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 IC50, CC50, 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. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).1.

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

This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, 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 T. cruzi. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).

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. In this work, we have investigated the function of T. cruzi histone deacetylase 4 (TcHDAC4). We show that, although TcHDAC4 is not essential for viability, metacyclic trypomastigote TcHDAC4 null mutants show a thin cell body and a round and less condensed nucleus located very close to the kinetoplast. Sixty-four acetylation sites were quantitatively evaluated, which revealed H2AT85ac, H4K10ac and H4K78ac as potential target sites of TcHDAC4. Gene expression analyses identified three chromosomes with overrepresented regions of differentially expressed genes in the TcHDAC4 knockout mutant compared with the wild type, showing clusters of either up or downregulated genes. The adjacent chromosomal location of some of these genes indicates that TcHDAC4 participates in gene expression regulation during T. cruzi differentiation.