Abstract
Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01), coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of their gene expression control.
Highlights
T. cruzi is the causative agent of Chagas disease, known as American trypanosomiasis, a parasitosis that affects more than 8–10 million people in the endemic areas of 21 Latin American countries [1]
Isolation of cell cycle stages using HU synchronization and RNA preparation In order to obtain parasite cultures synchronized at different cell cycle stages, we carried out a HU exposure protocol described previously [32], using T. cruzi epimastigote cultures of an unnamed TcI lineage strain
When we performed the analysis done by Kelly et al [66] using the T. cruzi information instead, we found a similar proportionality between cell cycle gene expression and the location of the genes in the polycistron, where mRNA abundance increases with the distance to the polycistron start for G2/M, and decreases for S-phase genes (S3 Fig)
Summary
T. cruzi is the causative agent of Chagas disease, known as American trypanosomiasis, a parasitosis that affects more than 8–10 million people in the endemic areas of 21 Latin American countries [1]. Further differences in the canonical cell division, including a closed mitosis, the absence of centrioles, and the existence of the kinetoplast, account for substantial divergence in the mechanisms of proliferation of trypanosomatids in comparison to their mammalian hosts. This has led to the proposal of the protozoan parasite cell cycle as a relevant target for drug development against the diseases [7,8,9,10]. The discovery of the proteins that are responsible for the control and progression of the replicative cycle in T. cruzi, as well as those necessary for the acquisition of infectivity, has been foreseen as an essential step for the development of rational drug design [11, 12]
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