Molecular characterization of a specific L-proline permease from Trypanosoma cruzi

SILBER AM; SAYE M; MIRANDA MR; CAMARA MM; PEREIRA CA

Caxambu, Minas Gerais

Congreso; XXVIII Annual Meeting of the Brazilian Society of Protozoology; 2012

Sociedade Brasileira de Protozoologia (SBPZ)

In trypanosomatids, proline is a key amino acid since constitutes a main carbon and energy source. Recently, new roles in the parasite biology have been assigned to this metabolite. In Trypanosoma cruzi, proline also provides the energy to support the host-cell invasion process in metacyclic trypomastigotes and the infection progression, particularly participating in the differentiation from intracellular epimastigotes to trypomastigotes. It is also noticeable that the accumulation of free proline constitutes a defense mechanism against oxidative stress. This accumulation is determined mainly by the rate three independent events: degradation, biosynthesis and transport. In this work we identified a proline permease called TcAAP1, which belongs to the Amino Acids/Auxin Permeases superfamily (TcAAAP). This protein of 476 aa, has 9-10 transmembrane spanners, with an N-terminal domain of 90 aa completely variable in terms of primary structure. This feature constitutes a characteristic of all the TcAAAP family. Using deletion mutant yeast strains, lacking the general amino acid permease (GAP1) and the proline permease (PUT4), and the plasmid pDR196-TcAAP1, complementation assays were performed. Yeasts transformed with TcAAP1 were able to grow in minimal medium using proline as the single nitrogen source. In addition, yeast transformed with TcAAP1 showed a proline transport rate up to 10-folds higher than controls. Finally, a TcAAP1 overexpression model was constructed in T. cruzi epimastigotes. In this model the subcellular localization of TcAAP1 will be established using the endogenous tri-FLAG tag, and also the parasites will be tested for in vitro differentiation assays and for oxidative stress resistance.