Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei.

The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has been suggested that glycolysis is the sole source of ATP in all bloodstream forms. However, earlier results indicated that in the mitochondria of the transitional intermediate/short stumpy bloodstream forms, the biochemical pathways are present that could allow intra-mitochondrial production of ATP. Using a high mannitol buffer to enhance permeability, we confirm previous observations showing that transitional forms maintain motility and respiratory activity with 2-oxoglutarate as the sole substrate. Using a luminometer to measure intracellular ATP levels via the luciferin/luciferase chemiluminescence assay, we show that these same transitional forms, but not long slender forms, maintain high levels of intracellular ATP in the presence of 2-oxoglutarate. Further, in the presence of bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, ATP levels are reduced with subsequent death and lysis of the cells when 2-oxoglutarate, but not glucose, is used as sole substrate. These data are direct evidence of ATP production by transitional bloodstream form mitochondria.

Herbicides to curb human parasitic infections: in vitro and in vivo effects of trifluralin on the trypanosomatid protozoans.

Leishmaniasis is a major tropical disease for which current chemotherapies, pentavalent antimonials, are inadequate and cause severe side effects. It has been reported that trifluralin, a microtubule-disrupting herbicide, is inhibitory to Leishmania amazonensis. In this study, the in vitro effect of trifluralin on different species of trypanosomatid protozoans was determined. In addition to L. amazonensis, trifluralin is effective against Leishmania major and Leishmania tropica, which cause cutaneous infections, Leishmania donovani, which causes visceral disease, Leishmania panamensis, which may cause mucocutaneous infection, and Trypanosoma brucei, an important human and veterinary pathogen. Moreover, most encouragingly, trifluralin is effective in vivo as a topical ointment against L. major and Leishmania mexicana murine cutaneous leishmaniasis. Thus, trifluralin is a promising lead drug for several related, prevalent tropical diseases: leishmaniasis, trypanosomiasis of animals, and, possibly, African trypanosomiasis in humans.

N-n-alkyl-3,4-dihydroxybenzamides as inhibitors of the trypanosome alternative oxidase: activity in vitro and in vivo.

On the basis of our previous demonstration of the high inhibitory activity of a series of p-n-alkyloxybenzhydroxamic acids and n-alkyl esters of 3,4-dihydroxybenzoic acid against the trypanosome alternative oxidase in a cell-free mitochondrial preparation of Trypanosoma brucei brucei, we synthesized a series of N-n-alkyl-3,4-dihydroxybenzamides for evaluation as inhibitors of this enzyme. This class of compounds was selected with the expectation of their having similar inhibitory activity to but greater solubility than the esters and hydroxamic acids noted above and greater resistance to serum hydrolases in vivo. We predicted that such properties would allow an inhibitor of the trypanosome alternative oxidase to be coadministered with glycerol as a means of providing treatment for infections by African trypanosomes. As expected, such benzamides were both more soluble and more stable, some being more active against the target enzyme than the corresponding ester. One, N-n-butyl-3,4-dihydroxybenzamide, was selected for evaluation in vivo against T. brucei brucei. When combined with glycerol, this benzamide was found to be curative. A regimen wherein 450 mg of N-n-butyl-3,4-dihydroxybenzamide per kg and 15 g of glycerol per kg were given hourly in three divided doses cured 17 of 19 mice with established T. brucei brucei infections. This combination is more active in vivo than any other designed to block simultaneously both the unique respiratory electron transport system and the anaerobic glycolytic pathways of these pathogenic protozoa.

Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor.

Here we report the presence of a trans-sialidase on the surface of Trypanosoma brucei culture-derived procyclic trypomastigotes. The enzyme is not detected in lysates of bloodstream trypomastigotes enriched for either stumpy or slender forms. The trans-sialidase catalyzes the transfer of alpha(2-3)-linked sialic acid residues to lactose. beta-galactopyranosyl residues are at least 100 times better acceptors for sialic acid than alpha-galactopyranosyl residues. In the absence of efficient acceptors, the purified enzyme transfers sialic acid to water, i.e., it acts as a sialidase. Although the T. cruzi and T. brucei trans-sialidases have very similar donor and acceptor specificities, they are antigenically distinct. Sodium dodecyl sulfate-polyacramide gel electrophoresis under nonreducing conditions and silver staining of the purified trans-sialidase reveals a single band of 63 kD. When the surface membrane of live procyclic trypomastigotes is trans-sialylated, using radioactive sialyllactose as the donor substrate, it appears that the only sialylated surface molecule is procyclin. Pronase treatment of live parasites removes only part of the surface sialic acid, in agreement with recent data showing that the glycosylphosphatidylinositol anchor of procyclin is sialylated (Ferguson, M. A. J., M. Murray, H. Rutherford, and M. J. McConville. 1993. Biochem. J. In press).

Trypanosoma (Nannomonas) congolense: changes in respiratory metabolism during the life cycle.

All four life cycle stages (bloodstream, procyclic, epimastigote, and metacyclic) of Trypanosoma congolense IL 3000 were assayed with an oxygen electrode (polarograph) for the presence of terminal oxidases and carbon-source preference. In addition, these stages were used for histochemical analysis of mitochondrial activity using rhodamine 123, nitroblue tetrazolium, and diaminobenzidine. Morphometry was used to compare mitochondrial volumes and surface area among the different life cycle stages. It was found that in contrast to epimastigote forms, which were metabolically almost identical to procyclic forms, metacyclic forms showed characteristics of, and seemed preadapted to, differentiation into the bloodstream stage. While mitochondrial NAD+ diaphorase activity and an electrochemical potential were detected in all life cycle stages, metacyclic metabolism was glucose-based and terminal oxidase activity was primarily dependent upon the trypanosome alternative oxidase with the contribution of cyanide-sensitive respiration accounting for only 20-30% of the total respiratory capacity.

Differential expression of the oligomycin-sensitive ATPase in bloodstream forms of Trypanosoma brucei brucei.

We report the differential expression of the oligomycin-sensitive mitochondrial ATPase in pleomorphic bloodstream forms of Trypanosoma brucei brucei as observed with enzymatic assays and electron microscope histochemistry. As the cells differentiate from long slender to short stumpy forms, total specific activity of the mitochondrial ATPase in a crude mitochondrial fraction doubles and the oligomycin-sensitive specific activity increases 5-fold. Upon in vitro differentiation to procyclic forms, there is a further doubling of total specific activity and a further tripling of oligomycin-sensitive specific activity. The oligomycin-insensitive ATPase activity remained essentially constant throughout differentiation. We have attempted to characterize this oligomycin-insensitive activity utilizing inhibitors of several other ATPases.

Mitochondrial development in Trypanosoma brucei brucei transitional bloodstream forms.

Intermediate and short stumpy bloodstream forms of Trypanosoma brucei brucei are transitional stages in the differentiation of mammal-infective long slender bloodstream forms into the procyclic forms found in the midgut of the tsetse vector. Although the mitochondria of the proliferative long slender forms do not accumulate rhodamine 123, the mitochondria of the transitional forms attain this ability thus revealing the development of an electromotive force (EMF) across the inner mitochondrial membrane. The EMF is inhibited by 2,4-dinitrophenol, rotenone and salicylhydroxamic acid but not by antimycin A or cyanide. Consequently, NADH dehydrogenase, site I of oxidative phosphorylation, is the source of the EMF and the plant-like trypanosome alternative oxidase (TAO) supports the electron flow serving as the terminal oxidase of the chain. Although the TAO is present in the long slender forms as well, it serves only as the terminal oxidase for electrons from glycerol-3-phosphate dehydrogenase. The data presented here, combined with older data, lead to the conclusion that the mitochondria of transitional intermediate and short stumpy forms likely produce ATP. This putative production is either by F1F0 ATPase driven by the complex I proton pump or by mitochondrial substrate level phosphorylation, or most likely by both. These conclusions contrast with the previously held dogma that all bloodstream form mitochondria are incapable of ATP production.

Respiration of bloodstream forms of the parasite Trypanosoma brucei brucei is dependent on a plant-like alternative oxidase.

CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants. The parasites contain 206 ng of CoQ9 mg protein-1 which co-sediments with respiratory activity. The redox state of this CoQ responds in a manner consistent with respiratory function: 60% being in the reduced form when substrate is available and the oxidase is blocked; 13% being in the reduced form when the oxidase is functioning and there is no substrate. The addition of CoQ to aceton-extracted cells stimulates salicylhydroxamic acid-sensitive respiration by 56%. After inhibition of respiration by digitonin-mediated dispersal of the electron transport components, liposomes restore 40% of respiratory activity while liposomes containing CoQ restore 66% of this activity. A less hydrophobic analogue, reduced decyl CoQ, serves as a direct substrate for the trypanosome oxidase supporting full salicylhydroxamic acid-sensitive respiration. After digitonin disruption of electron transport, the nonreduced form of this synthetic substrate can reestablish the chain by accepting electrons from dispersed sn-glycerol-3-phosphate dehydrogenase and transferring them to the dispersed oxidase. Similarities between the alternative oxidase of plants and the oxidase of the trypanosome respiratory system include: mitochondrial location, lack of oxidative phosphorylation, linkage of a dehydrogenase and an oxidase by CoQ, lack of sensitivity to a range of mitochondrial inhibitors, and sensitivity to a spectrum of inhibitors which selectively block transfer of electrons from reduced CoQ to the terminal oxidase but do not block electron transfer to the cytochrome bc1 complex of the mammalian cytochrome chain.

Trypanocidal CoQ analogues: their effect on other mitochondrial systems.

1. A comparative study of compounds which inhibit the respiration of the infective form of the protozoan parasite Trypanosoma brucei brucei, such as salicylhydroxamic acid, other substituted benzhydroxamic acid, esters of 2,3- and 3,4-dihydroxybenzoic acid and structurally related compounds, showed that they have a remarkable degree of selectivity for the trypanosome as compared to rat liver mitochondria even though they are putative CoQ analogues and both respiratory systems are dependent on CoQ. 2. The minimal inhibition of mammalian mitochondrial function could not be assigned to inhibition of ubiquinone function in these mitochondria. 3. CoQ-reducing mitochondrial dehydrogenases from rat liver, trypanosomes and skunk cabbage (Symplocarpus foetidus) were insensitive to these inhibitors. 4. The alternative oxidase of skunk cabbage mitochondria was sensitive to a spectrum of trypanosome respiration inhibitors suggesting a similarity to the oxidase of the trypanosome although differing degrees of sensitivity and differing responses to alterations in the molecular structure of the inhibitors indicate that the milieu of the active sites are dissimilar.

Effects of the ornithine decarboxylase inhibitors DL-alpha-difluoromethylornithine and alpha-monofluoromethyldehydroornithine methyl ester alone and in combination with suramin against Trypanosoma brucei brucei central nervous system models.

Two ornithine decarboxylase inhibitors, DL-alpha-difluoromethylornithine (eflornithine; DFMO) and a-monofluoromethyldehydroornithine methyl ester (delta MFMO X CH3) were compared in their ability to cure two distinct Trypanosoma brucei brucei central nervous system murine model infections. Both inhibitors cured the TREU 667 and LUMP 1001 isolates if used in combination with a single (20 mg/kg) injection of suramin, a trypanocide in current clinical use. The curative dose of delta MFMO X CH3 in combination with suramin was 1.09 g/kg/day, administered in the drinking water for 14 days; used with suramin, the curative dose of DFMO was 5.3 g/kg/day for 14 days (5 times the delta MFMO X CH3 dose required). In host animals, delta MFMO X CH3 was not toxic and was accumulated by trypanosomes 6-8 times faster than DFMO. Since DFMO by itself has been highly effective against T. b. gambiense infections in humans (12-15 g/day for 6 weeks) the present data suggest that delta MFMO X CH3 might be effective in a shorter regimen and at lower doses.

Esters of 3,4-dihydroxybenzoic acid, highly effective inhibitors of the sn-glycerol-3-phosphate oxidase of Trypanosoma brucei brucei.

Alkyl esters of 3,4-dihydroxybenzoic acid are inhibitors of the sn-glycerol-3-phosphate oxidase system of Trypanosoma brucei brucei in vitro and have significant trypanocidal activity in vivo when combined with glycerol. While the parent acid has little inhibitory activity in vitro, the esters are highly active with activity increasing as the chain length of the esterifying alcohol increases. The n-dodecyl ester was more than 400 times as active as salicylhydroxamic acid and 15 times more active than the corresponding p-n-alkyloxybenzhydroxamic acid, one of the most active sn-glycerol-3-phosphate oxidase inhibitors previously reported. When combined with glycerol (to block an alternative pathway of glycolysis) and tested in vitro against intact parasites, this ester was 100 times more effective than salicylhydroxamic acid and 10 times more effective than p-n-dodecyloxybenzhydroxamic acid. It was also active against T. b. brucei in mice when combined with glycerol whereas the latter compound was not. Esters of 3,4,5-trihydroxybenzoic acid (gallic acid) were also highly active while those of 2,3-dihydroxybenzoic acid were much less inhibitory and those of 2,5-dihydroxybenzoic acid were inactive. A related compound, 2',4',5'-trihydroxybutyrophenone, was also active as predicted by its structure but was too toxic to be of interest as a drug candidate.

p-Alkyloxybenzhydroxamic acids, effective inhibitors of the trypanosome glycerol-3-phosphate oxidase.

Energy production in bloodstream forms of African trypanosomes of the genus Trypanosoma involves two pathways unique to the parasite and which can be blocked by a combination of salicylhydroxamic acid (SHAM) and glycerol. Although this leads to rapid parasite destruction both in vitro and in vivo, the toxicity of SHAM precludes practical use of SHAM/glycerol as a therapeutic regimen. Based on our hypothesis that SHAM operates by interfering with ubiquinone, we attempted to develop this approach by synthesizing and screening a series of hydroxamic acids which more closely resemble ubiquinone: the p-n-alkyloxybenzhydroxamic acids. We also examined a variety of mono-, di- and trisubstituted benzhydroxamic acids together with a selected group of secondary heterocyclic hydroxamic acids. We found an increase in activity of the p-n-alkyloxy compounds with increasing chain length up to 12 carbon atoms with longer chains offering little advantage. The most active compound, p-n-tetradecyloxybenzhydroxamic acid, had an apparent Ki of 0.43 microM indicating a specific activity 70 times greater than SHAM. Although this represents a vast improvement, the low water solubility of these compounds reduces their bioavailability to the point where they are not practical substitutes for SHAM. Consequently, improvement in the SHAM/glycerol approach to chemotherapy appears to lie with improving solubility by altering lipophilicity of the alkyl side chain.

New drug combination for experimental late-stage African trypanosomiasis: DL-alpha-difluoromethylornithine (DFMO) with suramin.

Using a previously described mouse model of late-stage African trypanosomiasis (i.e., involvement of the central nervous system), we demonstrate that a combination of DL-alpha-difluoromethylornithine (DFMO) and suramin is curative. In the curative protocol, DFMO is given as a 2% solution in the drinking water for 14 days and suramin is administered as a single dose (20 mg/kg intravenously) on day 1 of DFMO administration. Since: 1) DFMO has very low toxicity, 2) suramin is one of the least toxic of the presently used trypanocides, and 3) suramin and DFMO act synergistically in mouse models of both acute and late stage trypanosomiasis, we conclude that this combination offers special promise in the treatment of African trypanosomiasis in man.