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The causative agent of Chagas disease, Trypanosoma cruzi, metabolizes glucose through two major pathways: glycolysis and the pentose phosphate pathway. Glucose is taken up via one facilitated transporter and its catabolism by the glycolytic pathway leads to the excretion of reduced products, succinate and l-alanine, even in the presence of oxygen; the first six enzymes are located in a peroxisome-like organelle, the glycosome, and the lack of regulatory controls in hexokinase and phosphofructokinase results in the lack of the Pasteur effect. All of the enzymes of the pentose phosphate pathway are present in the four major stages of the parasite's life cycle, and some of them are possible targets for chemotherapy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase are present, but there is no reserve polysaccharide.

b Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC 50 ) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH-and NADH-dependent reductases.C hagas' disease, caused by the protozoan Trypanosoma cruzi, affects 11 million people in Latin America (1). The parasite is usually transmitted to humans and other mammals by triatomine bugs. However, transmission can also be oral (through contaminated food), congenital, or transfusional or by organ transplantation, even in countries where the disease is not endemic (2). The course of infection includes an acute phase followed by an indeterminate phase without symptoms, which lasts throughout life in most infected people. Approximately 30% of patients can develop a chronic phase characterized by cardiac and/or digestive pathologies that can lead to death (3).Benznidazole (Bz) and, to a lesser extent, nifurtimox (Nx) are the drugs currently used for treatment of T. cruzi infection (4). Although the mechanism of action of these nitroheterocyclic compounds is not clearly understood, it is known that, to exert their trypanocidal effects, they need to be activated through the reduction of their nitro group (5). Several studies have proposed that an NADH-dependent trypanosomal type I nitroreductase (NTR I) is a key enzyme which catalyzes Bz and Nx activation in vivo (6,7,8,9). However, evidence indicates that although Bz is reduced to its anion radical, redox cycling may not be relevant for its mode of action, as ...

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We found that, as in African trypanosomes, endogenous phospholipase A1 (Plase A1) activity can catalyse extensive deacylation of phospholipids upon cell death in all life stages of Trypanosoma cruzi. A major lysosomal Plase A1 was purified and characterized. The enzyme products can explain the lesions surrounding degenerating T. cruzi cells in host tissues. Thus Plase A1 emerges as a target to block pathogenesis in trypanosomal infections.

The non-invasive technique of 3C-nuclear magnetic resonance was applied to study glucose metabolism in vivo in Trypanosoma cruzi, the causative agent of American trypanosomiasis (Chagas' disease). It was found that under anaerobic conditions [l-13C]glucose undergoes a glycolytic pathway whose main metabolic products were identified as [3-13C]alanine, [2-13C]succinate and ph~sphoryl[l-'~C]choline; [2-' 3C]alanine was also a minor metabolite. The addition of 70% 'HzO to the incubation mixture led to the formation of [3-13C, 3-'H]alanine derived from the prior incorporation of 'H+ into pyruvate. The existence of a [3-13C, 3-2H]pyruvate precursor, although not isolated, could be inferred from the formation of [2-13C, 2-2H]succinate in the same experiment. The latter derives from the C 0 2 fixation reaction on pyruvate or phosphocnolpyruvate to give malate, which is then converted to succinate through the fumarate intermediate step. The presence of [2-13C]alanine must be traced to a randomization of label at the malate-fumarate stage. Both creted by C. fasciculata into the incubation medium, and to establish that the primary C 0 2 fixation reaction on phosphoenolpyruvate is mediated by phosphoenolpyruvate carboxykinase. Recent studies [3, 81 have indicated that in T. cruzi an important final product of glucose catabolism is L-alanine, which may be also linked to NADH oxidation [3]. The same studies, employing enzymatic assays of catabolites in culture [2,8] We have therefore applied the non-invasive technique of 13C-NMR to the study of [l-'3C]glucose catabolism by intact T. cruzi cells. The results presented in this paper confirm succinate and L-alanine as the main catabolites of glucose, without production of glycerol or ethanol. Small amounts of acetate, arising from glucose, are also produced. Using sodium [13C]bicarbonate together with [l-'3C]glucose, it was shown that C 0 2 is fixed on a C3 acid which arises from glucose.A new and yet unreported COz reduction reaction to give phosphorylcholine was also detected. By using the deuteration technique mentioned above it was possible to establish that in T. cruzi there are two different L-alanine pools; the metabolites of one of them have free access to exchange with 'H20, while in the other no incorporation of deuterium could be detected.

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