Development of molecular tools for the characterization and functional analysis of Trypanothione Sinthetase (TryS) in Trypanosoma cruzi

MESIAS AC; PIÑEYRO MD; ROBELLO, C; ZAGO MP

Mar del Plata

Congreso; X Congreso de protozoología y enfermedades parasitarias; 2014

Sociedad Argentina de Protozoología

The antioxidant system of Trypanosoma cruzi is a sophisticated network of metabolic pathways, completely different to its human counterpart. Relying on thiols such as trypanothione, the system maintains redox homeostasis, deals with detoxification and respiratory burst, among other likely functions. It has also been proposed to have a role in drug resistance phenomena, ribonucleotides synthesis regulation and possibly in parasite persistence, avoiding the triggering of apoptosis in the infected tissue as part of the homeostatic clearance in the host. Trypanothione syntethase (TryS), encoded by a single copy gene, is a bifunctional enzyme in charge of trypanothione synthesis from glutathione and spermidine. Its essentiality has been confirmed in other organisms from the trypanosomatids. Its low abundance and substrate versatility makes it an ideal target for drug design. A direct correlation between overexpression of antioxidant enzymes, including TryS, and parasite virulence has formerly been shown in T. cruzi; moreover TryS is upregulated during metacyclogenesis. The aim of this work is the development of molecular tools to study TryS deeply. We propose the generation of parasites with different expression levels of this enzyme using two polar isolates, an attenuated strain(TCC) and a virulent one(Sylvio), both belonging to DTUI. In the TCC strain, we aim to overexpress TryS using the pTREX system. In the Sylvio strain, we attempt to knock down/out one or both alleles through the Gateway® technology. We have also designed two functional mutants in order to affect syntethase and amidase domains by a site directed mutagenesis strategy. Here we present the design and development of these tools, which will allow us to address the relationship among TryS expression and virulence or pathogenicity and parasite persistence as well. These parasites will be useful also for the study of other roles attributed to TryS(drug resistance, ribonucleotide synthesis, among others).