Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi’s genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a ‘core compartment’ and a ‘disruptive compartment’ which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes.
Trypanosoma cruzi, the causative agent of the Chagas disease, has a complex life cycle alternating between replicative and noninfective forms with nonreplicative and infective forms of the parasite. Metacyclogenesis is a process that takes place in the invertebrate host, comprising morphogenetic transformation from a noninfective form to an infective form, such that parasites acquire the ability to invade human cells. We analyze here the metacyclogenesis process by 2D electrophoresis coupled to MALDI-TOF MS. A large proportion of unique proteins expressed during metacyclogenesis were observed. Interestingly, 50% of the spots were found to differ between epimastigotes and trypomastigotes. We provide a 2D map of the infective metacyclic trypomastigotes. Sixty six protein spots were successfully identified corresponding to 43 different proteins. We analyzed the expression profiles for the identified proteins along metacyclogenesis and classified them into three groups according to their maximal level of expression. We detected several isoforms for a number of proteins, some displaying differential expression during metacyclogenesis. These results suggest that posttranslational modifications may be a fundamental part of the parasite's strategy for regulating gene expression during differentiation. This study contributes to the identification of relevant proteins involved in the metacyclogenesis process. The identification and molecular characterization of these proteins will render vital information about the steps of the parasite differentiation into the infective form.
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