Trypanocidal effect of SKF525A, proadifen, on different developmental forms of Trypanosoma cruzi.

SKF525A, an inhibitor and inducer of cytochrome P450, was tested on different developmental stages of Trypanosoma cruzi. Growth, motility and structure of epimastigotes, motility and infectivity of trypomastigotes, and infectivity of trypomastigotes to Vero cells in culture were abolished by the drug at 10-100 microM concentrations. When blood from infected mice was treated with the drug, and used to infect 8 day-old mice, no parasites were observed at 0.6-1 mM, and all animals survived. Blood cell morphology was well preserved, and the sleeping time of pentobarbital-treated mice inoculated with the same amount of drug was not increased. The present results suggest that SKF525A or other related inhibitors of cytochrome P450 coned be tested as an additive for blood sterilization in blood banks.

Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi.

Three proteinase inhibitors, one peptidyl acyloxymethyl ketone (AMK), Z-Phe-Lys-CH2-OCO-(2,4,6-Me3)Ph.HCl, and two diazomethyl ketones (DMKs), Z-Phe-Phe-DMK and Z-Phe-Ala-DMK, have been studied for their effects in vitro on the four developmental stages of Trypanosoma cruzi. The three inhibitors penetrated living parasites and inhibited the major cysteine proteinase, cruzipain. The AMK was the most potent inhibitor of cruzipain itself and at 20 microM caused lysis of epimastigotes and trypomastigotes. When at lower concentrations, however, it had little effect on epimastigote growth but reduced metacyclogenesis. The DMKs had no effect against epimastigotes but inhibited differentiation to metacyclics. All three inhibitors markedly reduced infection of Vero cells by the parasite and the multiplication of the intracellular amastigotes, whereas release of trypomastigotes was almost entirely prevented. The results confirm the importance of cysteine proteinases in the life cycle of T. cruzi, and suggest that the differentiation steps are the most susceptible to cysteine proteinase inhibitors.

Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine on Trypanosoma cruzi.

The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes of Trypanosoma cruzi, at concentrations lower than 100 microM, and motility and infectivity of the bloodstream trypomastigote form at 200 microM. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 microM TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.

Chromosomal localization of seven cloned antigen genes provides evidence of diploidy and further demonstration of karyotype variability in Trypanosoma cruzi.

The karyotype of Trypanosoma cruzi was studied by pulsed field gel electrophoresis (PFGE) in conditions that allowed 20-25 chromosome bands to be detected. However, several of these bands were present in non-equimolar amounts, suggesting that the total chromosome number is considerably higher. The patterns obtained with the different cloned and uncloned strains were unique, suggesting that the karyotype of T. cruzi is highly variable. The chromosomal localizations of seven cloned genes were determined by Southern blotting of PFGE-separated chromosomes. Three of the clones gave rise to similar patterns and mapped on a chromosome or a family of chromosomes larger than 1.6 Mb. Two clones mapped on either single or pairs of chromosomes, which in some cases differed considerably in size between the different strains tested, suggesting that extensive chromosome rearrangements occur in T. cruzi. Another clone hybridized to several chromosomes in most strains and probably represents a family of genes. Lastly, one clone hybridized to nearly all chromosomes. Many of the clones hybridized to pairs of restriction fragments in the different strains, suggesting that they are allelic. For one of the clones it was possible to provide further evidence for the allelic nature of the fragments by establishing detailed restriction maps around them and by showing that the two fragments in a pair hybridized to chromosomes which differed slightly in size. Taken together, the results infer that the genome of T. cruzi epimastigotes is diploid.

Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi.

A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.

On the regulatory properties of the pyruvate kinase from Trypanosoma cruzi epimastigotes.

The pyruvate kinase from Trypanosoma cruzi epimastigotes was activated by fructose 2,6-diphosphate ((A) 0.5 = 0.17 microM), through a decrease in (S) 0.5 and an increase in Vmax for both substrates. The enzyme was 50% inhibited by 0.9 mM ATP or 0.5 mM Pi in the presence of 30 mM MgCl2; these inhibitions were completely counteracted by 1.5 microM fructose 2,6-diphosphate. Both facts suggest that the effects are allosteric, and not due to chelation.

Interaction of benznidazole reactive metabolites with nuclear and kinetoplastic DNA, proteins and lipids from Trypanosoma cruzi.

Epimastigotes of Trypanosoma cruzi (Tulahuen strain Tul 0 stock) biotransform benznidazole (N-benzyl-2-nitro-1-imidazole acetamide) to reactive metabolites that bind covalently to DNA, proteins and lipids of the parasite. These effects might be related to the trypanocidal action of benznidazole, a chemotherapeutic agent against Chagas' disease.

Aerobic glucose fermentation by Trypanosoma cruzi axenic culture amastigote-like forms during growth and differentiation to epimastigotes.

Axenic culture amastigote-like forms of Trypanosoma cruzi, grown at 28 degrees C, reach a stationary phase after two generations, and differentiate to epimastigotes, which then resume growth. Axenic culture amastigotes readily ferment glucose to succinate and acetate, and do not excrete NH3; they have high activities of hexokinase and phosphoenolpyruvate carboxykinase, and very low citrate synthase activity; cytochrome o is absent, and cytochrome b-like is present at a very low level. Epimastigotes catabolize glucose and produce succinate and acetate at a considerably lower rate; they exhibit lower levels of hexokinase and carboxykinase, and much higher levels of citrate synthase and cytochromes o and b-like. They catabolize amino acids, as shown by excretion of NH3 to the medium. The results suggest that axenic culture amastigotes have an essentially glycolytic metabolism, and they acquire the ability to oxidize substrates such as amino acids only after differentiation to epimastigotes.

End products and enzyme levels of aerobic glucose fermentation in trypanosomatids.

Trypanosoma cruzi (epimastigotes), Crithidia fasciculata and Leishmania mexicana (promastigotes) were grown in a brain-heart-tryptose medium supplemented with heat-inactivated fetal calf serum. T. cruzi and C. fasciculata utilized glucose completely during the log phase of growth, whereas L. mexicana used significant amounts of the carbohydrate only at the end of the log phase and at the beginning of the stationary phase. In all cases glucose consumption resulted in excretion of succinate, and much smaller amounts of acetate. C. fasciculata and L. mexicana produced very small amounts of pyruvate. C. fasciculata produced ethanol, which was taken up again and metabolysed after glucose was exhausted. Lactate and malate were not produced. The cells were disrupted by sonic disintegration, and the activities of some key enzymes of carbohydrate and amino acid catabolism were assayed in the whole homogenates. Phosphoenolpyruvate carboxykinase was present in the three organisms; L. mexicana presented the highest specific activity. The activity of this enzyme was maximal during glucose consumption, and slightly decreased after glucose was exhausted. This suggests that the role played by the enzyme is glycolytic and not gluconeogenic; the latter is the case in most higher organisms. Hexokinase and pyruvate kinase presented their highest levels in C. fasciculata and T. cruzi during glucose consumption. L. mexicana, which was in active glycolysis during the whole experimental period, presented the highest specific activities of both enzymes. Citrate synthase, on the other hand, increased in C. fasciculata and, to a lesser extent, in T. cruzi, after glucose was exhausted; the enzyme could not be detected in L. mexicana. The NAD-linked glutamate dehydrogenase increased considerably in C. fasciculata and T. cruzi after glucose was exhausted, suggesting a catabolic role for the enzyme. This increase coincided with an increase in NH3 production by both organisms after glucose consumption. The NADP-linked glutamate dehydrogenase, on the other hand, presented a maximum about the time when glucose was exhausted, and then decreased again, which suggests a catabolic role for the enzyme. Both glutamate dehydrogenases had low activities in L. mexicana; this fits in well with the low NH3 production throughout the culture of this organism. The results are in good agreement with current ideas on the mechanism of aerobic glucose fermentation by trypanosomatids, and suggest that, under the experimental conditions used, both T. cruzi and C. fasciculata used glucose perferentially over amino acids for growth.

Inhibition by suramin of oxidative phosphorylation in Crithidia fasciculata.

1. The ADP plus Pi-stimulated oxidation of succinate by mitochondria from the insect trypanosomatid Crithidia fasciculata was maximally inhibited (64%) by suramin at a concentration (60 microM) which did not affect the electron transport uncoupled by FCCP. Inhibition of the latter required a considerably higher concentration of the drug, 50% inhibition being attained at about 0.8 mM. 2. ATP synthesis by mitochondrial particles was inhibited by suramin, 50% inhibition being attained at about 50 microM. This inhibition was strictly competitive towards ADP, but it was not linearly competitive, since a secondary plot of apparent Km values vs concentration of the drug was strongly concave upwards. 3. The FCCP-stimulated ATPase activity of the mitochondrial particles was completely abolished either by oligomycin (20 micrograms/ml) or by 200 microM suramin. 4. The results suggest that oxidative phosphorylation may be a primary target for the trypanocide effect of suramin on organisms having, like C. fasciculata, a well-developed respiratory chain.