Identification and characterization of an aldo-keto reductase from Trypanosoma cruzi and its putative role in the trypanocidal activity of quinines

GARAVAGLIA, PA; CANNATA, JB; RUIZ, AM; MAUGERI, D; DURAN, R; GALLEANO, M; LAVERRIERE, M; GARCIA, GA

Mar del Plata

Congreso; IX Congreso de Protozoología y Enfermedades Parasitarias.; 2011

Soc. Argentina de Protozoologia

Identification and characterization of an aldo-keto reductase from Trypanosoma cruzi and its putative role in the trypanocidal activity of quinones Garavaglia PA1, Cannata JJB2, 3,.Ruiz AM1, Maugeri D2, Duran R4, Galleano M5, Laverrière M2 and García GA1 1INP “Dr. Mario Fatala Chaben”. 2IIB-INTECH, UNSAM-CONICET. 3CEFYBO Facultad de Medicina-UBA. 4Instituto Pasteur de Montevideo. 5Fisicoquímica-PRALIB Facultad de Farmacia y Bioquímica-UBA Drugs currently used for treatment of Trypanosoma cruzi infection, the ethiological agent of Chagas’ disease, have shown side effects and variable efficiency. With the aim to describe parasite enzymes involved in the mechanisms of action of trypanocidal drugs and since it has been reported that reductases are crucial in their metabolism, we attempted to identify novel NADPH-dependent oxido-reductases from T. cruzi. The percolation of a soluble fraction of epimastigote lysates through a Cibacron Blue-Sepharose column followed by elution by NADPH yielded a predominant protein with an apparent molecular weight of 32 kDa. This protein was identified by MALDI-TOF as an aldo-keto redutase (AKR) and hence denominated TcAKR. TcAKR was mainly localized in the cytosol and was also present in trypomastigote and amastigote lysates. The recombinant TcAKR (recTcAKR) showed NADPH-dependent reductase activity with the AKR substrates 4-nitrobenzaldehyde (4-NBA) and 2-dihydroxyacetone (2-DHA). The saturation curves for both substrates were consistent with the Michaelis-Menten model. As many naphthoquinones (NQ) have displayed important trypanocidal activity, we also tested the hypothesis that TcAKR may reduce these compounds. recTcAKR did not show quinone oxido-reductase (QOR) activity with ρ-NQ, such as 5-hydroxy-1,4-NQ, 2-hydroxy-1,4-NQ, α-lapachone and menadione. However, it showed QOR activity with the following ο-NQ: 1,2-naphthoquinone (1,2-NQ), 9,10-phenanthrenquinone (9,10-PQ) and β-lapachone with concomitant generation of reactive oxygen species (ROS). ROS production was evidenced by the following facts: 1) TcAKR reduction of 9,10-PQ and 1,2-NQ lead to formation of superoxide anion detected by reduction of acetylated cytochrome C and 2) Electron spin resonance experiments revealed that hydroxyl radical was formed after reduction of 9,10-PQ and -lapachone. The substrate saturation curve with ο-NQ fitted to a sigmoidal curve, suggesting that recTcAKR presents a cooperative behavior. In accordance with this, three peaks assigned to monomers, dimers and tetramers were obtained when recTcAKR was submitted to a Superose 12 gel chromatography column. In addition, monomeric and dimeric forms were also detected in epimastigote lysates separated under native conditions by BN-PAGE and analyzed by western blot confirming the oligomeric nature of this enzyme in vivo. In order to study if TcAKR participates in the mechanisms of action of o-NQ in parasites, we evaluated the effect of these drugs in epimastigotes over-expressing the recombinant enzyme. TcAKR gene was cloned in the tetracycline-inducible expression vector for Trypanosoma cruzi pTcINDEX and this construction was then used to transfect epimastigotes that constitutively express the T7 RNA polymerase and the tetracycline repressor. After 72 h of the addition of tetracycline, the level of TcAKR detected by western blot increased substantially and this correlated with about 2 to 4-fold increase of the enzyme specific activity using 9,10-PQ or 4-NBA as substrates. We then studied the survival of both tetracycline- and non-induced TcAKR transfected parasites upon the treatment with the o-NQ used as substrates by the TcAKR. Tetracycline-induced parasites showed similar IC50 values than non-induced controls when they were treated with 1,2-NQ or 9,10-PQ. However, upon the treatment with -lapachone the IC50 value shifted from 7.9 µM to 5.1 µM for TcAKR over-expressing epimastigotes (P < 0.05), indicating they were more susceptible to this drug and suggesting TcAKR may be involved in the trypanocidal effect of -lapachone. The physiological role of the TcAKR in the parasite remains to be elucidated. Although the TcAKR amino-acid sequence shows the highest percentage of identity with the prostaglandin F2 synthase from Trypanosoma brucei, we could not detect NADPH reduction when prostaglandin H2 was used as substrate for recTcAKR, which suggests that this enzyme may not be involved in prostaglandin synthesis. On the other hand, the use of 2-DHA as substrate and its similarity to a glycerol dehydrogenase from Saccharomyces cerevisiae allow us to hypothesize that TcAKR may fulfill a related function in T. cruzi, therefore at this time we are trying hard to test this possibility. In conclusion, we report the identification, cloning and biochemical characterization of the TcAKR, a novel NADPH-dependent oxido-reductase from T. cruzi and the first member of the AKR superfamily described in this parasite. In addition, we show evidence that this enzyme may participate in the mechanisms of action of trypanocidal drugs such as ο-NQ with the subsequent generation of free radicals.