Cytochrome P450 reductases in Trypanosoma cruzi. TcCPR-B confers increased drug resistance.

PORTAL, P.; FERNÁNDEZ VILLAMIL, S.; DE VAS, M.; ALONSO, G.; FLAWIÁ, M.M.; TORRES, H.N.; PAVETO, C.

Mar del Plata, Buenos Aires

Congreso; XLIII Reunión Anual de SAIB; 2007

SAIB

Cytochrome P450 enzymes (CYPs) are involved in reactive oxygen species detoxification cascade and in the biosynthesis of endogenous compounds. The flavoenzyme Cytochrome P450 reductase (CPR) is the electron donor for the CYP catalytic activities. Three sequences codifying for respective proteins homologous to CPRs, named TcCPR-A, TcCPR-B and TcCPR-C, all of them showing the FMN, FAD and NADPH domains characteristic of reductases superfamily were found in the Trypanosoma cruzi genome database. Their aminoacidic sequences share 11% identity and differ mainly in the amino-terminal region. They were cloned, expressed and purified from bacterial systems. The recombinant proteins demonstrated a NADPH-dependent cytochrome c reductase activity, partially inhibited by a flavoprotein inhibitor (DPI). Native enzymes expression was demonstrated by Northern blot in epimastigotes CL Brener strain. Kinetical behaviour of the recombinant TcCPRs resemble the previously reported NADPH-dependent cytochrome c reductase activity demonstrated in T. cruzi by biochemical approaches. Stable transfection of TcCPR-B, was confirmed in T.cruzi epimastigotes by Southern blot. Overexpression was demonstrated by Western blot, augmented specific activity and IFI. These overexpressing parasites showed increased resistance to the anti-trypanosomal drugs Nifurtimox and Benznidazole.