starting point, has made considerable progress through the concerted action of more than 20 laboratories, most of them in the South. A brief overview of the current state of the project is given.

Characterization of a Trypanosoma cruzi genomic fragment complementary to several species-specific mRNAs but different from the spliced leader sequence

Foi isolado um clone recombinante (lambda 3b2-5) de ADN genômico de Trypanosoma cruzi, o qual contém seqüências que estäo reiteradas no genoma. Análise através da técnica de "Northern blot" mostrou que o clone 3b2-5 hibridiza com diferentes espécies de ARNs mensageiros de 1.200 a 5.000 bases. O número de espécies de ARNs mensageiros que hibridiza com o clone 3b2-5 excede sua capacidade codante, sugerindo que este clone apresenta seqüências que säo comuns a muitas espécies de ARNs mensageiros e conservadas no ARN poliadenilado. Estas seqüências näo säo homológas a seqüência líder presente nos ARNs de T. cruzi, pois, o clone 3b2-5 näo hibridiza com um nucleotídeo sintético complementar a seqüência líder. O clone 3b2-5 näo hibridiza com o ADN e ARN de diferentes gêneros de tripanosomatídeos e outras espécies de tripanosomas indicando que ele apresenta seqüências específicas para o T. cruzi