Disruption of the inositol phosphorylceramide synthase gene affects Trypanosoma cruzi differentiation and infection capacity.

Sphingolipids (SLs) are essential components of all eukaryotic cellular membranes. In fungi, plants and many protozoa, the primary SL is inositol-phosphorylceramide (IPC). Trypanosoma cruzi is a protozoan parasite that causes Chagas disease (CD), a chronic illness for which no vaccines or effective treatments are available.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx).

Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants.

CRISPR/Cas9 technology has been used to edit genomes in a variety of organisms. Using the GP72 gene as a target sequence, we tested two distinct approaches to generate Trypanosoma cruzi knockout mutants using the Cas9 nuclease and in vitro transcribed single guide RNA. Highly efficient rates of disruption of GP72 were achieved either by transfecting parasites stably expressing Streptococcus pyogenes Cas9 with single guide RNA or by transfecting wild type parasites with recombinant Staphylococcus aureus Cas9 previously associated with single guide RNA.

Editing the Trypanosoma cruzi genome with zinc finger nucleases.

Gene function studies in Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, have been hindered by the lack of efficient genetic manipulation protocols. In most organisms, insertion and deletion of DNA fragments in the genome are dependent on the generation of double-stranded DNA break (DSB) and repair. By inducing a site-specific DSB, zinc finger nucleases (ZFNs) have proven to be useful to enhance gene editing in many cell types.

Development of a dual reporter system to identify regulatory cis-acting elements in untranslated regions of Trypanosoma cruzi mRNAs.

In trypanosomatids, transcription is polycistronic and gene expression control occurs mainly at the post-transcriptional level. To investigate the role of sequences present in the 3'UTR of stage-specific mRNAs of Trypanosoma cruzi, we generated a new vector, named pTcDUALuc, containing the firefly and Renilla luciferase reporter genes. To test this vector, sequences derived from the 3'UTR plus intergenic regions of the alpha tubulin gene, which is up-regulated in epimastigotes, and amastin, which is up-regulated in amastigotes, were inserted downstream from the firefly reporter gene and luciferase activity was compared in transient and stable transfected parasites.