In cellulo and in vivo assays for compound testing against Trypanosoma cruzi.

Here, we describe a combined in cellulo and in vivo approach to identify compounds with higher potential for efficient inhibition of Trypanosoma cruzi. Phase I of in cellulo assays is designed to exclude inactive or toxic compounds, while phase II is designed for accurate IC, CC, and selective index (SI) determination. Compounds showing high SI are tested using in vivo infection models in parallel with benznidazole to assess their efficacy relative to a reference drug used for Chagas disease treatment.

Dissecting Trypanosoma brucei RRP44 function in the maturation of segmented ribosomal RNA using a regulated genetic complementation system.

Trypanosoma brucei belongs to a group of protozoans presenting fragmented large subunit rRNA. Its LSU rRNA equivalent to the 25S/28S rRNA of other eukaryotes is split into six fragments, requiring additional processing for removal of the extra spacer sequences. We have used a genetic complementation strategy to further investigate the T.

Trypanosoma brucei RRP44: a versatile enzyme for processing structured and non-structured RNA substrates.

Rrp44/Dis3 is a conserved eukaryotic ribonuclease that acts on processing and degradation of nearly all types of RNA. It contains an endo- (PIN) and an exonucleolytic (RNB) domain and, its depletion in model organisms supports its essential function for cell viability. In Trypanosoma brucei, depletion of Rrp44 (TbRRP44) blocks maturation of ribosomal RNA, leading to disruption of ribosome synthesis and inhibition of cell proliferation.

Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in .

This protocol outlines a new genetic complementation strategy to investigate gene function in , the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in .

Species-selective targeting of pathogens revealed by the atypical structure and active site of Trypanosoma cruzi histone deacetylase DAC2.

Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks.

Metacyclogenesis defects and gene expression hallmarks of histone deacetylase 4-deficient Trypanosoma cruzi cells.

Trypanosoma cruzi-the causative agent of Chagas disease-like other kinetoplastids, relies mostly on post-transcriptional mechanisms for regulation of gene expression. However, trypanosomatids undergo drastic changes in nuclear architecture and chromatin structure along their complex life cycle which, combined with a remarkable set of reversible histone post-translational modifications, indicate that chromatin is also a target for control of gene expression and differentiation signals in these organisms. Chromatin-modifying enzymes have a direct impact on gene expression programs and DNA metabolism.

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi.

Background: Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms.

Results: Here we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect's life stages using RNA-seq and ribosome profiling.

mRNA localization mechanisms in Trypanosoma cruzi.

Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution.

Molecular characterization of the Trypanosoma cruzi specific RNA binding protein TcRBP40 and its associated mRNAs.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected disorder that affects millions of people in the Americas. T. cruzi relies mostly upon post-transcriptional regulation to control stage specific gene expression.