Overexpression of citochrome P450 reductases in Trypanosoma cruzi. Subcellular localization and drug resistance.

DE VAS MATÍAS; PORTAL PATRICIO; FERNÁNDEZ VILLAMIL SILVIA; ALONSO GUILLERMO; TORRES HÉCTOR; FLAWIÁ MIRTHA; PAVETO CRISTINA

Rosario. Santa Fe, Argentina.

Congreso; VIII Congreso Argentino de Protozoologia y Enfermedades Parasitarias.; 2008

NADPH-dependent cytochrome P450 reductases (CPRs) belong to a large family of electron transfer flavoproteins that utilize both FAD and FMN as tightly bound prosthetic groups. CPRs catalyze the electron transfer from NADPH to cytochrome P450 (CYP). These enzymes have an important role in reactions involved in dotoxification mechanisms of endogenous and xenobiotic compounds as well as biosynthesis of compounds such as ergosterol. In our laboratory we have reported the identification and characterization of three CPRs of T. cruzi: TcCPR-A, TcCPR-B y TcCPR-C. The predicted proteins have an aminoacidic sequence similarity of 11% with a partial difference in the N-terminal region, and present the characteristic FMN, FAD and NADPH binding domains. Through the expression vectors pTREX and pTEX-GFP, we have generated the following constructions: pTREX-TcCPR-B, pTREX-TcCPR-C and pTEX-TcCPR-C-GFP, which then were utilized for transfecting epimastigotes of the strain CL Brenner. The two first constructions integrate in the parasite genome, while the GFP fusion plasmid remains as an episome. The transgenic parasites pTREX-TcCPR-B and pTREX-TcCPR-C have been selected and confirmed the presence of the constructions by Southern blot as well as respective gene overexpression by Northern blot. The expression of pTEX-TcCPR-C-GFP was confirmed by fluorescent microscopy. Subcellular localization will be analyzed by confocal microscopy. At present we are establishing the redox state and trypanocidal drug resistance in these parasites.