Discussion

Trypanosoma cruzi, the causative agent of Chagas disease, has three distinct developmental stages that are programed to rapidly respond to environmental changes within the vertebrate and invertebrate hosts. Its population is comprised of a highly heterogeneous pool of strains that exhibit a wide-range of biological characteristics such as distinct morphology, growth rate, curves of parasitemia, virulence and sensitivity to drugs. Unlike other eukaryotes, T. cruzi protein-coding genes are transcribed into polycistronic pre-mRNAs that are subsequently processed into mature, monocistronic mRNAs through coupled “trans-splicing” and poly-adenylation reactions. Because of this, control of gene expression relies on post-transcriptional mechanisms mainly affecting steady-state levels and translation rates of mature mRNAs. We performed whole transcriptome analysis comparing in vitro cultured epimastigotes, trypomastigotes and intracellular amastigotes from two cloned strains, named CL Brener and CL-14, which showed highly distinct virulence phenotypes. Our RNA-seq data revealed common changes in gene expression in both parasites strains, reflecting their capacity to adapt their energy metabolism, oxidative stress responses, cell cycle control and interactions with cellular components within the distinct host environments. However, differences observed in the transcriptomes from both strains also indicated that the avirulent phenotype of  the CL-14 strain may be due to reduced expression of genes encoding surface proteins that are associated with intracellular amatigote-trypomastigote differentiation. Since RNA-seq analyses also revealed significant changes in the expression of genes encoding RNA binding proteins (RBPs), we decided to characterize two novel RBPs that may act as regulatory factors involved in post-transcriptional control of gene expression during the life cycle of the two T. cruzi strains. Transcript levels of one RBP, named TcRBP99, are up-regulated 25-fold in epimastigotes of the CL Brener strain whereas the second RBP, named TcRBP300, presents constitutive expression in the virulent CL Brener but is highly up-regulated in trypomastigotes of the avirulent CL-14 clone. To investigate the role of these RBPs, we generated CL Brener epimastigote cell lines  in which the TcRBP99 gene was disrupted as well as CL-14 knockout (KO) mutants for TcRBP300 gene. Analyses of mutant parasites revealed a role for TcRBP99 as a main factor controlling the expression of genes involved with epimastigote proliferation and differentiation since TcRBP99 KO cells grew slowly and presented increased capacity to differentiate into metacyclic trypomastigotes compared to wild type CL Brener epimastigotes. RNA-seq analyses of TcRBP99 KO mutants showed reduced levels of epimastigote-specific transcripts compared to wild type parasites and co-immunoprecipitation assays confirmed the mRNA binding capacity of TcRBP99 to a transcript encoding a protein involved with differentiation. A role for the TcRBP300 related to parasite virulence is currently been investigated in infection assays with CL-14 KO mutants as well as with transfected CL Brener cell lines over-expressing this gene.