Ornithine Decarboxylase and Trypanothione Reductase Genes in Leishmania braziliensis guyanensis

. Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC I. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania .     

Identification and functional characterization of thioredoxin from Trypanosoma brucei brucei

Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.     

Trypanosomatidae codon usage and GC distribution.

A study of Trypanosomatidae GC distribution and codon usage is presented. The codon usage patterns in coincidence with the phylogenetical data are similar in Crithidia and Leishmania, whereas they are more divergent in Trypanosoma brucei and T. cruzi. The analysis of the GC mutational pressure in these organisms reveals that T. brucei, and to a lesser extent T. cruzi, have evolved towards a more balanced use of all bases, whereas Leishmania and Crithidia retain features of a primeval genetic apparatus. Tables with the approximated GC mutational pressure in homologous genes, and codon usage in Trypanosomatidae are presented.     

Overexpression of the putative thiol conjugate transporter TbMRPA causes melarsoprol resistance in Trypanosoma brucei

Melaminophenyl arsenical drugs are a mainstay of chemotherapy against late-stage African sleeping sickness, but drug resistance is increasingly prevalent. We describe here the characterization of two genes encoding putative metal-thiol conjugate transporters from Trypanosoma brucei. The two proteins, TbMRPA and TbMRPE, were each overexpressed in trypanosomes, with or without co-expression of two key enzymes in trypanothione biosynthesis, ornithine decarboxylase and gamma-glutamyl-cysteine synthetase. Overexpression of gamma-glutamyl-cysteine synthetase resulted in a twofold increase in cellular trypanothione, whereas overexpression of ornithine decarboxylase had no effect on the trypanothione level. The overexpression of TbMRPA resulted in a 10-fold increase in the IC50 of melarsoprol. The overexpression of the trypanothione biosynthetic enzymes alone gave two- to fourfold melarsoprol resistance, but did not enhance resistance caused by MRPA. Overexpression of TbMRPE had little effect on susceptibility to melarsoprol but did give two- to threefold resistance to suramin.     

Trypanothione-dependent synthesis of deoxyribonucleotides by Trypanosoma brucei ribonucleotide reductase

Trypanosoma brucei, the causative agent of African sleeping sickness, synthesizes deoxyribonucleotides via a classical eukaryotic class I ribonucleotide reductase. The unique thiol metabolism of trypanosomatids in which the nearly ubiquitous glutathione reductase is replaced by a trypanothione reductase prompted us to study the nature of thiols providing reducing equivalents for the parasite synthesis of DNA precursors. Here we show that the dithiol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione, is a direct reductant of T. brucei ribonucleotide reductase with a K(m) value of 2 mm. This is the first example of a natural low molecular mass thiol directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryparedoxin, a 16-kDa parasite protein with a WCPPC active site motif. The K(m) value of T. brucei ribonucleotide reductase for T. brucei tryparedoxin is about 4 micrometer. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regulation of deoxyribonucleotide synthesis by the redox state of the cell. The trypanothione/tryparedoxin system is a new system providing electrons for a class I ribonucleotide reductase, in addition to the well known thioredoxin and glutaredoxin systems described in other organisms.     

The thiol-based redox networks of pathogens: unexploited targets in the search for new drugs

Hydroperoxide metabolism in diverse pathogens is reviewed under consideration of involved enzymes as potential drug targets. The common denominator of the peroxidase systems of Trypanosoma, Leishmania, Plasmodium, and Mycobacterium species is the use of NAD(P)H to reduce hydroperoxides including peroxynitrite via a flavin-containing disulfide reductase, a thioredoxin (Trx)-related protein and a peroxidase that operates with thiol catalysis. In Plasmodium falciparum, thioredoxin- and glutathione dependent systems appear to be linked via glutaredoxin and plasmoredoxin to terminal thioredoxin peroxidases belonging to both, the peroxiredoxin (Prx) and glutathione peroxidase (GPx) family. In Mycobacterium tuberculosis, a catalase-type peroxidase is complemented by the typical 2-C-Prx AhpC that, in contrast to most bacteria, is reduced by TrxC, and an atypical 2-C-Prx reduced by TrxB or C. A most complex variation of the scheme is found in trypanosomatids, where the unique redox metabolite trypanothione reduces the thioredoxin-related tryparedoxin, which fuels Prx- and GPx-type peroxidases as well as ribonucleotide reductase. In Trypanosoma brucei and Leishmania donovani the system has been shown to be essential for viability and virulence by inversed genetics. It is concluded that optimum efficacy can be expected from inhibitors of the most upstream components of the redox cascades. For trypanosomatids attractive validated drug targets are trypanothione reductase and trypanothione synthetase; for mycobacteria thioredoxin reductase appears most appealing, while in Plasmodium simultaneous inhibition of both the thioredoxin and the glutathione pathway appears advisable to avoid mutual substitution in co-substrate supply to the peroxidases. Financial and organisational needs to translate the scientific progress into applicable drugs are discussed under consideration of the socio-economic impact of the addressed diseases.     

Enzymatic inhibitory activity and trypanocidal effects of extracts and compounds from Siphoneugena densiflora O. Berg and Vitex polygama Cham

Hexanic, methanolic, and hydroalcoholic extracts, and 34 isolated compounds from Vitex polygama Cham. (Lamiaceae, formely Verbenaceae) and Siphoneugena densiflora O. Berg (Myrtaceae) were screened for their trypanocidal effects on bloodstream forms of Trypanosoma cruzi and T brucei, as well as for their enzymatic inhibitory activities on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and trypanothione reductase (TR) enzymes from T cruzi and adeninephosphoribosyl transferase (APRT) enzyme from Leishmania tarentolae. In general, polar extracts displayed strong effects and some of the tested compounds have shown good results in comparison to positive controls of the bioassays.     

Evolution of codon usage and base contents in kinetoplastid protozoans

In this study we analyze and compare the trends in codon usage in five representative species of kinetoplastid protozoans (Crithidia fasciculata, Leishmania donovani, L. major, Trypanosoma cruzi and T. brucei), with the purpose of investigating the processes underlying these trends. A principal component analysis shows that the G+C content at the third codon position represents the main source of codon-usage variation, both within species (among genes) and among species. The non- Trypanosoma species exhibit narrow distributions in codon usage, while both Trypanosoma species present large within-species heterogeneity. The three non-Trypanosoma species have very similar codon-usage preferences. These codon preferences are also shared by the highly expressed genes of T. cruzi and to a lesser degree by those of T. brucei. This leads to the conclusion that the codon preferences shared by these species are the ancestral ones in the kinetoplastids. On the other hand, the study of noncoding sequences shows that Trypanosoma species exhibit mutational biases toward A + T richness, while the non- Trypanosoma species present mutational pressure in the opposite direction. These data taken together allow us to infer the origin of the different codon-usage distributions observed in the five species studied. In C. fasciculata and Leishmania, both mutational biases and (translational) selection pull toward G + C richness, resulting in a narrow distribution. In Trypanosoma species the mutational pressure toward A + T richness produced a shift in their genomes that differentially affected coding and noncoding sequences. The effect of these pressures on the third codon position of genes seems to have been inversely proportional to the level of gene expression.      

Glutathionylation of trypanosomal thiol redox proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.     

Functional and physicochemical characterization of the thioredoxin system in Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses a single thioredoxin that has an unusually high pI value of 8.5 and lacks a conserved aspartyl residue claimed to be involved in catalysis in other thioredoxins. Despite these peculiarities, T. brucei thioredoxin behaves like classical thioredoxins. It is reduced by thioredoxin reductases from different species, serves as donor of reducing equivalents for the ribonucleotide reductase of the parasite, and catalyzes the reduction of protein disulfides. The redox potential of -267 mV was obtained from protein-protein redox equilibration with Escherichia coli thioredoxin. The pK value of T. brucei thioredoxin was determined by two different methods. Carboxamidomethylation of the reduced protein yielded a pK value of 7.4 and generated mono-alkylated protein. The thiolate absorption at 240 nm resulted in a pK of 7.6 and, based on the extinction coefficient of 11.6 mm- 1 cm-1, there are two (or three) cysteines titrating with very similar pK values. A thioredoxin reductase has not yet been detected in any organism of the order Kinetoplastida. T. brucei thioredoxin is spontaneously reduced by trypanothione (bis(glutathionyl)spermidine). Obviously, a specific thioredoxin reductase is not required as thioredoxin reduction can be conducted by the parasite-specific trypanothione/trypanothione reductase system.     

Comparative codon and amino acid composition analysis of Tritryps-conspicuous features of Leishmania major

Comparative analyses of codon/amino acid usage in Leishmania major, Trypanosoma brucei and Trypanosoma cruzi reveal that gene expressivity and GC-bias play key roles in shaping the gene composition of all three parasites, and protein composition of L. major only. In T. brucei and T. cruzi, the major contributors to the variation in protein composition are hydropathy and/or aromaticity. Principle of Cost Minimization is followed by T. brucei, disregarded by T. cruzi and opposed by L. major. Slowly evolving highly expressed gene-products of L. major bear signatures of relatively AT-rich ancestor, while faster evolution under GC-bias has characterized the lowly expressed genes of the species by higher GC12-content.     

Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic machanism

Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.     

Synthesis and evaluation of 9,9-dimethylxanthene tricyclics against trypanothione reductase, Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani

Derivatives of 9,9-dimethylxanthene were synthesised and evaluated against trypanothione reductase (TR) and in vitro against parasitic trypanosomes and leishmania. High in vitro antiparasitic activity was observed for some derivatives with one compound showing high activity against all three parasites (ED50 values of 0.02, 0.48 and 0.32 microM, for Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani, respectively). The lack of correlation between inhibitory activity against TR and ED50 values suggests that TR is not the target.     

Methylglyoxal metabolism in trypanosomes and leishmania

Methylglyoxal is a toxic by-product of glycolysis and other metabolic pathways. In mammalian cells, the principal route for detoxification of this reactive metabolite is via the glutathione-dependent glyoxalase pathway forming d-lactate, involving lactoylglutathione lyase (GLO1; EC 4.4.1.5) and hydroxyacylglutathione hydrolase (GLO2; EC 3.2.1.6). In contrast, the equivalent enzymes in the trypanosomatid parasites Trypanosoma cruzi and Leishmania spp. show >200-fold selectivity for glutathionylspermidine and trypanothione over glutathione and are therefore sensu stricto lactoylglutathionylspermidine lyases (EC 4.4.1.-) and hydroxyacylglutathionylspermidine hydrolases (EC 3.2.1.-). The unique substrate specificity of the parasite glyoxalase enzymes can be directly attributed to their unusual active site architecture. The African trypanosome differs from these parasites in that it lacks GLO1 and converts methylglyoxal to l-lactate rather than d-lactate. Since Trypanosoma brucei is the most sensitive of the trypanosomatids to methylglyoxal toxicity, the absence of a complete and functional glyoxalase pathway in these parasites is perplexing. Alternative routes of methylglyoxal detoxification in T. brucei are discussed along with the potential of exploiting trypanosomatid glyoxalase enzymes as targets for anti-parasitic chemotherapy.     

Elevated levels of polyamines and trypanothione resulting from overexpression of the ornithine decarboxylase gene in arsenite-resistant Leishmania

The levels of trypanothione, a glutathione-spermidine conjugate, are increased in the protozoan parasite Leishmania selected for resistance to the heavy metal arsenite. The levels of putrescine and spermidine were increased in resistant mutants. This increase is mediated by overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Gene overexpression is generally mediated by gene amplification in Leishmania but, here, the mRNA and the enzymatic activity of ODC are increased without gene amplification. This RNA overexpression is stable when cells are grown in the absence of the drug and does not result from gene rearrangements or from an increased rate of RNA synthesis. Transient transfections suggest that mutations in the revertant cells contribute to these elevated levels of RNA. Stable transfection of the ODC gene increases the level of trypanothione, which can contribute to arsenite resistance. In addition to ODC overexpression, the gene for the ABC transporter PGPA is amplified in the mutants. The co-transfection of the ODC and PGPA genes confers resistance in a synergistic fashion in partial revertants, also suggesting that PGPA recognizes metals conjugated to trypanothione.     

Targeting the polyamine biosynthetic enzymes: a promising approach to therapy of African sleeping sickness, Chagas’ disease, and leishmaniasis

Summary. Trypanosomatids depend on spermidine for growth and survival. Consequently, enzymes involved in spermidine synthesis and utilization, i.e. arginase, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase, trypanothione synthetase (TryS), and trypanothione reductase (TryR), are promising targets for drug development. The ODC inhibitor α-difluoromethylornithine (DFMO) is about to become a first-line drug against human late-stage gambiense sleeping sickness. Another ODC inhibitor, 3-aminooxy-1-aminopropane (APA), is considerably more effective than DFMO against Leishmania promastigotes and amastigotes multiplying in macrophages. AdoMetDC inhibitors can cure animals infected with isolates from patients with rhodesiense sleeping sickness and leishmaniasis, but have not been tested on humans. The antiparasitic effects of inhibitors of polyamine and trypanothione formation, reviewed here, emphasize the relevance of these enzymes as drug targets. By taking advantage of the differences in enzyme structure between parasite and host, it should be possible to design new drugs that can selectively kill the parasites.     

Molecular studies on trypanothione reductase, a target for antiparasitic drugs

Trypanosoma and Leishmania are parasitic protozoa that cause a variety of diseases, which include African sleeping sickness and oriental sore. Attempts to determine pharmaceutically exploitable differences between host and parasite biochemistry have identified the unique trypanothione pathway as a possible target. This pathway includes the enzyme trypanothione reductase, the parasite analogue of glutathione reductase.     

A second class of peroxidases linked to the trypanothione metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, has three nearly identical genes encoding cysteine homologues of classical selenocysteine-containing glutathione peroxidases. The proteins are expressed in the mammalian and insect stages of the parasite. One of the genes, which contains a mitochondrial as well as a glycosomal targeting signal has been overexpressed. The recombinant T. brucei peroxidase has a high preference for the trypanothione/tryparedoxin couple as electron donor for the reduction of different hydroperoxides but accepts also T. brucei thioredoxin. The apparent rate constants k(2)' for the regeneration of the reduced enzyme are 2 x 10(5) m(-1) s(-1) with tryparedoxin and 5 x 10(3) m(-1) s(-1) with thioredoxin. No saturation kinetics was observed and the rate-limiting step of the overall reaction is reduction of the hydroperoxide. With glutathione, the peroxidase has marginal activity and reduction of the enzymes becomes limiting with a k(2)' value of 3 m (-1) s(-1). The T. brucei peroxidase, in contrast to the related Trypanosoma cruzi enzyme, also accepts hydrogen peroxide as substrate. The catalytic efficiency of the peroxidase studied here is comparable with that of the peroxiredoxin-like tryparedoxin peroxidases, which shows that trypanosomes possess two distinct peroxidase systems both dependent on the unique dithiol trypanothione.     

Polyamine metabolism in <Emphasis Type="Italic">Leishmania</Emphasis>: from arginine to trypanothione

Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)₂) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.